0.35, x = p/p0 , allows to obtain the monolayer capacity (Nm). In the above N is the amount of adsorbed water vapour, and CBET is a constant. Next, the surface area is calculated from the dependence: S=N ·M-1·L·σ where L is the Avogadro number (6.02·1023 molecules per mole), M is the molecular weight of gas or vapour (gram per mole) and σ is the molecule cross-sectional m
166
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
area for gas or vapour (10.8·10 -20 m2 for water molecule). The total carbon content was measured using TOC Analyzer Multi N / C 2000, HT 1300.
serve as sorption sites for water molecules. Furthermore, it is worth to mention that organic matter may absorb water molecules in deeper layer of soil or organic matter particles. In this case, higher values of SSA are referred to several processes associated with changes of structure, absorption or with both.
Results and Discussion
REFERENCES
(1) Chiou, C.T.; J-F Lee; Boyd, S.A. Envron. Sci. Technol. 1990, 24, 1164-1166. (2) Churchman, G. J.; Burke, C.M. J. Soil Sci. 1991, 42, 463-478. (3) Feller, Ch.; Schouller, E.; Thomas, F.; Rouiller, J.; Herbillon, A.J. Soil Sci. 1992, 153, 293-299. (4) Torres Sanchez, R.M.; Falasca, S. Z. Pflanz. Bodenk. 1997, 160, 223-226. (5) Sokołowska, Z.; Józefaciuk, G.; Sokołowski, S.; Urumova-Pleszeva, A. Clays a. Clay Minerals. 1993, 41, 346-352. (6) Yijie, M.; Chaoliang, Y. Soil Res. Rep. 1989, 20, 112. (7) Sikora, L.J.; Filgueira, R.R.; Fournier, L.L.; Rawls, W.J.; Pachepsky, Ya.A. Int. Agrophys. 2002, 16, 289-295. (8) Tester, C.F. Soil Sci. Soc. Am. J. 1990, 54, 827-831. (9) Sokołowska, Z.; Borowko, M.; Reszko-Zygmunt, J.; Sokołowski, S. Geoderma. 2002, 107, 33-54.
Figure 1. Exemplary correlations for Hplic Luvisol and
Hplic Podzol with addition of HA (Fluka), sodium salt of HA and fertilizer. S is the surface area, TC means total carbon content.
Figure 1 presents examples of correlations between specific surface area and total carbon content. The influence of the amount of organic matter on the SSA values was confirmed. The specific surface areas were higher for mixtures with Hplic Luvisol than Hplic Podzol. They varied in the range from 5.31 to 143.16 m2·g-1 for Hplic Podzol and from 15.88 to 151.23 m2·g-1 for Hplic Luvisol. For all samples the volume of vapor adsorbed was significantly higher for higher addition of organic matter. The linear relationship between the values SSA and the amount of organic matter was observed in each case. Almost linear correlation (R2>0.99) means that the studied systems can be treated as mechanical mixtures, with small interactions between components. The adsorption of water vapour is associated with the presence of various kinds of the polar and nonpolar functional groups which are present mostly in soil organic matter and organic additives. The most important are the functional groups containing oxygen, such as carboxyls, phenolics and carbonyls. These groups
167
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Fouling formation and chemical control in drip-irrigation systems using treated wastewater
S. Katz *, J. Tarchitzky, Y. Chen
The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, P.O.B 12, Israel * Corresponding author e-mail: [email protected] Keywords: Biofouling, NOM, Deposit, Drip-irrigation, Chlorine. Abstract: Drip irrigation systems (DIS) are clogged due to chemical precipitation and natural organic matter (NOM) fouling. In this research a novel approach was applied aiming to compare different maintenance treatments designed to prevent the NOM development caused by biofilm, and the proper function of DIS. Chlorination and acidification strategies showed that daily chlorination and periodic acidification may prolong proper function of the drippers by maintaining a normal flow-rate (FR) [(up to ±7%) of nominal FR] and coefficients of variation (CV) index (<7%) in correlation with low fouling accumulation in the pipeline (<0.01 mg deposit/cm2pipe). Current recommendations for DIS treatments were found to be insufficient. Chemical analyses of the fouling inside the DIS showed that biofilm can survive inside the dripper under harsh environmental conditions, even when the pipeline stays clean. These results shed light on biofilm growth and survival mechanisms and may pave the way to developing novel effective treatments.
Research on the characterization of deposits inside the dripper is therefore lacking. Evaluation of the efficacy of the various available treatments is also lacking. This research aims to characterize the differences between various preventive treatments designed to reduce the phenomena of NOM and chemical fouling and their damage to the drip-irrigation system using TWW.
Introduction Dissolved and suspended components in treated waste water (TWW) can be classified into four main groups: Suspended mineral particles; Suspended and soluble organic matter; Soluble salts and Biological populations. Biological fouling and the extracellular polymeric substances produced by the fouling bacteria are the main causes of damage to irrigation systems utilizing TWW. Interactions between these components affect their features and behavior in solution under different conditions (electrolyte-concentration, ionic-strength or pH values). NOM and mineral particles Interactions such as aggregate size, stability as well as in the survivability of biological activity can be change by environmental conditions. These processes have a critical impact on the nature of the water treatment required for DIS function control. Bacteria attached to surfaces or bound to organic substances can prevail and/or continue to grow in harsh environmental conditions. They can grow through porous aggregates of clay minerals, or in large organic aggregate. Once biofilms have formed, they are durable under a variety of environmental conditions, and continuous treatment is required to prevent the biofilm development and growth. To maintain proper function of the DIS, chemicals are needed on a regular basis. At present, in order to remove chemical deposits, it is recommended that the pH of the water be lowered (to pH 6), and to remove biological deposits, it is recommended that treatment with oxidizers or biocides—usually hypochlorite, be used. Previous studies led to two treatment options: (1) a single onetime treatment; and (2) a periodical treatment. Although deposits in DIS are a known phenomenon reported in the literature, only a few of these publications to date have reported on quantifying and characterizing the deposits, and comparing their impacts on FR and uniformity (CV). Scientific publications in recent years have not included chemical characterizations of localized deposits in the pipes.
Method A model system was established in a wastewater treatment plant in Nir Ezion, Israel in 2011. The system was designed to compare the FR, CV, fouling accumulation and fouling composition in laterals and drippers subjected to different treatments. Deposit characterization included deposit weight, the content of NOM, the ionic composition, dissolved organic carbon (DOC) and C/N ratio, as well as identification of functional groups (FTIR) and SEM. In addition binocular photos of the internal parts of the drippers were taken . Results and Discussion Based on the model system, the current research showed that partial DIS malfunction (when using TWW for irrigation) that are not treated, are more severe than in systems receiving regular treatments of chlorine and acid. Deposit accumulation was higher and much faster in the control (C) pipes (1.4–3.6 mg deposit/cm2 pipe), causing a decrease in FR and increase CV. In the chlorinated treatments (OCl), no significant deposit development has been observed in the pipes (0–0.2 mg deposit/cm2 pipe). Rapid formation of deposits and a rapid decrease in FR were found after stopping the treatment with chlorine, suggesting a similar formation mechanism in different systems and the need for ongoing treatments. A high correlation (R2 = 0.65–0.88) was found between the deposit weight and the change in FR (Fig. 1).
168
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
flocculation value and d spacing of individual component., Our observations suggest that interacting components such as clay and OM create an environment favoring bacterial and biofilm growth. These features changed as the biological activity was eliminated by oxidizers.
Fig 1. Change in flow rate (FR) relative to the nominal FR and fouling weight per cm2 of pipe as a function of operation time. C – Control; OCl-D-A – Daily chlorination and periodic acidification.
The chlorination strategy recommended by us are continuous treatment at the end of each irrigation cycle and periodically acidification (each six weeks). The composition of the deposit was generally the same in all treatments, except for the carbon and nitrogen, which were at higher concentrations in the non-chlorinated treatments compared to the chlorinated ones. Higher carbon content was found in the control treatment and it correlated well to the deposit weight (200–300 µg/cm2 pipe; 25% of deposit weight). No carbon was detected in the chlorinated treatment. Most of the carbon and all of the nitrogen were of organic nature. The NOM content was found to be 40% to 60% of the deposit weight in the middle of the lateral for most deposits in the experiment. The strongest impact was detected at the end of the lateral lines. The FR at the end section was up to 10 times lower than that at the beginning of the lateral. The deposit weight per cm2 of pipe was higher at the beginning of the lateral (2 mg deposit/cm2 pipe), compared to the end (0.4 mg deposit/cm2 pipe). NOM content was higher at the end of the lateral (75%) than at the beginning (50%), suggesting more biological activity, which increased dripper clogging. Dripper location and flow velocity have an influence on the accumulation of deposits and FR. These results demonstrate the need for ongoing treatment to prevent deposit formation. The dripper deposits from all treatments were analyzed and the samples exhibited similar compositions except for the amount of carbon, measured as dissolved organic carbon (DOC), which was higher in the control treatment (60 µg/cm2 dripper) than in the chlorinated one (20 µg/cm2 dripper). DOC per cm2 of drippers was 2 to 10 times higher than in the pipes. Deposited salts weight per cm2 of drippers was higher than that in the pipes for the chlorinated treatment, and vice versa in the control treatment. These results suggest that the dripper structure facilitates deposit formation. Our results may also imply better bioavailability of the chlorinated OM inside the drippers. Indeed, binocular photos taken inside a dripper demonstrated the formation of water path based on preferential flow (Fig. 2) induced by biofilm formation. In laboratory experiments, we showed that the bacteria-clay interaction can change their features when interacted jointly in suspensions, compared to the individual effect on of each component on the
Fig. 2. Binocular photos of the water flow path inside the drippers. The red line indicates the water-flow path and the brown area is the fouling material accumulated in the dripper.
REFERENCES
Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284, 1318-1322. (1) De Beer D, Srinivasan R, Stewart PS (1994) Direct measurement of chlorine penetration into biofilms during disinfection. Applied and Environmental Microbiology 60, 4339-4344. (2) Flemming HC (2002) Biofouling in water systems— cases, causes and countermeasures. Applied Microbiology and Biotechnology 59, 629-640. (3) Flemming HC, Schaule G, Griebe T, Schmitt J, Tamachkiarowa A (1997) Biofouling—the Achilles heel of membrane processes. Desalination 113, 215-225. (4) Li JS, Chen L, Li YF, Yin JF, Zhang H (2010) Effects of chlorination schemes on clogging in drip emitters during applications of sewage effluent. Applied Engineering in Agriculture 26, 565-578. (5) Ravina I, Paz E, Sofer Z, Marcu A, Shisha A, Sagi G (1992) Control of emitter clogging in drip irrigation with reclaimed wastewater. Irrigation Science 13, 129-139. (6) Tarchitzky J, Rimon A, Kenig E, Dosoretz CG, Chen Y (2013) Biological and chemical fouling in drip irrigation systems utilizing treated wastewater. Irrigation Science 31, 1277-1288..
Acknowledgments: The authors gratefully acknowledge the financial support of the BMBFGermany and the MOST-Israel, in the project: “On-line detection, treatment and reduction of biofilm and chemical scaling in irrigation systems utilizing treated wastewater (TWW)"; we wish to extend our thanks to the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development financialy supportin this project.
169
IHSS 2014 IOANNINA, GREECE 1-5 SEPTEMBER
Study of pH Effect on Solid Phase Extraction (SPE) of Suwannee River Dissolved Organic Matter (SR DOM) Y. Li (a), N. Hertkorn (a)*, S. Dvorski (a), M. Lucio(a), M. Harir (a), P. Schmitt-Kopplin(a,b) (a)
Helmholtz Zentrum München, German Research Center for Environmental Health, Research Unit Analytical Biogeochemistry (BGC), Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany (b) Chair of Analytical Food Chemistry, Technische Universität München, 85354 Freising, Germany * Corresponding author e-mail: [email protected] Keywords: pH, SPE, SR DOM, FTICR MS, NMR, statistical analysis Abstract Solid phase extraction (SPE) combines ease of use with acceptable carbon yield and has become an established method of dissolved organic matter (DOM) isolation from water. Here, PPL cartridges were used to process solutions of Suwannee River dissolved organic matter (SR DOM) at different pH. Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and nuclear magnetic resonance (NMR) data revealed that SR DOM obtained at pH 2 after SPE contained the most representative assembly of organic compounds. At lower pH, side reactions were commonly observed, whereas at increasing pH, ever larger proportions of compounds were not retained on the cartridges. Statistical analysis suggested that molecular compositions with lower O/C ratios correlate with higher pH. The objective of this was to investigate the effect of pH variation on the yield of Suwannee River dissolved organic matter and SPE-DOM molecular composition. We have utilized PPL cartridges for isolation of SPEDOM, and have applied the high resolution FTICR MS to assess molecular composition and 1H NMR spectroscopy for quantification of key substructures.
Introduction Dissolved organic matter (DOM) is a collection of organic compounds with ~50% carbon content and variable proportions of heteroatoms such as oxygen, nitrogen, sulphur and phosphorus (1). DOM is one of the Earth’s largest active carbon pools and actively involved in biodegradation, photodegradation, microbial metabolism like nitrogen assimilation and fatty acids catabolism. DOM is also one of the most complex mixtures of organic molecules known on Earth. The elucidation of DOM on a molecular composition and structure is imperative to understand global carbon cycle (1). However, due to its compositional heterogeneity and low concentration, DOM is difficult to extract from water, which challenges the molecular analysis (1, 2). A considerable number of methods have been developed to extract and isolate DOM such as dialysis, ultrafiltration, reverse osmosis and the combination of reverse osmosis and electrodialysis (2). Among those, solid phase extraction (SPE) turns out to be one of the most widely used techniques due to its convenience, especially in field studies, and pleasing carbon recovery in the range of ~50% (1-3). SPE of DOM depends on interactions between dissolved heterogeneous molecules and a stationary phase, and is therefore prone to chemical selectivity depending on extraction conditions. Critical variables are pH, solvents used for conditioning and elution, concentration of samples and flow rate (3, 4). Moreover, there are considerable works on pH effects on the molecular composition or optical properties of DOM (5, 6). For example, Tfaily et al. evaluated the acidification of DOM and found that DOM was sensitive to pH, and that oxygen-rich compounds were newly formed after acidification (5). But pH effect on SPE of NOM has not been systematically investigated yet.
Experimental The Suwannee River water was collected on site in 2012. After sampling, SR DOM was immediately filtered and stored at 0 oC until use. SR DOM was subjected to SPE processing according to the methods described by Dittmar et al. (3). After conditioning 100 mg PPL cartridges with MeOH and water (pH 2), SR DOM solutions were loaded on the cartridges, and washed with water (pH 2) to get rid of salts. Then, the cartridges were dried, eluted with 1 mL of MeOH. The eluates were stored at -20 oC immediately after SPE. The FTICR MS and NMR spectra were acquired as described in Hertkorn et al. (7). The data analysis was carried out by using RStudio 0.98.501 (RStudio, Boston, USA) and Simca 13.0.3 (Umetrics, Umea, UHEA, Sweden). Results and Discussion Analytes collected after eluting the cartridges: Significant differences in molecular compositions were observed by comparing the SR DOM eluates obtained at different loading pH, shown in [Figure 1 and Figure 2]. Notably, a more representative complement of SR DOM molecules was obtained at low pH (pH 1 and 2) favoring especially CRAM. Conversely, the proportion of CRAM remarkably decreased as the loading pH increased. At pH 7, the proportion of peptides and aliphatic compounds in the SPE DOM euate seems considerably enlarged. At low pH values, only a small portion of compounds was lost
170
IHSS 2014 IOANNINA, GREECE 1-5 SEPTEMBER
during loading and washing steps, thus making it possible that more compounds were eluted by MeOH.
summary, pH explains 40% of the peaks (770 peaks in all 1911 peaks) that are strongly correlated. [Figure 3] revealed that positively correlating masses were separated from the negatively ones primarily based on their elemental O/C ratios but also differed in relative instauration (H/C ratio). Most of the molecular composition whose mass peak amplitude correlated positively with pH showed O/C ratios < 0.5; whereas mass peak amplitudes correlating negatively with pH showed molecular compositions with O/C ratios > 0.5. Hence, lower O/C elemental ratios correlated with higher pH and vice versa.
Figure 1. FTICR MS derived van Krevelen diagrams of SR DOM molecular formulas
In [Figure 2], we can see that CRAM and methoxy groups and carbohydrates (at δ H ~ 3.5-5 ppm) were depleted with the increasing of loading pH of SR DOM while aliphatics increased in relative abundance. Moreover, special attention should be paid to methoxy protons in the chemical shift range δH ~ 3.5-3.7 ppm which appear at elevated concentration at low pH (from pH 1 to pH 3). These methoxy groups might be produced during SPE processes by esterification between SR DOM and MeOH. Similar findings were also reported by other authors. Flerus et al. detected esterification in MeOH SPE-DOM extract stored at 20 oC by using FTICR MS (4). Tfaily et al. discovered SPE-DOM had higher O/C ratios than organic matter obtained by dialysis (2). In both reports, no NMR data were available to confirm these hypotheses.
Figure 3. van Krevelen diagram illustrating the H/C and O/C ratios of the molecular compositions correlating strongly with pH.
REFERENCES
(1) Hertkorn, N. et al. Anal. Bioanal. Chem. 2007, 389, 13111327. (2) Tfaily, M. M. Anal. Bioanal. Chem. 2012, 404, 447-457. (3) Dittmar, T. et al. Limnol. Oceanogr. 2008, 6, 230-235. (4) Flerus, R. et al. Mar. Chem. 2011, 124, 100-107. (5) Tfaily, M. M. et al. Anal Chim Acta. 2011, 706, 261-267. (6) Yan, M. et al. J. Lumin. 2013, 142, 103-109. (7) Hertkorn, N.et al. Biogeoscience. 2013, 10, 1583-1624. (8) Roth, V.-N. et al. Geochim. Cosmochim. Ac. 2013, 123, 93-105.
Acknowledgments: The authors are thankful for the China Scholarship Council (CSC) for the financial support of Yan Li.
Figure 2. 500 MHz 1H NMR spectra of SPE-DOM obtained after eluting the cartridges with MeOH.
Redundancy analysis (RDA), one constraint ordination technique, was further applied to elucidate the changes of loading pH on the variables (e.g. molecular masses and formulas) in a single integrated analysis (8). From the dataset, molecular masses that exhibited strong correlation (R2 > |0.7|) with pH were extracted. We found 351 masses positively correlated with pH and 419 masses negatively correlating. In
171
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Humic Substances and Organic Matter δ13C Signature of a South Brazilian Cambisol After Charcoal Application O. A. Leal (a) *, D. P. Dick (a), A. D. B. Nebenzahl (a), V. G. Maciel González-Pérez(c), H. Knicker(c), N. T. Jiménez-Morillo(c)
(a)
, K. C. Lombardi
(b)
, J. A.
(a) Federal University of Rio Grande do Sul, Bento Gonçalves Avenue 9500, 91501-970, Porto Alegre, Brazil (b) State University of Centro-Oeste, PR 153 km, 784500-000, Irati, Brazil (c) Institute for Natural Resources and Agrobiology, Reina Mercedes Avenue, n. 10, 41012, Seville, Spain * Corresponding author e-mail: [email protected] Keywords: total carbon content, chemical fractionation, isotopic ratio Abstract Brazil is the world's greatest producer of charcoal, supplying mainly the steel industry. However, 15 % of the production is lost as charcoal fines. Thus, this residue has been proposed as a soil conditioner. Aiming to bring some light on the impact of charcoal on soil organic matter (SOM) composition and chemistry we investigated the effect of charcoal on SOM δ13C signature and on the humic substances distribution of a Cambisol. About 40 Mg ha-1 of charcoal were applied to the soil. After 20 months, soil samples were collected (0-5; 5-10; 10-20; 20-30 cm). The soil without charcoal was also sampled. The main results were observed in the top depth, where the charcoal increased the total carbon content, mainly in its less oxidized fraction (humin). SOM δ13C signature was lowered mainly in the 0-5 cm indicating that charcoal was not relevantly leached and/or incorporated to further depths. with a slight harrow. In September 2011 three field replicates were sampled in four soil depths: 0-5; 5-10; 10-20 and 20-30 cm. Each sample was a composite of three subsamples collected within each plot. Samples of soil without charcoal application were also collected (control). The samples were air dried and passed through a 2.00 mm sieve. The C content of the soil was determined by dry combustion. The SOM chemical fractionation (4) was performed in duplicate and one gram of soil was used. The following fractions were obtained: C as non humic substances (CNHS), humic acid (CHA), fulvic acid (CFA) and humin (CHU). The δ C signature of the charcoal and of the soil samples was determined (quatripletts) in a Flash 2000 HT elemental analyzer coupled to a Delta V Advantage IRMS (Thermo Scientific). Isotopic ratios are reported as 13 parts per thousand (‰) deviations from that of Pee Dee Belemnite. The results were evaluated by standard deviation.
Introduction Brazil is the world's greatest charcoal producer, producing annually 10 million tons, which supplies mainly the steel industry and the pig iron sector. However, approximately 15 % is lost as charcoal fines (1). Currently, there is a trend to use charcoal as a soil conditioner as well as a source of stable carbon (C). Pyrogenic carbon (PyC) behaviour in soils is usually assigned to its high-C content and aromaticity, which partially explains the PyC inherent biochemical recalcitrance (2). In this context, the PyC contained in the charcoal fines is supposed to increase the soil C content and also change the SOM composition. Such changes can be studied through the SOM chemical fractions, which are usually more sensitive than the whole SOM to environmental changes. Furthermore, the changes of the SOM δ13C signature can inform about the impact of the charcoal on the endogenous SOM (1). In Brazil several studies have been performed about the effects of charcoal application on soil properties. However, these studies usually focus on other issues(2)(3) and studies regarding the SOM fractionation and alterations after charcoal application are still needed. Thus, the objective of this study was to investigate the alterations in SOM chemical fractions and in the SOM δ13C signature after the application of charcoal fine residues.
Results and Discussion Total C (TC) content varied between 23.2 and 53.8 g kg-1 and tended to decrease with depth in both treatments (Table 1). Differences between treatments were observed until 20 cm, but were largest at the 0 - 5 cm depth, where the charcoal increased the TC content in 16.6 g kg-1. Despite the charcoal incorporation at 10 cm, probably a considerable amount of it remained at the soil surface. At 20 - 30 cm soil depth, the TC contents for 0 Mg ha-1 (23.2 + 0.21) and 40 Mg ha-1 (29.4 + 6.29) were similar (Table 1). This result shows that charcoal was not leached relevantly along the soil profile within 20 months of its incorporation. The CNHS content varied between 1.18 and 2.02 g kg-1 (Table 1) and did not differ between treatments. This fraction is mainly composed of hydrophilic and small compounds which are related to microbial activity and to the root exudation (5). Due to their biochemical
Experimental The field experiment was implemented in February 2010 at Unicentro, Irati, Brazil. The soil was classified as a Haplic Cambisol. The charcoal used in this experiment came from the fine residues of burned trees (over 45% of the particles smaller than 2 mm) and its main properties are: C content = 46.6 %; fixed C = 7.6 %; ash = 8.22 %; pH = 7.59. The dose of 40 Mg ha-1 of charcoal was applied first on the soil surface and thereafter it was incorporated at 10 cm
172
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
between -16.62 ‰ and -19.12 ‰ in the soil without charcoal, whereas in the soil with charcoal the δ13C varied between -16.41 and -23.42 ‰ (Table 1). Charcoal showed the lowest δ13C, -25.99 ‰. The most pronounced alteration of the SOM δ13C signature was observed at the 0 - 5 cm depth, where δ13C decreased from -19.12 to -23.42 ‰ (Table 1). This result confirms the relevant contribution of PyC to the TC content. Only a slight difference was observed at 10 20 cm depth, where the charcoal application decreased the δ13C signature in -1.18 ‰. The δ13C signature did not differ at the other soil depths, corroborating the assumption that charcoal did not migrate relevantly along the profile within 20 months after its application. This observation also suggests that the increase of CHA at 10 - 20 cm and at 20 - 30 cm depths is an indirect effect of the charcoal application.
lability and to their weak association to the soil -1 mineral matrix, NHS is usually found in lower proportions in soils. The CFA varied between 2.95 and 7.01 g kg (Table 1), representing 18.4 and 13 % of the TC, respectively. This chemical fraction-1 was not affected by the charcoal incorporation. Contrary to CNHS and CFA, the CHA differed between the treatments and varied between 4.14 and 10.3 g kg , which represents 15.1 and 27.7 % of the TC content, respectively. At 10 - 20 and 20 - 30 cm soil depth an increase of 3.67 and 3.42 g kg-1 of CHA, respectively, was observed after charcoal incorporation. Considering that after charcoal application only a slight increase of the TC content occurred at 10 - 20 cm and that differences of TC content at 20-30 cm was not observed, this result suggests that charcoal HU stimulated the formation of HA from the endogenous SOM. The outstanding difference between treatments was observed in the HU fraction. C varied between 13.5 and 35.5 g kg-1 and represented 58.4 and 66 % of the TC content, respectively. This difference was observed at 0 - 5 cm depth, where charcoal increased the CHU content in 16.3 g kg-1. Therefore, it can be concluded that the increase of the TC content at 0-5 cm depth occurred preferentially in the HU fraction. This is best explained by the hydrophobic character of the charcoal, in this way this material tends to accumulate mainly in the non-alkaline extractable fraction, HU. The charcoal is assumed to be more resistant to the degradation than the natural SOM due to its particular chemical composition which includes polycyclic aromatic structures. Higher contribution of charcoal to the HU and HA fractions than to the more labile and mobile fractions (NHS and FA) has been already described by other authors in anthropogenic soils (6)(7), who assigned it to the presence of PyC. Except for the soil without charcoal at 10 - 20 cm, the proportion of chemical fractions decreased as follows HU>HA>FA>NHS. The main effect of the charcoal on distribution pattern was revealed for the HU at the top depth, where the charcoal increased the proportion of HU from 51.5 to 66 %. The main difference in the (CFA+CHA)/CHU ratio was observed in the topsoil. The charcoal application decreased this ratio from 0.85 to 0.47 (Table 1), confirming the contribution of hydrophobic organic compounds to the SOM composition, which are typical of PyC (8). In contrast, the higher (CFA+CHA)/CHU ratio at the 10 - 20 and 20 30 cm confirms the preferential formation of more functionalized humic substances, which are attributed to endogenous HA. SOM δ13C signature varied
Conclusions: The charcoal application increased the total carbon content mainly in its more stable chemical fraction, humin. Indirectly, the charcoal also increased the CHA at 10 - 20 and 20 - 30 cm depth, suggesting that its presence affects the SOM dynamics. The charcoal changed the soil organic matter δ13C signature, mainly in the topsoil (0 - 5 cm). Despite the limited time span of the experiment, relevant leaching of charcoal along the soil profile was not evidenced by our data. Acknowledgments: The authors are greatfull to the CNPq and Capes for the research fellowship and to the UFRGS, Unicentro, IRNAS and the Mineco ( 2011BR0097) for supporting this project. REFERENCES
(1) Maia, C. M. B. F. Embrapa Florestas, 2010, 36p. (2) Steiner C, Teixeira, W. C.; Lehmann, J.; Nehls, T.; Macedo, J. L. V.; Blum, W. E. H.; Zech, W. Plant Soil. 2007, 291, 275-290. (3) Angelo, L. C.; Mangrich, A. S.; Mantovani, K. M.; Santos, S. S. J Soil Sediments. 2014, 14, 353-359. (4) Dick, D.P.; Gomes, J.; Rosinha, P.B. R. Bras. Ci. Solo. 1998, 22, 603-611. (5) Potes, M.L.; Dick, D.P.; Dalmolin, R.S.D.; Knicker, H.; Rosa, A.S. R. Bras. Ci. Solo. 2010, 34, 23-32. (6) Cunha, T. J. F.; Madari, B.E.; Benites, V. M.; Canellas, L. P.; Novotny, E. H.; Moutta, R. O.; Trompowsky, P. M.; Santos, G. A. Acta Amazonica, 2007, 37, 91 - 98. (7) Lima, H. N. PhD thesis. 2001, 176p. (8) Kramer, R.W.; Kujawinski, E.B.; Hatcher, P.G. Enviro. Sci. Tech, 2004, 38, 3387-3395.
Table 1. Total carbon content (TC), content of carbon as non humic substances (CNHS), humic acid (CHA), fulvic acid (CFA) and humin (CHU), (CFA+CHA)/CHU ratio and δ 13C SOM signature of a Cambisol after charcoal application. Charcoal Dose Mg ha0
40
Depth cm 0-5 5 - 10 10 - 20 20 - 30 0-5 5 - 10 10 - 20 20 - 30
TC
CNHS
CHA
CHU
(CFA+CHA)/ CHU
δ13C
10.3 + 1.50 8.43 + 1.12 4.14 + 1.05 4.67 + 1.11 9.73 + 0.20 8.18 + 0.09 7.81 + 0.12 8.09 + 1.75
19.2 + 1.97 20.1 + 2.86 16.5 + 1.68 13.5 + 0.28 35.5 + 3.04 21.7 + 1.34 16.8 + 3.21 15.4 + 5.76
0.85 0.67 0.56 0.56 0.47 0.71 0.81 0.81
-19.12 + 0.27 -19.28 + 0.70 -17.50 + 0.32 -16.62 + 0.18 -23.42 + 0.30 -20.21 + 0.27 -18.68 + 0.38 -16.41 + 0.96
CFA -
37.2 + 1.98 34.7 + 2.87 27.4 + 0.00 23.2 + 0.21 53.8 + 1.34 43.2 + 2.97 31.9 + 2.33 29.4 + 6.29
1.64 + 0.28 1.18 + 0.91 1.70 + 0.85 2.02 + 0.31 1.50 + 1.33 1.26 + 0.56 1.41 + 0.87 1.59 + 1.35
g kg 6.09 + 1.21 5.03 + 1.37 5.05 + 1.88 2.95 + 1.36 7.01 + 0.56 7.28 + 0.98 5.88 + 0.12 4.33 + 0.12
173
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characterization of cyanobacteria-derived intracellular organic matter and its release during cell oxidation J.A. Korak (a) *, E.C. Wert(b), F.L. Rosario-Ortiz(b)
(a) Department of Civil, Environmental and Architectural Engineering, University of Colorado, Boulder, CO 80309 USA (b) Southern Nevada Water Authority, Las Vegas, NV 89193 USA * Corresponding author e-mail: [email protected] Keywords: Cyanobacteria, Intracellular Organic matter, Fluorescence, Oxidation Abstract The objective of this study was to evaluate the feasibility of using fluorescence spectroscopy as a surrogate for cyanobacteria intracellular organic matter (IOM) released into a heterogeneous dissolved organic matter (DOM) matrix due to oxidation processes. IOM was isolated from three cyanobacteria species and characterized using fluorescence. IOM has a distinct optical signature by having a high fluorescence index (FI) greater than 2 and phycobiliproteins that fluoresce in the visible wavelength range. Interaction effects between IOM and DOM were evaluated and found that the highly fluorescent phycobiliproteins are quenched by the presence of DOM. Phycobiliproteins were also readily oxidized decreasing their fluorescence intensity. Both the quenching interactions and rapid oxidation limit their utility as an optical surrogate. FI proved to be the most successful surrogate for IOM release by monitoring compositional changes of the DOM through bench-scale oxidation studies. isolated by first rinsing and centrifuging the cells with buffer to remove extracellular organic matter (EOM). Cells were then lysed using freeze-thaw cycles and sonication3. The IOM was diluted in 10 mM phosphate buffer (pH=7.5) to concentrations of 2 mg C/L for MA and 1 mgC/L for OSC and LYG for oxidation studies. Each IOM was exposed to four different oxidants (ozone (O3), free chlorine (HOCl), chlorine dioxide (ClO2) and chloramine (ClA)) at oxidant:DOC mass ratios between 0 and 2. Chlorine based oxidants were quenched with 100 mg/L sodium thiosulfate after 120 min. Potential interactions between IOM and DOM were characterized by spiking IOM into Colorado River Water (CRW), which has a background DOC of about 2.5 mgC/L, at concentrations from 1 to 3 mgC/L. Finally, intact, rinsed cells were spiked into CRW. The suspension was oxidized with the same four oxidants5. Fluorescence excitation-emission matrices (EEMs) were measured after filtration (0.7 μm GF/F). Excitation wavelengths ranged from 250 to 700 nm (10 nm increments), and emission wavelengths ranged from 300 to 800 nm (2 nm increments). All fluorescence data were corrected and normalized to Raman units (RU) following published methods6. The fluorescence data were analyzed following two metrics. The fluorescence index (FI) was calculated as an indicator of compositional differences with high FI values (~2) as an indicator of less aromatic, microbial DOM and low values (~1.2) as an indicator of more aromatic, terrestrially derived DOM. Cyanobacteria phycobiliproteins were quantified by the overall pigment intensity defined as the sum of all intensities in the range: Ex=450-670 nm and Em=550-750 nm. Results and Discussion Fluorescence EEMs. EEMs of each isolated IOM in phosphate buffer showed two main regions with strong fluorescence signals. Figure 1 breaks the full EEM for OSC apart displaying each region separately. All three
Introduction Cyanobacteria blooms are an area of concern for watershed management and protecting drinking water source quality. Some cyanobacteria produce cyanotoxins, such as microcystin-LR, that may present a human health risk1. Other species produce the metabolites geosmin and 2-methylisoborneol that lead to taste and odor episodes2. In addition to excreting metabolites, cyanobacterial intracellular organic matter (IOM) contains high concentrations of metabolites along with disinfection byproduct precursors3. A number of drinking water treatment plants apply oxidants upstream of physical removal processes to meet various treatment objectives, which has the potential to damage cell integrity and release metabolite-containing IOM4,5. The release of IOM is difficult to quantify, because it is likely found at low concentrations compared to ambient dissolved organic matter (DOM) and may be biodegradable in the environment5. Monitoring the fluorescent components of IOM may be a valuable surrogate of interest. The objective of this study was to evaluate the feasibility of using fluorescence spectroscopy as a surrogate for IOM detection in a heterogeneous DOM matrix. An ideal fluorescence surrogate should exhibit several key characteristics: it has a unique fluorescence signature different from background DOM, its fluorescence signature is not impacted by interactions with DOM, and its signature is not lost upon oxidation in order to act as a conservative tracer. This study investigated each of these three characteristics for IOM extracted from three cyanobacteria species in ultrapure and natural water matrices. Experimental Microcystis aeruginosa (MA), Oscillatoria sp (OSC) and Lyngbya sp (LYG) were cultured in the lab under controlled conditions published elsewhere4. IOM was
174
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
IOM had strong fluorescence at the lower wavelengths (Figure 1a), which has been associated with phenolic and indolic functional groups. A common emission at 460 nm also coincided with the intensities used to calculate the FI. FI values for the IOM were greater (>2.3) than that of CRW, which was in the range of typical surface waters (1.4-1.6). This difference suggests that FI could be a useful indicator for IOM released into a water body. Figure 1b shows that IOM has a strong fluorescence signal in the visible range (between excitation 450-650 nm) indicative of the phycobiliproteins phycocyanin and phycoerythrin 7. The strong pigment fluorescence occurs in a region where DOM is weakly fluorescent and suggests that it may be a viable option for detecting IOM released into a water body.
Excitation Wavelength (nm)
450
OSC a)
FI = 2.65
400
FI
350 300 250 300 650
400
500
600
0.27 0.18 0.09 1 0
0.66
550 500 450 550
calibration to a standard solution would underestimate the presence of IOM. IOM Oxidation. The fluorescence response to oxidation was evaluated to determine the likelihood of measuring IOM in the bulk solution if released by an oxidation process. The more aggressive oxidants (O 3, HOCl and ClO2) decreased FI for the IOM but not CRW. Despite the decrease, FI for the IOM remained higher than that of CRW at all doses. There was no decrease in FI with ClA oxidation, indicating that this oxidant does not change the composition of fluorescent IOM. Despite a change in IOM composition with some oxidants, the IOM fluorescence signature is still different from CRW and a release of IOM may still be detected by measuring FI. The phycobiliproteins reacted readily with every oxidant and rapidly lost their fluorescent signature. Overall pigment intensity decreased by more than 90% at the lowest dose ratio applied for O3, HOCl and ClO2. Unlike FI, the pigment intensities decreased with ClA exposure by 45% to 80% at a dose ratio of 0.5. From a practical standpoint, these results indicate that FI may be a better tool for detecting IOM in a DOM background matrix compared to measuring pigment fluorescence. Cell Oxidation and Release of IOM. Bench scale oxidation experiments with cells suspended in CRW were conducted to determine if there was a fluorescence response upon cell oxidation. FI increases when MA is exposed to all 4 oxidants, indicating IOM release. LYG and OSC had small increases with exposure to free chlorine. No increase in pigment fluorescence was detected upon cell oxidation.
0.33 650 750 Emission Wavelength (nm)
0
Figure 1. EEMs of OSC IOM in the a) UV-Vis region and b) Visible region
REFERENCES
Interactions with DOM. Fluorescence interactions were evaluated by adding IOM to CRW. FI increased with increasing IOM concentrations in a non-linear relationship as shown previously8. While an increase in FI is not representative of the amount of IOM released into a natural water source, it may be a sensitive tool for detecting IOM release by measuring compositional differences. Fluorescence in the pigment region was analyzed to determine if the IOM pigment fluorescence changes in a DOM matrix. When phycocyanin and phycoerythrin standards were spiked into CRW, there was no indication of interactions with DOM that alter either the intensity or fluorescence wavelengths. When IOM was spiked into CRW, severe interactions were observed. The fluorescence intensity is quenched by up to a factor of 20, and fluorescence wavelengths are also altered. The single fluorescence peak observed in the IOM isolate breaks into three smaller peaks indicating a change in the pigment photophysics. Since these interactions were only observed with the IOM and not the pigment standards, it suggests that there is something fundamentally different about the IOM compared to the purchased standards. A practical implication is that phycobiliproteins may not be the best surrogate for detecting IOM release, and
(1) Hitzfeld, Höger & Dietrich. Environ Health Persp 108, 113 (2000). (2) Smith, Boyer & Zimba. Aquaculture 280, 5–20 (2008). (3) Wert & Rosario-Ortiz. Environ Sci Technol 6332– 6340 (2013). (4) Wert, Dong & Rosario-Ortiz. Water Research 47, 3752–3761 (2013). (5) Wert, Korak, Trenholm & Rosario-Ortiz. Water Research 251–259 (2014). (6) Murphy et al. Environ Sci Technol 44, 9405–9412 (2010). (7) MacColl J Struct Biol 124, 311–334 (1998). (8) Korak, Dotson, Summers & Rosario-Ortiz. Water Research 49, 327–338 (2014).
Acknowledgments: The authors acknowledge the Water Research Foundation (project number 4406) for their financial support of this project. JAK acknowledges the National Science Foundation for their support through the Graduate Research Fellowship Program (DGE 1144083). The authors thank Mei Mei Dong with the SNWA Water Quality Research and Development team for preparing the intracellular organic matter standards.
175
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Natural organic matter in urban atmospheric aerosols: profiling the water soluble components using comprehensive two-dimensional liquid chromatography J.T.V. Matos(a) *, R.M.B.O. Duarte(a), A.C. Duarte(a) (a)
Department of Chemistry and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal * Corresponding author e-mail: [email protected] Keywords: Comprehensive two-dimensional liquid chromatography; Complex organic mixtures; Non-target chromatography; Pattern representation; Organic aerosols; Water-soluble organic compounds Abstract The fraction of natural organic matter (NOM) of atmospheric aerosols can be the predominant fraction of the fine suspended matter mass and encompasses a huge variety of molecular structures with different physicochemical properties and sources. However, the lack of knowledge regarding the inherent complexity of their chemical nature is nowadays one of the major impairments to an improved understanding of atmospheric organic matter chemistry and composition, and predicts how these compounds affect the climate system. The use of LC×LC– DAD, combining the use of two independent separation mechanisms has shown a great potential for providing a deeper insight into the complexity of NOM. In this work, besides extending the range of NOM separation, the data obtained by LC×LC–DAD will combined with a proposed mathematical procedure that allows a graphical representation of the chromatographic and spectral profiles, thus providing valuable clues for unfolding the complexity of NOM in organic aerosols. Introduction Despite the enormous challenge that arises from unravelling the complexity of the organic aerosols into their individual components and structures, this challenge, when accomplished, will provide unparalleled rewards towards a better understanding of their role in various atmospheric processes (1). A common approach for resolving the inherent complexity of this organic fraction is to group the different organic components according to their physicochemical properties; being the solubility and affinity for water the most widely used property. The growing interest on the water-soluble organic compounds (WSOC) is fuelled by the realization that this organic fraction covers a highly variable amount (10–80%) of the total organic aerosol and can have an important role in several atmospheric processes (2). In this work, a comprehensive 2D liquid chromatography (LC×LC) coupled with a diode array detection (DAD) procedure will be applied for the first time to identify a profile of variation of NOM in atmospheric particles collected during different seasons and, therefore, improve the chemical resolution of NOM heterogeneity in these samples. The chromatographic conditions used in this study were based on the LC×LC method proposed by Duarte et al. (3) to improve the resolution and reduce the heterogeneity of two well-known complex organic mixtures: Suwannee River Fulvic Acids standard material and Pony Lake Fulvic Acids reference material from the IHSS. In their study, the authors combined the use of two independent separation mechanisms (per-aqueous liquid chromatography (PALC) and size-exclusion chromatography (SEC)) for mapping the hydrophobicity versus molecular weight distribution of the NOM samples using a DAD
operating at a single wavelength. This study, however, will take advantage of the full UV–VIS spectrum provided by the DAD. LC×LC–DAD is especially useful for non-target analysis and identification of patterns based on the information extracted from those complex samples. For this end, a procedure recently introduced by Matos et al. (4) will be used to deal with the large amount of data generated by these systems and provide a 3D fingerprinting for each sample, which alongside the other samples, can be used to identify different patterns associated with the specific properties of every sample under study. Experimental The PM2.5 aerosol samples were collected in an urban location near Aveiro, Portugal. The sampling time was 7 days to collect enough material for the analysis. A total of 20 aerosol samples were collected from November 2009 and March 2011. These aerosol samples were grouped together (groups of three and four samples), according to similar ambient conditions, on a total of five groups representative of different seasonal periods. Each collected filter was entirely extracted with 150mL of ultra-pure water by mechanic stirring during 5 min plus ultrasonic bath during 15 min. The final slurry so obtained was filtered through a membrane filter of 0.22µm pore size. The WSOC samples were then isolated and fractionated by adsorption onto a DAX-8 resin, according to existing protocols (5, 6). For the subsequent LC×LC–DAD analyses, the samples solutions were prepared by diluting each sample in 10% of the mobile phase (v/v) of the first dimension. The LC×LC experimental procedure used in this work is based on proposed by Duarte et al. (3). The first dimension using an Acclaim Mixed-Mode HILIC-1 column was operated in
176
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
isocratic mode using a mobile phase composition consisting of 20mM CH3COONH4 and 10% (v/v) CH3CN. The second dimension using a PSS Suprema 30Å analytical column was also operated in isocratic mode with a mobile phase composition consisting of 20mM NH4HCO3 and 11% (v/v) CH3CN. The effluent from the second column was connected to a DAD operating in a range between 230 and 300 nm.
2C and Figure 2E) and winter (Figure 2D and Figure 2F) seasons. From a chemical point of view, it may be suggested that the chromatographic profile of the winter sample, which shows a wider separation along the first dimension, is compatible with the presence of a larger number of components with higher hydrophobic character than those of the summer sample. Apparently, an increase in the hydrophobic nature of the organic components is accompanied by a decrease in their molecular weight, as suggested by the chromatographic profile of the winter sample in Figure 2B.
Results and Discussion Figure 1 shows the 2D chromatograms recorded at 254 nm of two aerosol WSOC hydrophobic acids samples collected during the same season (autumn) but in two consecutive years: 2009 (Figure 1A) and 2010 (Figure 1B). Overall, both chromatograms indicate that the two NOM samples exhibit fairly similar profiles in terms of molecular weight distribution and hydrophobicity for the same season in different years. A
A
B
C
D
E
F
B
Figure 1. Two-dimensional chromatograms of aerosol WSOC hydrophobic acids collected in (A) autumn 2009 and (B) autumn 2010, recorded by a DAD at 254 nm.
Furthermore, the degree of separation obtained for these two complex samples, underlines the potential of LC×LC technique, not only by showing a remarkable increase in resolution and in resolving the chemical heterogeneity of these complex unknown organic compounds, but also to by correlating two properties of the different organic components in a single chromatographic run, which would be impossible to attain using any of the one-dimensional separation techniques in a totally independent manner. The association of multichannel detectors with multidimensional separation techniques, such as LC×LC– DAD, can be of great help for accomplishing the chemical pattern recognition of NOM in atmospheric aerosols. Nevertheless, the extraction of useful information from large amount of multi-dimensional data still is one of the major drawbacks for a wider application of this technique (7). Figure 2 show the chromatographic profile using the total sum of intensities of the LC×LC–DAD data obtained for the aerosol WSOC hydrophobic acids samples collected during the summer (Figure 2A) and winter (Figure 2B) seasons. Despite the obvious differences between the profiles of the samples, using the total sum of intensities originates a loss of spectral information and, consequently, reduces the amount of information available for the characterization of such samples. In order to avoid this loss, a simple and fast procedure was recently introduced by Matos et al. (4) for identifying the three-dimensional regional maxima of each chromatographic peak generated in a LC×LC– DAD system: retention times at the peak maximum in the first- and the second-dimensions and the wavelength of the maximum UV absorption. As shown in Figure 2, a three-dimensional profile was produced for the samples collected in summer (Figure
Figure 2. Total sum of intensities for the LC×LC–DAD chromatogram of summer (A) and winter (B) samples. Twodimensional and three-dimensional representation of the regional maxima for the LC×LC–DAD data of summer (C and E, respectively) and winter (D and F, respectively) samples.
REFERENCES
(1) R.M.B.O. Duarte, A.C. Duarte, in: M. Simpson, A. Simpson (Eds.), NMR Spectroscopy: A Versatile Tool for Environmental Research, 2014, John Wiley & Sons, Ltd, Chichester, UK. (2) R.M.B.O. Duarte, A.C. Duarte, TrAC Trends Anal. Chem. 2011, 30, 1659–1671. (3) R.M.B.O. Duarte, A.C. Barros, A.C. Duarte, J. Chromatogr. A 2012, 1249, 138–46. (4) J.T. V Matos, R.M.B.O. Duarte, A.C. Duarte, Anal. Chim. Acta 2013, 804, 296–303. (5) R.M.B.O. Duarte, E.B.H. Santos, C.A. Pio, A.C. Duarte, Atmos. Environ. 2007, 41, 8100–8113. (6) R.M.B.O. Duarte, A.M.S. Silva, A.C. Duarte, Environ. Sci. Technol. 2008, 42, 8224–30. (7) J.T. V Matos, R.M.B.O. Duarte, A.C. Duarte, J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2012, 910, 31–45.
Acknowledgments: This work was supported by the Centre for Environmental and Marine Studies (PEsTc/MAR/LA0017/2013, University of Aveiro, Portugal) and the Portuguese Science and Technology Foundation (FCT), through the European Social Fund (ESF) and “Programa Operacional Potencial Humano – POPH”. João T.V. Matos also acknowledges FCT for a PhD grant (SFRH/BD/84247/2012). This work was also funded by FEDER under the Operational Program for Competitiveness Factors – COMPETE and by National funds via FCT within the framework of research project ORGANOSOL (FCOMP-01-0124FEDER-019913; PTDC/CTE-ATM/118551/2010).
177
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The Intra/Inter-Molecular Interactions of NOM Components: A Study of Self-Assembly Using Small Angle Neutron Scattering and PulsedField Gradient NMR C. Johnson-Edler (a) *, G. Chilom(a), L. Hoffman(a) , J. Rice(a)
(a) Department of Chemistry and Biochemistry, South Dakota State University, Box 2202, Brookings, SD 570070896 * [email protected] Keywords: NOM, Self-Assembly, Small Angle Neutron Scattering, Pulsed-Field Gradient NMR, Molecular Interactions Abstract Recent work has shown that natural organic matter (NOM) is a self-assembled material comprised of components of varying chemical composition including humic acid (HA0), a highly aromatic non-amphiphilic fraction (HA1), a lipid-like fraction (L1), and a strongly amphiphilic fraction (HA2) that self-assemble via a hierarchical aggregation process (1). Small Angle Neutron Scattering (SANS) was used to obtain information regarding interactions that occur between NOM components. SANS was also used to establish contrast match points for the lower level components from one material source for future investigation of structural differences between NOM components. Pulsed field gradient (PFG)-NMR data was acquired to generate Diffusion Ordered Spectroscopy (DOSY) spectra to determine the diffusion coefficients of the HA0, HA1, HA2, and L1 humic acid fractions, and the native composite of HA2 and L1 referred to here as L0, from two material sources. Introduction NOM has been shown to self-assemble via a multistep process that involves the interaction of amphiphilic (HA2) and lipid fractions (L1) (1). It has also been suggested that the organizational state of NOM is more closely correlated to NOM’s stability than its chemical characteristics. The hierarchical self-assembling nature of NOM allows the use of a “soft materials” chemistry approach to understand the interactions that may possibly be involved in its creation. Although NOM can be broken down into the three aforementioned fractions, the fractions themselves are still inherently complex. This complexity has been understood to allow NOM to resist enzymatic breakdown by bacteria. However, recently published data show that the assembled NOM materials are more readily biodegraded by bacteria than the disassembled materials (2) . A previous study using AFM and SEM has suggested that particles sizes for all the materials to be analyze range in size from ten to a few hundred nm (3). The shape and structure of these materials, however, is still poorly understood. This paper describes the results obtained from an ongoing study of HA2, L1 and reassembled L0 by SANS for one source material, and PFG-NMR data correlated to a DOSY plot for all fractions of two material types. The overall goal of this investigation is to develop an understanding of the intra- and intermolecular interactions that initiate NOM self-assembly and to develop a descriptive model to describe the organization of NOM’s components using a combination of PFG-NMR and neutron scattering techniques.
reference materials using a traditional alkaline extraction method. These two source materials were chosen for the differencesin their carbontype distributions. The IHSS Leonardite HA0 consists primarily of aliphatic and aromatic carbon types (4) while
Experimental Humic acid (HA0) was isolated from the International Humic Substances Society’s Leonardite (BS104L) and Elliott silt loam soil (BS102M) bulk
1
Figure 1. Flow diagram illustrating fractionation of NOM (the humin portion of NOM has been omitted and only the humic acid fractionation is included.
HA has soila the 0 Elliot typical humic acid carbon-type distribution (4). The lipid fraction L0, and a humic-like fraction HA1 were obtained from HA0 by Soxhlet extraction using a benzene:methanol azeotrope (3:1 v/v, Figure 1) (5). The L0 fraction is a composite material that is separated into HA2 and L1 using an additional alkaline extraction step. Portions of the HA0, HA1, and HA2 fractions of each material type were converted to the hydrogen-form by cation exchange using a column packed with Dowex® 50W-X H+ resin to ensure metals which may interfere with the NMR data acquisition had been removed from samples. H solution-state NMR spectroscopy data were obtained using a Bruker Avance 600 mHz instrument with a 5 mm inverse 1H-13C-15N TXI probe. 1H NMR spectra were obtained using 16 scans with a delay of 2
178
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
F1 [m2/ s * 1e-9]
The importance of these findings in understanding the nature of the self-assembled NOM components in each source material will be discussed. The application of the structural and molecular interaction information obtained in this investigation may contribute to enhanced practices (6) to increase sequestration of organic carbon in the soils and sediments of the Earth.
2.5
2.0
1.5
1.0
(a)
6
4
2
F2 [ ppm]
1.0
F1 [m2/s * 1e-9]
8
2.5
2.0
Results and Discussion Data collection is still ongoing for this project, however, Figure 2 contains DOSY spectra for IHSS Leonardite HA2 (a), and IHSS Elliott soil HA2 (b). These data show substantial differences not only in the composition of the components present in each fraction, but also indicate variations in diffusivity between material types. The Leonardite HA2 contains structural moieties that are mostly concentrated in the regions approximately around 0.5-1.5, 3.5, 4.5, and 7 ppm which indicates the presence of mostly hydrogen bonded to aliphatics, C=C, and phenols. All structure types exhibit polydispersity as evidenced by the large range of diffusivities. The mean diffusivity value for all peaks was calculated to be 9.882 x 10-10 m2/s using the Bruker® T1/T2 relaxation softeware after 29 iterations. Conversely, the Elliott HA2 has a larger variety of structural components with signals ranging widely from 1.0 - 7.5 ppm. The largest signal being in the aromatic region with subsequent signals correlating to the presence of hydrogen associated with aliphatic carbons, esters, ethers, and phenolic structures. Similar to the Leonardite, the Elliott soil structure types also exhibit polydispersity which is demonstrated by a large range of structural diffusivities. The mean difusivity value for all peaks in the Elliott soil spectrum was calculated to be 7.533 x 10-10 m2/s after 40 iterations.
differences are the result of differences in the geochemical history of the parent material.
1.5
seconds between pulses. PFG-NMR spectroscopy data were collected on all samples dissolved in either D2O or C6D6:CD3OD (3:1 v/v) using a Bipolar-Pulse Pair Longitudinal Eddy-current Delay (BPPLED) sequence. Data acquisition parameters consisted of sine-shaped gradient pulses ranging from 1.0 – 1.8 ms ( 2.0 – 3.6 ms bi-polar pulse pair) were used with a gradient strength from ~ 7 - 330 mT m-1 and diffusion time range of 100-180 ms at 295 K. The diffusion time and gradient length where optimized to achieve ~ 95% signal suppression at the maximum gradient strength. Diffusion decays were evaluated using the T1/T2 software included in the Bruker® system operating software using the SimFit algorithm. DOSY NMR spectra were then generated to directly correlate the diffusion coefficient to the proton chemical shift in a two-dimensional plot. Samples used for SANS analysis were dissolved in the benzene:methanol azeotrope with varying degrees of deuteration to determine contrast match points for the HA2 and L1 components of NOM fractions for Leonardite. Contrast match points for HA2 and L1, and were determined to be 50%/50% H/D and 85%/15% H/D, respectively. SANS data were then obtained for samples for authentic L0, “reassembled” L0, and a “reassembled” L0 isotopically labeled with deuterated palmitic acid. Data was collected for all samples at room temperature, atmospheric pressure, and a Q range of 0.0038 – 0.4 Å-1.
(b) 8
6
4
2
F2 [ ppm ]
Figure 2. DOSY spectra of HA2 component for (a) Leonardite and (b) Elliott Soil. Diffusivities on the y-axis are 2 -9 in m /s x 10
REFERENCES
(1) Chilom, G.; Bruns, A.S.; Rice, J.A. Org. Geochem. 2009, 40, 455-460. (2) Khalaf, M.M.R.; Chilom, G.; Rice, J.A Soil. Biol. Biochem. 2014, 73, 96-105. (3) Hoffman, L.; Chilom, G.; Venkatesan, S.; Rice, J.A.; Microsc Microanal 2014 ACCEPTED (4) International Humic Substances Society, 2008. (5) Chilom, G.; Rice, J.A.; Langmuir 2009, 25, 90129015. (6) Lal, R.; Phil. Trans. R. Soc. B 2008, 363, 815-830
Acknowledgments: This project has been supported by the Department of Education Graduate Assistance in Areas of National Need (GAANN) program, and partly by the National Science Foundation through grant number 1012648. PFG-NMR data were collected at the NMR facility at South Dakota State University. SANS data were collected at The Lujan Neutron Scattering Center at Los Alamos National Laboratory.
SANS data demonstrate a definite difference between similar fractions of two different NOM types. PFGNMR data will continue to be collected on all fractions of the two material types to further investigate if these
179
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Investigation of humic-aminorganosilanes interaction using smallangle X-ray scattering AB Volikov
(a)
(b)
*, A Gutsche , SA Ponomarenko
(c)
(a)
, IV Perminova
(a) Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia; (b) Institute for Mechanical Process Engineering and Mechanics, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany; (b) Enikolopov Institute of Synthetic Polymeric Materials of Russian Academy of Sciences, Moscow, Russia; * Corresponding author e-mail: [email protected] Keywords: Humic Acid, silanol derivativies, SAXS, fractal Abstract Modification of humic substances is an important task. Thus the introduction of surface-active function allows their use in ecology. We have developed a method of introducing a silanol functions into humic substances. To introduce new function in native humic materials, alkoxy-silylation was used in aquatic medium. However, the mechanism of their interaction with humic substances is unknown and difficult to study. This research demonstrate how the non-destructive methods such as small-angle X-ray scattering can solve this problem. Model of fractals are used to describe this system, it was found that with the passage of time the fractal dimension of the particles was growth. During the research were conducted to study of the kinetics of these processes as a function of various parameters, that allowing to directly study the reaction mechanism. Introduction In nature, many functional nanoarchitectures are selfassembled due to supramolecular interactions of natural organic materials, generically known as humic substances (HS), and clay-size minerals. Those nanoarchitectures play key roles in natural attenuation processes taking forms of colloids, colloidosomes, and hybrid porous materials. Hybrid porous materials, represented by soil aggregates, play a crucial role in both protecting organic carbon and water retention capacity of soils [1]. Hence, getting control over interactions of humic materials with mineral surfaces may contribute in developing accelerated natural attenuation technologies Incorporation of Si-containing functional groups into humic macromolecules is a promising direction of the research for tailoring humic molecules with high mineral affinity. This makes possible the use of humic substances in various environmental technologies, such as permeable reactive barrier, posing in situ for the detention of various toxicants [2]. However, the mechanism of interaction of organosilanes with humic substances in the aquatic medium has not been elucidated. In the interval since a fractal nature for humic materials was first reported, there have been few papers to actually exploit the fractal concept to better understand these enigmatic substances. Rice and Lin [3]. and Malekani and Rice [4] have used SAXS to demonstrate that. Two types of fractals - mass fractals and surface fractals are relevant to these studies. Thus, the definition of fractal dimension gives an indication of the nature of the substance insitu. In connection with the foregoing, for the optimal investigation of humic-aminoorganosilane system is to use a technique that allows to monitor in situ the growth and formation of clusters of SiO2 in the medium of humic substances by using small angle X-Ray scattering (SAXS). Elucidation of the
mechanism and conditions affecting the reaction will optimize the process and receive hybrid humic organosilane compositions with known properties. Experimental Leonardite humic acid (CHP) and peat humic acid (PHA) were used for the experiments. For the preparation of solutions weighed humic substances dissolves by lithium hydroxide and then adjusted by hydrochloric acid to a desired pH value and then adjusted to the required concentration with distilled water. In the experiments solutions with concentration of 1 to 10 g / l and a pH of from 4 to 10 was used. 3amino-propyltriethoxy-silane (APTES) was used for treatment of CHP. To the reaction was carried the organosilane added dropwise to solution of humic acids under stirring and then acidified to the desired values of pH. The SAXS experiments were conducted with a camera constructed on a base of a Katky camera. The photosensitive imaging used as detector. The samples were placed in a quartz capillary and irradiated for three minutes. A detailed description of the camera and the data evaluation can be found in [5]. TEM images were acquired using a Philips CM 12 microscope operating at 120 kV. For these measurements carbon-coated grids were briefly dipped into the solution and dried under air. Re sults and Discussion In our experimentation has been studied the interaction of humic substances with the organosilane in the water. 3-amino-propyltriethoxy-silane (APTES) was used for treatment of humics. We have developed method of in aqueous media by hydrolysis of the functional organosilane in the presence of humic substances (Fig. 1). The product of this reaction has the ability to communicate with the mineral matrix is
180
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
close to previously obtained alkoxy derivatives of humic substances [6]
Figure 1. Scheme of interaction o f hu mic substances with a 3-aminopropyltrimethoxysilane in aquatic medium
Silanol derivatives of humics were obtained by its interaction with APTES. The reaction was run at different APTES-to-humics ratios, from 1:4 to 2:1 by weight. To the system was prepared according to the kinetic parameters for various: reagent ratio, pH, concentration, and the effect of adding calcium chloride to the system. As a typical example of the behavior of the system, consider the change SAXS curves over time for a mixture APTES and CHP ratio of one to one with a concentration of 5 g/l at pH 7 (Fig. 2)
Figure 3. TEM images of silanol derivative of humic substances in 15 minutes, 1, 8, 24 hours after mixing
REFERENCES
(1) Six, J., Elliott, E.T., Paustian, K. So il Bio logy and Biochemistry. 2000. 32. 2099. (2) Balcke, G.U., Georg i, A., Woszidlo, S., Kopinke, F.D., Poerschmann, J. Use of Hu mic Substances to Remediate Polluted Environ ments: Fro m Theory to Practice, NATO Science Series: IV: Earth and Env iron mental Sciences, 52, Springer, Dordrecht, 2005, 203. (3) Rice, J.A.,, Lin, J.S. Env iron. Sci. Technol. 1993, 27, 413 (4) Malekani, K., Rice, J.A. Soil Sci. 1997, 162, 333. (5) Goert z, V., Gutsche, A., Dingenouts, N., Nirschl, H. J. Phys. Chem. C 2012, 116, 26938−26946 (6) Perminova, I.V., Karpiouk, L.A., Pono marenko, S.A., Hatfield, K., Konstantinov, A.I., Hertkornd, N., Muzafarov, A.M. Colloids and Surfaces A: Physicochem. Eng. Aspects 2012, 396, 224.
Figure 2. Small angle X-ray scattering curves for the 5 g/l mixtu re 1:1 o f CHP and APTES with pH 7 after various times after mixing
Acknowledgments: A.B. Volikov would like to express his gratitude to the program of academic exchanges of the Karlsruhe Institute of Technologies and Lomonosov Moscow State University for support of his fellowship at the Institute of Mechanical Processes of KIT (Germany). This research was partially supported by the Russian Foundation for Basic Research (grant # 13-04-0185313).
As we can see, the slope of the curve changes with time from 2.37 to 3.76. Thus complexes of humic substances and organosilane transformed from a mass fractal to surface fractal. This phenomenon can be explained by the fact that the organosilane gradually polymerized in water, and over time the particles become more and more dense. We have also obtained TEM images of solution at different times after the start of the reaction. As can be seen from Figure 3, TEM data are in good agreement with the data obtained by SAXS, thus, over time the particles are transformed into a dense structure. We have found that the growth rate of the fractal dimension depends on various parameters of solution. Thus, as the concentration of the solution and the mass ration APTES:HS growth rate also increased, which agrees wells with laws of physical chemistry. Dependence of the rate of the pH is more complex, because, at low and high speed high pH and at neutral pH of the growth rate drops significantly. We can assume that this is due to the fact that the formation of siloxane bond catalyzed by acid and alkaline medium.
181
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Effects of land use change on soil organic matter (SOM) characteristics: a comparison of crops, young forests and old forests P. Rovira (a) *, A. Sala(a), A. Merino(b)
(a) CEMFOR-CTFC, Carretera de Sant Llorenç de Morunys, km 2, 25280 Solsona, Spain (b) Unit of Sustainable Forest Management, University of Santiago de Compostela, 27002 Lugo, Spain * Corresponding author e-mail: [email protected] Keywords: Land use change, Carbon sequestration, Crop abandonment, Physical protection, Recalcitrance Abstract Changes in SOM characteristics were studied in a sequence of crop abandonment and substitution by secondary pine stands, and compared with old pine stands on never cropped sites, in Catalonia (NE Spain). Crop replacement by forests result in significant increases in soil C stock in the litter horizons and at the mineral soil surface (first 5 cm depth); below this treshhold crop abandonment may result in C losses, particularly in deep layers (>15 cm depth), which apparently can be recovered in the very long term. With crop replacement by secondary forest, SOM becomes less stable and more vulnerable to microbial attack, particularly at the soil surface. This increased vulnerability, which occurs in spite of the increased biochemical recalcitrance of forest- relative to crop soils, seems due to the decreased physical protection of SOM: the % of total SOM associated to the organomineral complex (< 20 µm) decreases with crop abandonment. Introduction The abandonment of crop lands and its replacement by secondary vegetation (shrublands or forests, either man-made or subspontaneous) is widespread in many areas of Spain. Since crop soils are usually poor in C, this phenomenon should result in an increased C stock in spanish soils, i.e. a net C sequestration. This is expected to occur in the long term: in the short term crop abandonment has been shown to result often in C losses, not gains. Crop soils receive yearly inputs of organic residues: the interruption of these regular inputs implies that for a time the soil will only loss C by respiration, until new inputs appear, when the new vegetation type (shrublands, secondary forests, manmade stands) becomes plenty functional in so an essential point such as the release of dead residues to the soil (litter, roots). Crop abandonment, thus, may not have an immediate positive effect on the soil C budget. In addition to these constraints, SOM features are expected to change in the transit from croplands to other vegetation types. These changes are expected to affect its distribution with depth – SOM in crops is much more evenly distributed in depth than in any other vegetation type –, and also relevant features such as the physico-chemical features which, finally, determine its stability in soil: physical protection – e.g., by size fractionation –, and biochemical quality or abundance of refractory forms of SOM – which can be measured by the resistance to acid hydrolysis. Here we present a dataset in which these processes of change in total SOM stock, its physico-chemical characteristics and its stability against the microbial attack are studied. The study has been carried out in a complex of active crops, abandoned crops (covered by secondary forests) and old forests. It may be envisaged as representative of the kind of changes expected in the soils, in a context of agricultural abandonment.
Experimental The study was carried out in the agricultural zone of Cardona (near Barcelona, NE Spain), widely terraced for agriculture, and developed over Tertiary marls. Many croplands were abandoned in the 60s: these crops were replaced by secondary stands of Pinus nigra, grown from adjacent forests of the same species. We studied a total of 12 plots: (i) Crops (CR). 3 plots. Cropped in 1956 and today. (ii) New forests 1 (NF1). 4 plots. Cropped in 1956, forests today. (iii) New forests 1 (NF2). 2 plots. Forests in 1956 and today, but clear signs of ancient agriculture (manmade terraces). Probably older than 100 years. (iv) Old forests (OF). 3 plots. Forests in 1956 and today. No signs of ancient agriculture anywhere. Soils were sampled (5 cores/plot) down to 30 cm depth. Cores were divided in depth layers (0-5, 5-15 and 15-30 cm). These samples were air-dried, sieved to 2 mm and studied for organic C, size fractionation, acid hydrolysis, and submitted to incubation under standard conditions (25ºC, optimal humidity). In the forest plots, the litter layers (L, F and H) were also sampled and quantified. Results and Discussion Overall C stock. [Figure 1] The replacement of crops by forests results in an increase in soil C stock, but also in a relevant redistribution of C along the profile: the net sequestration in surface horizons is partially compensated by C losses in deep layers. The oldest new forests (NF2) still accumulate much less C than the old forests (OF): this suggest a high potential for C sequestration for many years.
182
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
0
2
4
6
8
0
2
4
Biochemical recalcitrance [Figure 3]. Here we measure recalcitrance by the resistance to acid hydrolysis with HCl in a two-stage protocol (1M HCl, followed by 6M HCl). The unhydrolyzable fraction is higher under forest stands, particularly in surface horizons. The replacement of crops by forest results in a general decrease in the biochemical quality of SOM.
6
Organic horizons
Unhydrolyzable Carbon (% of total OC)
Depth (cm)
-10
Crops New forest 1 New forest 2 Old forest
0
25
30
35
40
45
50
55
Litter Mineral soil
10
0 - 5 cm 20
Crops New Forest 1 New Forest 2 Old forest
30
Figure 3. SOM biochemical recalcitrance. 5 - 15 cm
Stability against the microbial attack [Figure 4]. SOM (un)stability is measured by the fraction of SOM lost by microbial respiration upon incubation under optimal conditions (25ºC, optimal humidity). SOM is clearly more stable in crop soils. The organic matter is also more stable in deep layers.
15 - 30 cm 0
1
2
3
4
kg C m-2
Figure 1. Carbon stocks, given in kg C m-2.
Physical protection of SOM: [Figure 2] In crop soils, more than 70% of the total C is found in the organomineral complexes (< 20 µm). In forest soils this percentage clearly drops, particularly in the soil surface. C in fractions < 20 µm (% of total OC) 0
50
60
70
80
90
100
Figure 4. Stability of SOM against the microbial attack.
Depth (cm)
5
10
Overall view. The replacement of crops by secondary forests incraeses soil C stock, but the new, sequestered C is less stable against the microbial attack than that of the original, agricultural soil. Apparently the reason is a decrease in the degree of physical protection: in forest soils a greater portion of SOM is in form of unprotected fragments (particulate organic matter: POM). The increased biochemical recalcitrance apparently does not affect SOM stability.
Crops New Forest 1 New Forest 2 Old forest
15
20
25
Acknowledgments This research has been carried out in the framework of the CRONOCARB project, financed by the spanish Ministry of Science and Innovation.
30
Figure 2. Physical protection of SOM, as measured by the percent of total C in the organomineral complexes (< 20 µm).
183
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Selectivity and limitations of solid phase extraction of dissolved organic matter from fresh water samples J. Raeke (a) *, T. Reemtsma(a)
(a) Helmholtz Centre for Environmental Research – UFZ, Department Analytical Chemistry, Permoserstrasse 15, 04318 Leipzig, Germany * Corresponding author e-mail: [email protected] Keywords: dissolved organic matter, solid phase extraction, high resolution mass spectrometry Abstract Solid phase extraction is often used as an enrichment procedure before the analysis of dissolved organic matter with high resolution mass spectrometry. Different SPE cartridges were tested for their extraction efficiency and selectivity. The extraction behaviour of 18 model compounds of low molecular weight and the influence of extraction parameters on the mass spectrometric data set of a DOM-rich freshwater sample were studied. Introduction Dissolved organic matter (DOM) is a complex mixture of diverse compounds, which mainly consists of products from natural decay processes and polymerization reactions. Fulvic and humic acids represent a significant percentage of DOM, though smaller molecules are also present. A powerful tool for the molecular characterization of DOM is ultrahigh resolution Fourier transform ion cyclotron mass spectrometry (FT-ICR MS). With this analytical technique several thousand signals can be obtained for the molecular species in a complex sample. The exact masses can be used to gain information about the molecular composition of the sample, with the relative signal intensities serving as an indicator for the concentration of the respective compounds (1). Prior to analysis with FT-ICR MS the DOM is usually enriched, also to remove dissolved salts and other unwanted compounds and to standardise the sample matrix. Several possibilities for enrichment exist. A method frequently used for clean-up and enrichment is solid phase extraction (SPE). For that matter C18 cartridges but also modified copolymers are employed (2, 3). However, this method of extraction is also selective and incomplete. Extraction efficiencies between 20 % and 60 % are described in literature (2, 3). The comparison of the molecular composition of a C18 extracted sample and a directly injected sample showed that highly oxidized tanninlike substances and aliphatic amines/amides are hardly enriched and therefore are underrepresented in the extract (4). Systematic studies on the influence of solid phase extraction conditions on the composition of the DOM were rarely published so far. Although, for the assessment and comparison of data obtained with FT-ICR MS it is essential to be aware of the method’s selectivity and in particular of the critical boundary parameters which have to be respected.
Experimental Different SPE cartridges were tested for their extraction efficiency and selectivity. The presented results are limited to the Bond Elut PPL and the Oasis HLB cartridge (properties shown in Table 1). Table 1: Properties of SPE cartridges cartridge
material
Bond Elut PPL (Agilent) Oasis HLB (Waters)
modified SDVB copolymer copolymer
particle size (µm) 125
pore size (Å) 150
bed mass (mg) 500
Vol
30
80
500
6
(ml) 6
For this purpose 18 low molecular weight substances of different compound classes were selected as model compounds. Most of these substances contained at least one carboxyl group, which is the most common functional group in dissolved organic matter. The selected model compounds are shown in a van Krevelen diagram in Figure 1. A DOM-rich fresh water sample (DOC ~ 5 mg/l) was separated into two aliquots. One aliquot was spiked with 0.5 µmol/l per model compound and 0.05 µmol/l of sodium dodecyl sulphate and 12hydroxydodecanoic acid respectively (spiked before extraction = sbe). A volume of 50 ml of this spiked sample (acidified with HCl to pH 2) was passed through the cartridge. After rinsing with acidified water (pH ~ 2) and drying under nitrogen for 10 min, the sample was eluted with 4 ml of methanol and finally filled up to a volume of 5 ml with a resulting enrichment factor of 10. The second aliquot of the sample was extracted in parallel and then spiked with the model substances in a concentration of 5 µ mol/l or 0.5 µmol/l respectively according to the enrichment factor (spiked after extraction = sae). The concentration of model compounds was determined using LC-Q-TOF-MS. With these results the
184
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
extraction efficiency was calculated according to the following equation:
with any of the cartridges. The extraction efficiency for naphthalene-1,5-disulfonic acid was also very low. The model compounds gallic acid, glutaric acid and catechol, which were also comparatively early eluting, could clearly be better extracted with the HLB cartridge. For the other substances the extraction efficiency of the HLB cartridge was 7 % to 50 % poorer than with the PPL cartridge. Naphtalene-2sulfonic acid and sodium dodecyl sulphate could not be extracted with the HLB cartridge. Mass spectrometric dataset In Figure 3 the CHO compounds extracted from a DOM-rich freshwater sample are plotted in a van Krevelen diagram. 614 molecular formulae out of 1281 could be assigned in the extracts of both cartridges. The major portion of the molecular formulae specific for the PPL cartridge is situated in the region of the more unsaturated, polar compounds with a low H/C and a high O/C ratio. The molecular formulae which could only be extracted with the HLB cartridge are more spread over the entire van Krevelen diagram.
Figure 1: Van Krevelen diagram of model compounds (♦) and different compound classes
Additionally, the influence of the extraction parameters on the mass spectrometric dataset of a DOM-rich fresh water sample (DOC ~ 5 mg/l) was investigated. A volume of 50 ml of the fresh water sample was passed through a HLB and a PPL cartridge. The SPE extracts were directly injected with a nano electrospray source in a Thermo Orbitrap Fusion® mass spectrometer (resolution ~ 85000, FWHM, m/z 400). Spectra were acquired in the mass range 150 – 700 m/z. For one spectrum about 50 scans were coadded. Molecular formulae were calculated from the m/z based on the following parameters: 12C 413 C0-1, 1H4-100, 16O2-100, 14N0-3, 32S0-1, O/C ≤ 1.2, 100, 0.333 ≤ H/C ≤ 2.25, S/N ≥ 5, mass accuracy Δm ≤ 2.0 ppm.
2.5
2.0
H/C
1.5
1.0
0.5
0.0
extraction efficiency (%)
0.4
O/C
0.6
0.8
1.0
Regarding nitrogen containing compounds these seem to be favoured by the HLB cartridge. For the HLB cartridge 20 % of all assigned formulae were CHON compounds compared to only 10 % for the PPL cartridge. Taking everything into account no clear recommendation can be given for one of the cartridges, the choice may depend on the nature of the extracted material and the compound classes of interest. REFERENCES
120 100 80 60
(1) Reemtsma, T., J. Chromatogr. A 2009, 1216, 3687-3701 (2) Dittmar, T., Koch, B., Hertkorn, N.,Kattner, G., Limnol. Oceanogr. Meth. 2008, 6, 230-235 (3) Minor, E. C., Steinbring, C. J., Longnecker, K.,Kujawinski, E. B., Org. Geochem. 2012, 43, 1-11 (4) Sleighter, R. L.,Hatcher, P. G., Mar. Chem. 2008, 110, 140-152
40 20 Bond Elut PPL
0.2
Figure 3: Van Krevelen diagram with the CHO compounds extracted with HLB and PPL cartridge from a fresh water sample (DOC ~ 5 mg/l)
Results and Discussion Model Compounds The extraction efficiency of the different model compounds in the DOM-rich fresh water sample of the PPL and the HLB cartridge is displayed in Figure 1. The model compounds are ordered according to their elution time in liquid chromatography.
0
0.0
Oasis HLB
Figure 2: Extraction efficiency of the model compounds in a DOM-rich fresh water sample on PPL and HLB cartridge, order of model compounds: ■ sorbitol ■ glucuronic acid ■ cellobiose ■ naphthalene-1,5-disulfonic acid ■ gallic acid ■ glutaric acid ■ catechol ■ 4-hydroxybenzoic acid ■ phthalic acid ■ chlorogenic acid ■ vanillic acid ■ epicatechin ■ 2-hydroxy-2-phenylacetic acid ■ syringic acid ■ naphthalene-2-sulfonic acid ■ ferulic acid ■ 12-hydroxydodecanoic acid ■ sodium dodecyl sulfate
Acknowledgments This work was supported by the German Federal Ministry of Education and Research (BMBF) in the framework of the project “Loading of drinking water reservoirs with dissolved organic matter – prediction and prevention” (TALKO, FKz 02WT1290A)
Sorbitol, glucuronic acid and cellobiose, the most polar and sugar-like substances, could not be enriched
185
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Two Dimensional HETCOR Solid-State NMR Spectroscopy as a Tool to Improve our Understanding of the Chemical Alterations Caused by Charring of Humic Material H. Knicker 1*, M. Molina-Velasco1 , M. Lopéz-Martín1, J.M. de la Rosa1, A.E. Berns2 1
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Adva. Reina Mercedes, 10. 4012 Sevilla, Spain Institute of Bio- and Geosciences, Agrosphere (IBG-3), Forschungszentrum Jülich GmbH, Jülich, Germany
2
* Corresponding author e-mail: [email protected] Keywords: Soil organic matter, pyrogenic organic matter, black nitrogen, charring Abstract: The structure of pyrogenic organic matter (PyOM) is still a matter of controversial discussions. In order to obtain more detailed information 2-dimenstional (2D) 13C HETeronucleus CORelation (HETCOR) Nuclear Magnetic Resonance (NMR) spectroscopy was applied to model compounds, fire-affected and unaffected soils. Here, the C-signal intensity is modulated by the proton chemical shift via scalar coupling, which allows a more detailed correlation of protons to carbon groups. With those first experiments, we demonstrated the high potential of this technique for the study of complex organic samples. Our results supported the concept of PyOM being a heterogeneous mixture of partly thermally altered molecules, rather than a highly condensed polyaromatic network. For the first time, a 2D 15N HETCOR NMR spectrum of PyOM was acquired, evidencing that Black Nitrogen is indeed an integral part of the Black Carbon network and that some amide N groups can survive intense charring. the direct dimension t2. Solid-state heteronuclear correlation (HETCOR) NMR spectroscopy represents a 2D technique which has been already applied to NOM (3). It allows the determination which protons are interacting with which carbons. However, with respect to nitrogen, such 15N HETCOR spectra of NOM are still missing. In order to fill this gap and to demonstrate the potential of this technique, 2D HETCOR 13C NMR spectroscopy of model compounds, fire-affected and unaffected NOM was supplemented with a first 2D HETCOR 15N NMR spectra of isotopically labelled grass char.
Introduction During the last years, increasing evidences are provided that the common view of charcoal as a polyaromatic network is too much simplified. Experiments with model compounds indicated that it represents a heterogeneous mixture of thermally altered biomacromolecules with N, O and likely also S substitutions as common features. If produced from a N-rich feedstock, the so called black nitrogen (BN) (1) has to be considered as an integral part of the aromatic charcoal network. In order to study this network one-dimensional (1D) solid-state nuclear magnetic resonance (NMR) spectroscopy is often applied (2). However, this technique suffers from broad resonance lines and low resolution. Applying 2D techniques can help but until recently, this was unfeasible for natural organic matter (NOM) due to sensitivity problems and the high complexity of the material. On the other hand, during the last decade, the development of stronger magnetic field instruments and advanced pulse sequences has put them into reach for NOM research. Although 2D NMR spectroscopy has many different applications, all pulse sequences are based on the introduction of a preparation time during which the magnetization of a spin system is adjusted into a state appropriate to whatever properties are to be detected in the indirect dimension. Then, the spins are allowed to evolve with the given conditions and after their additional manipulation during a mixing period the modulated magnetization is detected. Assembling several 1D spectra with incrementing evolution time creates a data set which is two-dimensional in time (t1, t2). Fourier transformation of both dimensions leads to a 2D contour plot correlating the interactions detected in the indirect dimension t1 with the signals detected in
Experimental Samples derived from charred grass residues enriched in 15N (4) and charred lignin (1). In additions demineralised fire affected and unaffected soils from Southern Spain were analyzed. The solid-state 13C and 15N HETCOR NMR spectra were obtained with a Varian 7.05 T Unity Inova (300 MHz) and a Bruker Advance III 600 MHz NMR spectrometer at a magic angle spinning speed of 8 kHz and 15 kHz for the 13C NMR experiments. For the 15N NMR experiments, a spinning speed of 10 kHz was sufficient to avoid overlapping of signals with spinning side bands. Homonuclear coupling of the protons during cross polarization was avoided by applying phase-shifted Lee-Goldburg cross polarization (LG-CP) and continuous Lee-Goldburg decoupling was used. Since the high spinning speeds did not allow to rely on dipolar dephasing approaches (5), carbons with and without directly to protons were distinguished by acquiring 2D plots with increasing LG-CP time.
186
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
1
H dimension show that amides are still present. The survival of amides was confirmed by its 2D 15N HETCOR NMR spectrum. Here, additional cross peaks were identified proving the formation of Nheterocyclic N during charring. Correlation of signal intensity in the amide-N and pyrrole-N regions with the aromatic chemical shift region of the 1H dimension underlines earlier findings that BN has to be considered as an integral part of the Black Carbon network. The 2D 13C HETCOR NMR spectrum of the fireunaffected soils revealed that most of the carboxyl C occurs as ester or amide. Aside from cross peaks typically seen in spectra of NOM, the spectrum of the respective fire-affected counterpart shows additional signals assignable to PyOM.
Results and Discussion The solid-state 13C HETCOR NMR spectrum obtained with a LG-CP = 50 µs of a lignin char heated under oxic conditions for 4 min at 450°C [Figure 1] shows clear cross peaks indicating strong interactions between aromatic C with 1H, which confirms a low condensation degree already assumed from the atomic H/C ratio of 0.6. Increasing the LG-CP time to 1 ms increased the broadness of the aromatic cross peak from 1 ppm to 10 ppm of the 1H dimension demonstrating interaction those C with 1H located at a further distance (data not shown). Cross signals correlating alkyl C (20 to 13 ppm: terminal CH3) with 1 H from aromatic compounds indicate that those two groups are in close neighbourhood and that alkyl groups are part of the macromolecular char network. Clear cross peaks were detected for aryl-OH revealing that during thermal degradation, lignin undergoes a demthylation rather than demethoxylation.
REFERENCES (1) Knicker, H.; Org. Geochem. 2010, 41, 947-950. (2) Knicker, H.; Hilscher, A.; González-Vila, F. J.; Almendros, G.; Org. Geochem. 2008, 39,935-939. (3) Mao, J.D.; Xing, B.; Schmidt-Rohr, K.; Environ. Sci. Tech. 2001, 35, 1928-1935. (4) De la Rosa, J.M.; Knicker, H.; Soil Biol. Biochem. 2001, 35, 1928-1934. (5) Wilson, M.A: NMR Techniques and Application in Geochemistry and Soil Chemistry. Pergamon Press: Oxford, 1987.
Acknowledgments: The Ministry of Economy and Competitiveness of Spain together with the European Regional Development Fund (FEDER) are acknowledged for funding the project CGL201237041. Marta Velasco-Molina acknowledges the International Humic Substance Society for granting a training award. The JAE-Doc program co-financed by the European Social Fund (JAE-DOC-056) is thanked for support of J.M. de la Rosa. The “CITIUS” research center of the University of Seville (Spain) together with Bruker Española S.A. is thanked for granting an award allowing the performance of the time consuming 2D-NMR spectra.
Figure 1. a) 2D 13C HETCOR NMR spectrum of lignin
charred at 450°C for 4 min and b) grass residues charred at 350°C for 4 min.
[Figure 1b] shows the 2D 13C HETCOR NMR spectrum of a grass char produced at 350 °C under oxic conditions. The LG-CP time of 1 ms allowed interaction of 13C with 1H at a distance further than one to two bonds. The cross peaks at 55 and around 172 ppm in the 13C dimension with NCH region of the
187
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Isotopic exchange ultrahigh resolution mass spectrometry is a powerful tool for investigation of molecular structure of Dissolved Organic Matter Yury Kostyukevicha, Alexey Kononikhina, Alexander Zherebkerb, Igor Popova, Irina Perminovab and Eugene Nikolaeva
(a) Institute for Energy Problems of Chemical Physics Russian Academy of Sciences Leninskij pr. 38 k.2, 119334 Moscow, Russia (b) Lomonosov Moscow State University, Department of Chemistry, Leninskie Gory 1-3, 119991 Moscow, Russia * Corresponding author e-mail: [email protected] Keywords: FT ICR, DOM Abstract We report that isotopic exchange approach coupled to ultrahigh resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR MS) is the powerful tool for chemical and structural characterization of the individual molecules of Dissolved Organic Matter (DOM). In the present work we used in-ESI source Hydrogen/Deuterium (H/D) exchange for enumeration of acidic and hydroxyl hydrogens, acid catalyzed 16O/18O exchange for enumeration of labile oxygens and acid catalyzed H/D exchange for determination of labile back-bone hydrogens. We identified number of labile hydrogens in 450 individual molecules and number both of labile hydrogens and oxygens in 231 molecules composing DOM. Also we observed that several back-bone hydrogen atoms can be exchanged for deuterium under acidic conditions. Introduction DOM represents one of the largest reservoirs of active carbon on Earth. DOM is a molecular ensemble of thousands individual species generally produced by biodegradation of living organic. Due to the extreme complexity DOM is poorly separating even on fractions and separation on individual species cannot be performed at all no matter what separation technique is used (chromatography, dielectric focusing e.t.c)1. This is the reason why powerful physical methods such as NMR, Optical spectroscopy or X-ray spectroscopy cannot reveal molecular structure of individual compounds of DOM and can characterize DOM only in general2. Fourier Transform Ion Cyclotron Resonance Mass-Spectrometry (FT ICR MS)y3 used with ambient ionization techniques (ESI, APPI and etc.) proved to be an important tool for investigation of DOM4. But despite many attempts only unique FT ICR experiments (such as performed by Witt et. all.5 or Solouki et. all6) provided structural information about individual molecules of DOM. In this study we present the methodology to obtain chemical and structural information about each molecule of DOM using isotopic exchange techniques (H/D and 16O/18O) coupled to ultrahigh resolution FT ICR MS.
Figure 1. Workflow used in the present paper. H/D exchange in ESI source. SRDOM was dissolved in deuterated methanol and diluted to concentration of 1 g/L. To prevent back exchange in ESI source 300 ul of D2O were placed on a copper plate 3mm beneath ESI needle7,8. Acid catalyzed 16O/18O exchange. 5mg of SRDOM were dissolved in 200 ul of H218O. SRDOM is acid so there is no need in introducing another acid for catalyzing the exchange reaction. The vial was stored in thermostat at different temperatures (60 oC - 95 oC) for different time. The best degree of exchange was achieved when sample was incubated at 95 oC for 20 days. During this time at 10 day the vial was unsealed for picking 30ul of sample.
Experimental The methodology used in the present work is briefly described in Fig. 1. Samples and Instruments. Suwannee River Dissolved Organic Matter (SRDOM) was used as a sample. All chemicals were analytical grade or higher. All experiments were performed on LTQ FT Ultra
188
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Acid catalyzed back-bone H/D exchange. 5mg of SRDOM were dissolved in 200 ul of D2O. and 100 ul of TFA. Solution was placed in a glass vial which was sealed afterwards. The vial was stored in thermostat at 90 oC for 14 days.
(Firg. 3). We can see that molecules with different number forms clusters on those diograms. Also we performed acidic catalyzed exchange of back-bone hydrogen atoms for deuterium. Back-bone H/D exchange was observed in each DOM molecule.
Spectrum processing. Elemental composition was assigned to each peak in original spectrum using accurate mass in Xcalibur software (Thermo). All isotopic exchange spectra were processed semiautomatically. First for each compound presented in the original spectrum were found peaks in isotopic exchanged spectra that could be related to this compound. Precisely for mass M were chosen all peaks Mi such that:
Mi M k d E where k – is integer number, d– mass difference equals to 1.006277 for H/D exchange experiments and 2.004245 for 16O/18O exchange experiments, E is initial error set by user which was large enough to account for possible calibration drift or mass shifts caused by experimental conditions. Normally we used E=10 -3. Then among selected M i peaks were chosen those that form binomial isotopic distribution and which mass differ by md with best accuracy. The last step was performed manually for each peak. The main source of errors in spectra processing is the peak splitting leading to falsepositive identifications.
Figure 3. A – The 3D diagram for SRDOM ions. Color represents number of labile hydrogens (see Fig. 4), B – The 3D diagram for SRDOM ions. Color represents number of non-labile oxygens, C – Kedrik Mass Defect plot. Color represent number of labile hydrogens, D – Kendrik Mass Defect plot. Color represents number of non-labile oxygens. Mass of segment –(CH2)n – was set to be integer. REFERENCES (1) Chefetz, B.; Tarchitzky, J.; Deshmukh, A. P.; Hatcher, P. G.; Chen, Y. Soil Science Society of America Journal 2002, 66, 129. (2) Schmidt, M. W. I.; Knicker, H.; Hatcher, P. G.; KogelKnabner, I. European Journal of Soil Science 1997, 48, 319. (3) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrometry Reviews 1998, 17, 1. (4) Stenson, A. C.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Analytical Chemistry 2002, 74, 4397. (5) Witt, M.; Fuchser, J.; Koch, B. P. Analytical Chemistry 2009, 81, 2688. (6) Solouki, T.; Freitas, M. A.; Alomary, A. Analytical Chemistry 1999, 71, 4719. (7) Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Kharybin, O.; Perminova, I.; Konstantinov, A.; Nikolaev, E. Analytical chemistry 2013, 85, 11007. (8) Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Nikolaev, E. Analytical chemistry 2013, 85, 5330.
Figure 2. Van Krevelin diagram for SRDOM: A - 450 ions for which were determined the number of labile hydrogens. B,C – 231 compounds for which were determined number of labile hydrogens and non-labile oxigens. Number is coded by corresponding color (see A). Results and Discussion Compact visual representation of the obtained data can be performed using Van Krevelen diagram (see Fig. 2A,B,C) and Kendrick diagram
189
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Does light-screening by Humic Substances completely explain the retardation effect on contaminants photo-degradation? J. F. Leal (a) *, V. I. Esteves (b), E. B. H. Santos (b) (a, b)
Department of Chemistry and CESAM – Centre for Environmental and Marine Studies, University of Aveiro, 3810-193 Aveiro, Portugal * Corresponding author e-mail: [email protected] Keywords: Humic acids, Fulvic acids, XAD-4 fraction, photo-degradation, oxytetracycline, BDE-209 Abstract Humic substances – HS – have an important role on the environmental fate and behaviour of organic compounds, sensitizing or delaying their photo-degradation. The retardation effect is frequently attributed to lightscreening, but this does not completely explain the retardation effect of HS on the photo-degradation of organic contaminants. In this work, the quantum yield of oxytetracycline (OTC) photo-degradation in water and the outdoor half-life times of OTC in midsummer and midwinter days were estimated. The light-screening caused by each fraction of HS on OTC photo-degradation were also calculated. The results suggest that the delay effect on photodegradation of OTC (hydrophilic compound) is predominantly justified by light-screening caused by HS, but on photo-degradation of BDE-209 (very hydrophobic compound), the light-screening has a minor effect and the interaction with HS seems to be the predominant effect. Introduction Humic Substances – HS – are complex molecules with several structural components 1. The prevalence of each type of functional group or structural moiety varies with aquatic system and is influenced by the origin of HS2,3. Three fractions of HS soluble in water can be distinguished: humic acids (HA), fulvic acids (FA) and XAD-4 fraction. HA and FA, isolated with XAD-8 resin, are the more hydrophobic fractions of aquatic HS. XAD-4 fraction is the more hydrophilic fraction, isolated with XAD-4 resin assembled in series after XAD-8 resin4,5. Regarding to the role of HS on interactions of light with contaminants, two opposite effects must be considered: the photosensitisation effect and the retardation effect. On one hand, the light absorption by HS can promote a number of photochemical processes that begin with the excitation of HS. As a result, several reactive species can be produced, promoting the increase of environmental degradation of a contaminant. On the other hand, HS are able to absorb solar radiation6 and modify the spectrum of radiation penetrating into the water column. The retardation effect of HS on contaminants photo-degradation is frequently attributed to this light-screening (Sλ). But, does lightscreening completely explain the delaying effect of HS on contaminants’ photo-degradation? What is its contribution for the retardation on photo-degradation of different compounds? This work considered two completely different organic compounds to compare how light-screening caused by HS affects their photodegradation: the oxytetracycline (OTC), a very soluble compound in water, and the bis(pentabromophenyl) ether (BDE-209), a very hydrophobic compound. Both compounds are largely used as antibiotic (in veterinary medicine) or flame retardant, respectively.
Experimental Oxytetracycline hydrochloride was provided by Sigma Aldrich. For the preparation of all solutions, distilled water was used. The HS fractions – HA, FA and XAD-4 fraction – were isolated from river Vouga water (Carvoeiro, Portugal). The relative abundance of each fraction in the sample was 10.3 %, 69.4 % and 20.3 % for HA, FA and XAD-4 fraction, respectively. The HS used were isolated, purified and stored according to the procedure of IHSS. Irradiation experiments of OTC were performed on aqueous solutions (4 mg/L). For each irradiation time, and each replicate, two tubes of each solution were introduced in the sunlight simulator: one was exposed to radiation and the other wrapped in aluminum foil to protect from light (dark control). A simulator of solar radiation, Solarbox 1500 (Co.fo.me.gra, Italy) equipped with a 1500 W arc xenon lamp and outdoor UV filters was used for the irradiation experiments. Quantitative analysis of OTC was made by HPLCUV, using a New ACE C18 column-PFP. The mobile phase was 20 % acetonitrile and 80 % water acidified to pH 2 with formic acid. The detection was done at 350 nm. More details about Sunlight simulator and HPLC instrument are described in Leal et al. (2013)7. Results and Discussion Kinetics of OTC photo-degradation For the photo-degradation kinetic studies, the OTC aqueous solutions were irradiated during 20, 40, 60, 90 and 120 min. The results obtained were well fitted (R2=0.9755) by the equation C / C0 e kt , where k is the first-order rate constant, t is time and C0 and C are the concentrations of OTC when protected from light or exposed to it, respectively. These results suggest that OTC photo-degradation in water follows a pseudo first-order kinetics. The first-order rate constant obtained was 0.0198 ± 0.0005 min-1 and the half-life
190
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
time was 35.0 ± 0.9 minutes. The quantum yield (Φ) is a very relevant parameter which allows correctly compare results from different photochemical studies. The quantum yield of OTC was calculated to the range 290-450 nm and the value obtained was 4.22 x10-4 ± 0.33 x10-4. More details of calculation were previously described7. To the best of our knowledge, there are no other data for the quantum yield of OTC in water using simulated sunlight. An outdoor half-life time was also calculated assuming that the radiation used in these experiments correctly simulates the solar spectrum. The method used for this calculation is thoroughly explained in a previous work that we have published 7. The predicted outdoor half-life times for a midsummer day and for a midwinter day, at 40"N Latitude (Sea Level) under clear skies are 1.60 h and 3.67 h, respectively. Effect of HS on photo-degradation The photo-degradation experiments of OTC in water were performed in the absence and in the presence of each fraction of aquatic HS – HA, FA and XAD-4 – with concentrations of 8 mg/L and 16 mg/L, during 60 min. The photo-degradation percentage, at 60 min, obtained for the OTC in the absence of HS was 69.3 ± 2.5 %, which is not different from the photo-degradation percentage estimated (69.5 ± 0.9 %) based on first-order rate constant obtained from independent experiments on photo-degradation kinetics of OTC in water. In the presence of HS, the photo-degradation of OTC decreases (the values range between 45.9 ± 2.8 and 60.3 ± 1.1 in the presence of HA 16 ppm and XAD-4 8 ppm, respectively). The one-way ANOVA comparison of the mean values for the photo-degradation percentage in the presence and in the absence of HS allowed to conclude that any fraction of HS, at 8 mg/L and 16 mg/L significantly (p < 0.001) delays the OTC photo-degradation. The retardation effect caused by HS is frequently attributed to the light-screening. To verify this hypothesis, the overall screening factor (SƩλ) was calculated for each fraction of HS, for the wavelength range between 290 and 450 nm. No light-screening occur when Sλ = 1. The S Ʃλ values obtained range between 0.79 and 0.97 for the HA (16 ppm) and XAD-4 (8 ppm) fractions, respectively. More details of calculation were previously described7. From SƩλ obtained for each HS fraction it is possible to estimate the photo-degradation rate constants of OTC in the presence of these fractions. Using those estimates of the rate constants, the percentage degradation after 60 min of irradiation was estimated. Comparing the results obtained and estimated based on SƩλ (Fig.1A), the light-screening is not the unique effect of retardation because the photodegradation experimentally observed is lower than the photo-degradation estimated based on SƩλ values.
A
The results obtained for OTC were compared to those obtained in a previous study7 for BDE-209 (Fig 1B). The comparison between the photo-degradation percentages observed and estimated for the BDE-209 in the presence of each HS fraction suggest a big difference between these two sets of values. For the case of OTC, the observed photo-degradation values, although lower (8-15% difference) than the values estimated based on light-screening, follow the same graphic tendency, what suggest that, in this case, the light-screening effect caused by the presence of HS is the predominant delaying effect. Contrarily to what happens for OTC, the decrease of BDE-209 photo-degradation observed comparatively with photo-degradation estimated is high and does not follow the tendency that would be expected by the light-screening. For the BDE-209, the difference between the photo-degradation percentages is higher for the HA and FA fractions, which are the more hydrophobic fractions of HS. For the OTC, the delay is more evident in the presence of HA fraction, which is the fraction that presents the higher light-screening. The BDE-209 is a very hydrophobic compound, whereby its tendency to associate to the more hydrophobic fractions (HA and FA) is higher than to the hydrophilic fraction (XAD-4). The OTC is a compound soluble in water whereby is expectable that its tendency to associate with hydrophobic fractions is lower than for the BDE-209. Thus, the light-screening seems to be a minor effect on retardation of BDE-209 photo-degradation and the predominant effect may be the hydrophobic associations between BDE-209 and HS, as a result of its high hydrophobicity. For the case of OTC, the hydrophobic association with HS is not expectable due to the low hydrophobicity of the compound. However, OTC also suffers selfphotosensitized degradation and, in the presence of HS, a competition of HS by the reactive species produced by OTC, namely singlet oxygen, may occur, delaying the OTC photo-degradation. REFERENCES (1) Kordel, W.; Dassenakis, M.; Lintelmann, J.; Padberg, S. Pure & Appl. Chem 1997, 69, 1571-1600. (2) Esteves, V. I.; Otero, M.; Duarte, A. C. Org. Geochem. 2009, 40, 942-950. (3) Thurman, E. M. Organic Geochemistry of Natural Waters; Kluwer Academic Publishers, 1985. (4) Esteves, V. I.; Cordeiro, N. M. A.; Duarte, A. D. Mar Chem 1995, 51, 61-66. (5) Santos, M. E. B. H., PhD Thesis, University of Aveiro, 1994. (6) Sulzberger, B.; Durisch-Kaiser, E. Aquat Sci 2009, 71, 104-126. (7) Leal, J. F.; Esteves, V. I.; Santos, E. B. H. Environm Sci Technol 2013, 47, 14010-14017.7 Acknowledgments: This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEst-C/MAR/LA0017/2013. Joana Leal thanks FCT for her PhD grant (SFRH/BD/88572/2012).
B
Figure 1 – Relation between the % of photo-degradation observed and estimated based on SƩλ.
191
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Effects of pyrogenic carbon feedstock and pyrolysis temperature on the oxidation kinetic and benzene polycarboxilic acids formation W.V. Cerqueira(a) *, T.F. Rittl(b), M.A. Pinheiro(a), A.D. Pereira Netto(a), E.H. Novotny(c) (a)
Federal Fluminense University, Chemistry Institute, 24020-150 Niterói - RJ (Brazil) Wageningen University, Department Soil Quality, 6700 AA Wageningen (Netherlands) (c) Embrapa Soils, 22460-000 Rio de Janeiro - RJ (Brazil) * Corresponding author e-mail: [email protected] (b)
Keywords: oxidation kinetic; biochar; benzene polycarboxilic acids; pyrogenic carbon quantification Abstract Pyrogenic carbon (PyC) has a high potential to soil carbon sequestration. Among the methods used to quantify PyC, the method of benzene polycarboxilic acids (BPCA) is well-established. The oxidation step of this method is essential for a reliable PyC quantification. Up to now there are no studies on the influence of feedstock and pyrolysis temperature in the oxidation kinetic of PyC and BPCA formation; and then these are the purposes of this study. For this, different PyC were oxidised at 170 ºC during different times. The data showed similar kinetic curves for all PyC, but different BPCA production depending upon feedstock and pyrolysis temperature. All PyC showed maximum production of BPCA at 6-8 h under nitric acid oxidation at 170 ºC. We concluded that 8 h of nitric acid oxidation at 170 ºC produces a reliable data to PyC quantification, thus representing a robust method. The oxidation step is crucial for the BPCA method. The oxidation must assure that all aromatic structures will be converted to BPCA. Dittmar (3) observed a similar oxidation kinetic for three different samples (perylene, humic acid standard and marine dissolved organic matter) and a recovery of 92-100% for all samples after 9 h of oxidation. However, up to now there are no studies about how feedstock and the pyrolysis temperature affect oxidation kinetics and BPCA production. So, this study aimed to evaluate feedstock and pyrolysis temperature effects on the oxidation kinetic and BPCA production.
Introduction Pyrogenic carbon (PyC), known as biochar, may play an important role in the global C cycle. When added to the soil, PyC is expected to contribute to the recalcitrant C pool and decelerate the soil organic carbon decomposition. Among the available methods of PyC quantification in soil, the BPCA method (1,2) is the most well-established. The BPCA first-step includes the oxidation of PyC by nitric acid at 170 ºC for 8 h. During this oxidation, the polycyclic or aromatic structures present in the PyC are converted to BPCA (Figure 1).
Experimental Biochar samples were obtained after 1 h pyrolysis of eucalyptus (Eucalyptus dunnii) wood and sugarcane (Saccharum officinarum) bagasse at 450 ºC; and from water hyacinth (Eichhornia crassipes); eucalyptus (Eucalyptus dunnii); and pine (Pinus taeda) woods at 350 ºC. Aliquots of 5 mg of PyC were weighed and filled into 5 mL glass ampoules, which were sealed after addition of 0.5 mL HNO3 (65%). The ampoules were placed into microwave vessels and heated up to 170 ºC for 1; 2; 4; 6 and 8 hours using a laboratory oven. After oxidation, the ampoules were opened and the nitric acid evaporated under a gentle N2 flux. The samples were diluted up to 1 mL in the mobile phase. The samples were analysed by the liquid chromatography with UV-DAD detection (RRLC-UVDAD), using a Zorbax Eclipse C18 column
Figure 1. Chemical structures of BPCA (adapted from (1)).
192
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The oxidation kinetics were similar for pine, eucalyptus and water hyacinth pyrolysed at 350 ºC, however they differed in the BPCA production. Eucalyptus produced the highest amount of BPCA, followed by pine and the water hyacinth (Figure 4).
Results and Discussion The yields of BPCA produced from eucalyptus and sugarcane bagasse increased with longer nitric oxidation times (Figure 2). After 8 hours, the production of BPCA for the two PyC reached the maximal.
Figure 4. The kinetic oxidation of eucalyptus, pine and water hyacinth pyrogenic carbon (PyC) pyrolysed at 350 ºC. All PyC had the maximum production of benzene polycarboxilic acids (BPCA) at 8 h.
Figure 2. The kinetic oxidation of eucalyptus and sugarcane bagasse pyrogenic carbon (PyC) pyrolysed at 450 ºC. Both PyC had the maximum conversion of aromatic structures in benzene polycarboxilic acids (BPCA) at 8 h.
The water hyacinth produced a significant small concentration of BPCA, indicating that feedstock play an important role on BPCA formation. Similar kinetics curve were found for most of the studied PyC, despite the feedstock and pyrolise temperature, indicating similar reaction mechanisms for these PyC. Although the pyrolys temperature and feedstock used influenced BPCA production, all PyCs had the maximum BPCA production after 6-8 h. These results are similar to those previously presented (3). Therefore, considering our results and the previous ones, we conclude that 8 h of nitric acid oxidation at 170 ºC is enough for maximal PyC conversion to BPCA and that these are robust oxidation parameters for the PyC quantification.
The oxidation kinetics was similar for the eucalyptus PyC pyrolysed at different temperatures (Figure 3). The temperature did not change the oxidation kinetic, however, as expected, eucalyptus PyC obtained at 450 ºC produced higher amount of BPCA than eucalyptus PyC pyrolysed at 350 ºC.
REFERENCES
(1) Glaser, B.; Haumaier, L.; Guggenberger, G.; Zech, W. Org. Geochem. 1998, 29, 811. (2) Brodowski, S.; Rodionov, A.; Haumaier, L.: Glaser, B.; Amelung, W. Org. Geochem. 2005, 36, 1299. (3) Dittmar, T. Org. Geochem. 2008, 39, 396.
Acknowledgments: WVC and TFR thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for PhD scholarships. ADPN and EHN thank CNPQ for individual research grants. MAP thanks PIBIC-UFF for an undergraduate scholarship.
Figure 3. The kinetic oxidation of eucalyptus pyrogenic C (PyC) pyrolysed at 350 and 450 ºC. Both PyC had the maximum production of benzene polycarboxilic acids (BPCA) at 8 h.
193
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Soil application of a commercial humic product may improve nitrogencycling K. R. Little (a) *, H. M. Gan(b), M. T. Rose(a), W. R. Jackson(a), T. R. Cavagnaro (c), A. F. Patti(a) (a)
School of Chemistry, Monash University, Clayton, Victoria, 3800, Australia School of Science, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway 46150, Petaling Jaya, Selangor, Malaysia (c) School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB1 Glen Osmond, SA, 5064, Australia (b)
* Corresponding author e-mail: [email protected] Keywords: humic, microbial, nitrogen, ammonium, nitrate Abstract Commercially-available lignite-derived amendments have been promoted as plant growth stimulants leading to higher crop yields. These products are also claimed to promote beneficial soil bacteria. An incubator study was designed to investigate changes in the soil microbial community in a sandy, low organic matter soil, in response to application of a soluble humate product. The product was applied at 0, 20 and 300 kg/ha, and following a 6 week incubation period, the bacterial communities were investigated by 16S rDNA amplicon sequencing. With increasing product application rate, there was concurrent increase in the abundance of bacterial families involved in N-cycling. Notably, there was an increase in abundances of Rhodospirillaceae, Beijerinckiaceae and Bradyrhizobiaceae which increased by 31, 61 and 48 % respectively. In addition, there was a 133% increase in Nitrosomonadaceae, and a 77% increase in abundance of Xanthomonadaceae. This provides support for the claim that humate products increase bacterial abundance, and indicates that addition of this type of product to soil may improve N-cycling. Introduction The use of commercially-available humic products in agriculture has been associated with productivity benefits in terms of shoot and root growth, and pasture and crop yields. The extent of benefit is influenced by the source and form of the humic substance, the soil and crop/pasture type, and the product application rate [1]. The mechanisms for these benefits have not been fully explained. On one hand there is evidence to support a direct effect of humic acid on the plant, thereby influencing plant metabolism [2], however there is also evidence to support a nutritional effect due to the complexing ability of humic substances with soil nutrients, making essential nutrients more accessible to the plant [3]. Manufacturers of commercial humate products claim that these products promote beneficial soil bacteria however there is little scientific evidence to support this claim. To date, there has been very little work on the potential effects of humic acids on the microbial community and biogeochemical processes that are essential for soil and plant health. The application of soil amendments has been shown to have direct and indirect effects on soil microbes, with community response reflecting even minor changes within a short period of time [4]. Nitrogen availability is a key factor in the productivity of a cropping or pasture system. Nitrogen fertiliser costs represent a substantial proportion of a farmer’s input costs, and losses can not only affect profitability but can also result in a negative impact on the environment, an example of which is the contribution of N2O to greenhouse gas emissions.
The objective of this study was to investigate the impact of a commercial humic product on the soil microbial community, with particular emphasis on Ncycling bacteria Experimental An incubation experiment was conducted to assess changes in the soil microbial community in response to the addition of commercial soluble humate granules (SHG) applied at 0, 20 and 300 kg/ha. The soil had a sandy texture and was mildly acidic (pH 5.97), with a low organic matter content (1.6%). The humic component of the SHG product was humic acid extracted from Victorian lignite coal. Five replicates of each application rate were prepared. The SHG were thoroughly incorporated into the soil at the appropriate application rate and deionised water was added to the equivalent of 60% field capacity. The incubation containers were loosely covered to reduce moisture loss, and incubated at 25ºC in a dark environment for 6 weeks. Soil moisture was maintained by the addition of deionised water when required. Immediately following harvest, the soils were subdivided into two equal portions, with one portion stored at 4ºC for soil analysis and the other stored at -20ºC for DNA extraction. From the refrigerated portion of soil, ammonium and nitrate concentrations were determined on weighed sub-samples extracted with 1M KCl for 1 h. Ammonium was analysed by reaction with salicylate in the presence of nitroprusside, and absorbance measured at 650 nm. Nitrate was measured by vanadium (III) reduction and reaction with Greiss
194
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
reagents, with absorbance read at 540 nm, following color development. The DNA for each replicate sample was extracted using PowerSoil DNA Isolation Kit (MoBio Laboratories), following the manufacturer’s protocol. The extracted DNA was quantified and the purity verified with a Nanodrop spectrophotometer at 260 and 280 nm. From this DNA, amplification of 16S rDNA was achieved using primers S-D-Bact-0341-bS-17 and S-D-Bact-0785-a-A-21 containing Illumina adaptors and unique barcode. After normalisation and pooling of the PCR products, sequencing was performed on the MiSeq sequencer. Raw reads were overlapped to generate sequences of approximately 464 bp to be used for subsequent analysis. After chimera removal and OTU identification using UPARSE, the microbial community composition was analysed using QIIME. All data was normalised by arcsin transformation and then analysed by ANOVA. Where significant differences were found, pairwise comparisons were made using Tukey’s honestly significantly difference (HSD).
0 kg/ha a ab
5 4
20 kg/ha
b
300 kg/ha a ab
b
3
b Rhodospirillaceae
0
Beijerinckiaceae
1
ab a
b
ab
a
Xanthomonadaceae
2
b
b
a
Bradyrhizobiaceae
6
Nitrosomonadaceae
Abundance (%)
identification of N-cycling genes and measurement of N2O emissions.
Bacterial family
Figure 1. Abundance of N-cycling bacterial families at
0, 20 and 300 kg/ha application rates of a commerciallyavailable soluble granule humate product (SHG). Values allocated the same letter were not significantly different at the P<0.05 level as assessed by Tukey’s HSD. Table 1. Ammonium and nitrate in soils incubated for 6 weeks at 25ºC following amendment with 0, 20 and 300 kg/ha soluble humate granules (SHG). SHG application rate (kg/ha) Nitrogen pool (±SE) 0 20 300 NH4+ (ug/g dry soil) 6.64 (1.4) 6.35 (0.8) 3.51 (0.3) NO3- (ug/g dry soil) 291 (24) 275 (8) 274 (12) References 1. Rose, M.T., et al., Chapter Two - A Meta-Analysis and Review of Plant-Growth Response to Humic Substances: Practical Implications for Agriculture, in Advances in Agronomy, L.S. Donald, Editor. 2014, Academic Press. p. 3789. 2. Nardi, S., et al., Physiological effects of humic substances on higher plants. Soil Biology and Biochemistry, 2002. 34(11): p. 1527-1536. 3. Chen, Y., C. Clapp, E, and H. Magan, Mechanisms of plant growth stimulation by humic substances: the role of organoiron complexes. Soil Science and Plant Nutrition, 2004. 50(7): p. 1089-1095. 4. Bünemann, E.K., G.D. Schwenke, and L. Van Zwieten, Impact of agricultural inputs on soil organisms—a review. Soil Research, 2006. 44(4): p. 379-406. 5. Madigan, M., S.S. Cox, and R.A. Stegeman, Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae. Journal of Bacteriology, 1984. 157(1): p. 73-78. 6. Cavagnaro, T.R., et al., Short-term population dynamics of ammonia oxidizing bacteria in an agricultural soil. Applied Soil Ecology, 2008. 40(1): p. 13-18. 7. Heylen, K., et al., The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. Environmental Microbiology, 2006. 8(11): p. 2012-2021. 8. Falk, S., B. Liu, and G. Braker, Isolation, genetic and functional characterization of novel soil nirK-type denitrifiers. Systematic and Applied Microbiology, 2010. 33(6): p. 337-347. Acknowledgments: KRL gratefully acknowledges a Brown Coal Innovation Australia (BCIA) scholarship and an Australian Postgraduate Award scholarship. TRC gratefully acknowledges the Australian Research Council for supporting his research through the Future Fellowship program (FT120100463). The support of BCIA and partners (International Power, Clean Coal Victoria and Bass Coast Landcare) for a larger project on commercially-available humate products is gratefully acknowledged.
Results and Discussion The application rate of SHG had a significant effect on the abundance of several bacterial families whose function is associated with N-cycling in soil (Figure 1). Comparing the unamended soil (0 kg/ha application rate) to that amended with 300 kg/ha of SHG, the largest change in abundance was seen in the bacterial family Nitrosomonadaceae, which increased by 133.9%. The abundance of the bacterial families Rhodospirillaceae, Beijerinckiaceae, Bradyrhizobiaceae and Xanthomonadaceae increased by 31.3, 60.9, 47.6 and 77.7% respectively. Rhodospirillaceae, Beijerinckiaceae and Bradyrhizobiaceae are associated with fixation of atmospheric N [5]. Nitrosomonadaceae includes nitrifying bacteria which facilitate the nitrification of ammonium to nitrate [6]. The family Xanthomonadaceae, consist of denitrifying bacteria which facilitate the transformation of nitrate to atmospheric N [7]. As well as functioning as an N fixer, Bradyrhizobiaceae may also act as a denitrifier [8]. While emission of N2O was not investigated here, an increase in abundance of denitrifying bacteria could reduce N2O emissions by facilitating denitrification directly to atmospheric N. While not significant by ANOVA analysis, with increasing application rate of SHG there was a decreasing trend in ammonium and nitrate in the postincubated soils (Table 1). Ammonium decreased by 47.1% between the 0 and 300 kg/ha application rates, and nitrate decreased by 5.8%. Along with the increase in abundance of N-cycling bacteria, this may suggest improved nitrogen cycling in the soil. In summary, the application of SHG at increasing application rates significantly increased the abundance of N-cycling bacteria. This along with decreases in soil ammonium and nitrate may infer improved Ncycling in the soil. Further investigation is required to link abundance changes in the microbial community with functional changes. This could include the
195
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Lead binding to soil fulvic and humic acids: NICA-Donnan modeling and XAFS spectroscopy
Juan Xiong,† Luuk K. Koopal,†,‡ LiPing Weng⊥ and WenFeng Tan,*,†,§ †College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China ‡Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands ⊥Department of Soil Quality, Wageningen University, P.O. Box 8005, 6700 EC, Wagneningen, The Netherlan §State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling, Shaanxi 712100, China
* Corresponding author e-mail: [email protected] Keywords humic acid(HA), Fulvic acid(FA), Pb binding, NICA-Donnan model, generic parameters, EXAFS
Abstract Lead binding to FA and HAs was studied by Pb binding isotherms and XAFS spectroscopy. The NICA-Donnan model described Pb binding to HS satisfactorily. Milne’s generic parameters didn’t provide a prefect prediction for Pb binding to soil samples. For the soil HAs, the Pb binding prediction with generic parameters can be improved significantly by using the value nPb1=0.92 instead of generic value nPb1=0.60. The nPb1/nH1 ratios obtained were relative high, indicating mono-dentate Pb binding to carboxylic-type groups. The nPb2/nH2 ratios depended somewhat on the method of optimization, but the values were distinctly lower than the nPb1/nH1 ratios, especially 2+ when the optimization was based on Pb bound vs log [Pb ]. These low values indicate bi-dentate binding to the phenolic-type groups at high Pb concentration. The EXAFS results at high Pb loading suggested that Pb was bound in bidentate complexes of one carboxylic and one phenolic group or two phenolic groups in ortho position. Introduction Pb−HS samples were collected with the fluorescence FA and HA are believed play an important role in mode; the reference materials were measured in controlling the fate of metal ions in environment transmission mode. All samples were recorded at the Pb because it are ubiquitous and the large number of acidic L3-edge (E=13035eV)[3]. functional groups which can interact with metal ions. Results and Discussion The interaction between HS and metal ions vary with NICA-Donnan Modeling of Pb Binding Table1 Non-ideality parameters for FA and HAs environmental conditions, such as pH, ionic strength JGFA GFA a PAHA JGHA JLHA GHA b etc. Therefore, it is a challenge to reliably predict metal Pb binding parameters for log Pb 2+-log adsorbed Pb 2+ ion binding to HS using chemical speciation model as the NICA-Donnan model[1, 2]. In fact, NICA-Donnan nH1 0.66 0.64 0.88 0.80 0.67 0.81 n Pb1 0.95 0.60 0.92 0.91 0.94 0.60 model with generic parameters succeeded in giving nH2 0.88 0.76 0.64 0.83 0.90 0.63 reasonable predictions for metal ions such as Cu and nPb2
Cd, but it failed in predicting Pb binding to soil HS. Lead is a widespread metal pollutant whic h can be toxic to plants, animals, and humans. A good
0.79
0.69
0.53
0.85
0.72
0.69
2+
Pbnbinding parameters for log0.81 Pb -amount Pb2+ 0.72 0.90 of adsorbed 0.68
understanding of Pb binding to soil HS and confidence in the NICA-Donnan model to describe Pb bound by soil HS is important. The X-ray absorption fine structure (XAFS) spectroscopy which is a promising technique can unravel the Pb binding mechanisms to understand the interaction of Pb and HS. Combining NICA-Donnan model with XAFS on Pb binding to soil HS leads to a better understanding of the molecular structure of Pb−HS complexes and characteristics of Pb binding to soil HS. The aims of the study are (1) improve the generic parameters to obtain a better description of Pb bound by soil HS (2) to investigate the coordination environment of Pb binding to HS by XAFS; (3) to derive material-specific parameters and to compare the model results with spectroscopic results.
n H1
0.94
0.93
0.97
0.71
nH2
Pb1
0.95
0.83
0.92
0.94
nPb2
0.66
0.49
0.52
0.44
1
-1
log Pb sorbed (mol kg )
0 -1 pH7
-2
pH6
2+
-3 pH7
-4
-6 1
-1
log Pb sorbed (mol kg )
0
PAHA
pH6
JGFA
pH4 -3 -2
pH4
PAHA
-1 pH7 -2 -3
2+
Methods and materials
JGFA
pH6
-5
pH4
JGFA and JGHA were extracted from the mountainous meadow soil and JLHA was extracted from the brown soil. PAHA was purified from commercial Aldrich humic acid collected from lignite. Pb binding to HS was measured at ionic strengths 0.05M and three pHs (4, 6 and 7) using Pb titration. The free Pb concentration in solution was measured by Pb-ion selective electrode (Pb-ISE) in combination with an Ag/AgCl reference electrode. The Pb-HS complexes were prepared at pH 5.5 in 0.1M KNO 3. The final equilibrium concentration of Pb2+ was about 0.1mM and the amount of Pb bound was about 1.3mol/kg for the HAs and 1.8mol/kg for JGFA. The XAFS spectra of HS loaded with Pb and reference materials were measured at 25oC on the 1W1B beamline at the Beijing Synchrotron Radiation Facility (BSRF). Spectra for
-4 -5
JGFA
pH7 pH6 pH6 pH4
-6 -11 -10 -3 -2 pH4 pH4
-9
-8
-7
-6
JLHA
-1
2+ lo g Pb (mol L )
-5
-4
pH4 -3 -11 -3 -2
-10 pH4
-9
PAHA NICA-Donnan model
-8
-7
2+
-6
-1
lo g Pb (molL )
-5
-4
-3
Figure PAHA 1 Predictions of for Pb binding to HS. pH7 pH7 Solid symbols: experimental data; open symbols: results calculated using material-specific parameters; Red curves: predictions of NICA-Donnan model using the full generic parameters; Blue curves: predictions of NICA-Donnan model using the generic pH7 pH7 parameters of HA w ith parameter n Pb1 a djusted to 0.92 .
The Pb binding isotherms are presented in Figure1 and JLHA JLHA the optimized NICA-Donnan parameters are tabulated in Table1. The goodness of fit can be observed in Figure1 where the open symbols represent the NICA-Donnan fits to data. As expected, the amount of Pb bound by HS, at a given Pb equilibrium concentration, decreases with decreasing pH due to H+−Pb2+ competition for binding sites and the weaker
196
pH6
pH6
pH4
pH4
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
electrostatic attraction. At high Pb concentrations the amounts of Pb bound by HS level off due to the decrease of electrostatic attraction and the site occupancy by Pb, and consequently the effects of pH become smaller. Coordination Environment of Pb 3
k x( k)
a
4
3
Pb(NO3)2
2
JGFA JGHA
Fourier transfer magnitude
1
JLHA
0
PAHA
-1 3
4
5
6
7
k (Å ) -1
8
9
10
b
Pb-O
Pb-C
Pb(NO ) JGFA
3 2
JGHA JLHA PAHA
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
R (Å )
Figure2The k3-weighted (a) and Fourier transformed (b) EXAFS for 0.1 M aqueous Pb(NO3)2 and for Pb binding to HS at pH 5.0. Table 2 parameters for EXATS optimizations. sample Atomic R(Å) σ2(Å2) CN ΔE0 (ev) Rf backscatter JGFA Pb-O 2.40 0.0190 3.73 -11.40 0.0072 Pb-C 3.25 0.0167 2.49 -11.40 0.0057 PAHA Pb-O 2.37 0.0196 4.17 -13.50 0.0076 Pb-C 3.17 0.0193 3.36 -13.50 0.0098 JGHA Pb-O 2.38 0.0197 4.01 -10.75 0.0076 Pb-C 3.21 0.0226 2.85 -10.75 0.0091 JLHA Pb-O 2.40 0.0237 4.73 -8.95 0.0237 Pb-C 3.27 0.0149 2.77 -8.95 0.0117
The EXAFS spectra for Pb-HS complexes and of aqueous Pb(NO3)2 are depicted in Figure2a and the 3 k -weighted Fourier transforms of EXAFS spectra are depicted in Figure 2b. The vertical dashed lines indicate the peak positions for the first and second oscillation, i.e. Pb−O and Pb−C coordination. The results indicated that Pb is bound to HS by inner-sphere complexation with a C−O−Pb structure, then the EXAFS spectra were fitted and parameters are listed in Table2.The results revealed that Pb was coordinated by 3.73−4.73 O at a distance of 2.37−2.40 Å in the first shell, and by 2.49−3.36 C at a distance of 3.17−3.27 Å for the second shell. It is generally accepted that carboxylic and phenolic groups of HS are quantitatively the most important binding sites for metals ions. In addition, the five-membered and six-membered rings containing a C−O−Pb−O−C structure are stable[4]. It conclusion emerges that, at high Pb loading, Pb tend to be predominantly complexed by phenolic and/or carboxylic groups located in ortho-position. Pb Binding Structure The observed nPb1/nH1 ratios are high (1.05−1.44), indicating the monodentate Pb binding to carboxylic groups. The nPb2/nH2 ratios of soil HS are substantially smaller (0.80−1.02) and indicate that part of the phenolic groups is involved in bidentate Pb binding. The Pb complex structures inferred from the above n Pbj/nHj ratios present a different picture with the spectroscopic results. Note that the present fit is based on a comparison of log[bound Pb] vs log[Pb2+], a relatively high weight has been given to low Pb
loadings, whereas the spectroscopic results are obtained at relatively high Pb binding. In the new fitting procedure, the NICA-Donnan model is fitted to the binding data using a linear scale for the bound amounts for giving the relatively high weight to high Pb loadings. The new n Pb1/nH1 ratios were still somewhat larger than unity and indicated monodentate Pb binding to the carboxylic groups. The new nPb2/nH2 ratios were in the range of 0.47−0.69, clearly indicateing that the average number of Pb bound by the phenolic groups is relatively low. Therefore, the NICA-Donnan model predicts that, at high Pb loading, Pb is bound by the phenolic groups in a bidentate structure. In view of the fact that the model take no account of the mixed complexes involving both carboxylic and phenolic group, the partial agreement of the model results with the spectroscopic results, which indicated that at high loading Pb was bound in bidentate complexes of one carboxylic and one phenolic group or two phenolic groups, is satisfying. Comparison with Predictions Using the Generic NICA-Donnan Parameters In Figure1 a comparison between the measured Pb binding to HS and the predictions of the NICA-Donnan model using the generic parameters[5] are presented. It implied that Milne’s generic parameters didn’t provide a prefect prediction for Pb binding, except PAHA. The Pb-FA data sets used to optimize generic parameters contained eight data sets, of which six were limited to relative high Pb concentrations (>10−7M) and five of the FAs were extracted from podzol soils. The HA-Pb data sets used by Milne et al. were mainly based on PPHA, purified HA extracted from Irish moss peat[5]. Undoubtedly, the parameters of Pb binding to FA and HA are constrained by the properties of podzol soils FA and PPHA. The parameter values of soil HS scatter around the generic parameter values, except for nPb1, which is similar for both soil samples but much higher than the generic value. In view of the wrong initial slope also here the relatively low generic nPb1 value is the first parameter that should be suspected. The sensitivity tests showed that for soil HAs the model prediction with generic parameters could be strongly improved only when the generic nPb1 (0.60) was replaced by the specific value of nPb1 (0.92). For JGFA, only when nPb1, logKPb1, and logKPb2 of generic parameters were replaced by JGFA material-specific values was an improvement achieved. This is probably due to the fact that JGFA is a rather exceptional FA, also its proton binding behavior deviates strongly from JGFA [2]. References [1] Kinniburgh D.G., van Riemsdijk W.H., Koopal L.K., Borkovec M., Benedetti M.F., Avena M.J., Colloids Surf., A 1999, 151, 147-166. [2] Tan W., Xiong J., Li Y., Wang M., Weng L., Koopal L.K., Colloids Surf., A 2013, 436, 1152-1158. [3] Xiong J., Koopal L.K., Tan W., Fang L., Wang M., Zhao W., Liu F., Zhang J., Weng L., Environ. Sci. Technol. 2013, 47, 11634-11642. [4] Basolo F., Johnson, R.C., Coordination chemistry, New York, 1986. [5] Milne C.J., Kinniburgh D.G., Tipping E., Environ. Sci. Technol., 2003, 35, 2049-2059.
197
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Size distribution of humic substancesevaluated with flow field-flow fractionation and itssize-dependent cation binding Y. Yamashita (a) *, T. Saito(b) (a)
University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan Japan Atomic Energy Agency, 2-4 Shirakatashirane, Tokai, Ibaraki 31901195, Japan * Corresponding author e-mail: [email protected] (b)
Keywords: hydrodynamic size, flow-field flow fractionation, cation binding, pH-buffering agents AbstractSize distributions of humic substances (HS) with different pH-buffer agents and pH were evaluated by using a flow field-flow fractionation (flow-FFF). In addition, its size-dependency of multivalent cation binding was measured with an ICP-MS connected downstream of the flow-FFF. A certain type of pH-bufferingagent clearly affected the size distribution, while the fractogram with others was similar to that without buffer. Change of pH (6.5 to 8.5) in the same buffer exerts little influence on the size distribution. The size-dependent cation binding demonstrated that larger molecules have greater affinity for all cations tested at the present study. UO22+and Eu3+in the presence of MOPS at pH 7.5 for metal binding study. Using an asymmetrical flow-FFF system(AF2000 FOCUS, Postnova Analytics)that equipped with flow channel made of 1,000 Da polyethersulfone membrane,which is termed an accumlation wall,we evaluatedthe size distribution of the HS.The flow-FFF system allows us to fractionate and subsequently eject the HS samples in ascending order of hydrodynamic diameter. In principle, retention time of the samplecorresponds to its hydrodynamic diameter. The detection of the amount ofeluted HSwas performed withabsorbance at 255 nm of UV/Vis detector (SPD20A, Shimadzu).Only for the metal binding study, the eluted samplewerefurther flowed intoan ICPMS(7500cx, Agilent) connected downstream of the UV detector.
Introduction In soil and aquatic environments, humic substances (HS) can associate with toxicants and nutrients, especially cationic metal ions [1], and thereby can behave as carriers of them [2]. To better understand the fate of such ions binding by HS, simultaneous evaluation of their hydrodynamic properties and cation binding behavioris strongly required.Schimpf and Petteys [3] have demonstrated that a flow field flow fractionation system (flow-FFF) has advantage in study ofsize of HS.Siripinyanond et al. [4] proposed that flow-FFF connected withinductively-coupled plasma mass spectroscopy (ICP-MS)is promising approach for investigation of metal-HS interaction. In the present study, we determined size distribution of several IHSS standard samples and a commercial oneby usingflow-FFFin the presence of different pHbuffer agents and evaluated their effect on their size distribution. Moreover,we studied size-dependent metal binding by using ICP-MS connected with flowFFF system when low metal concentration was loaded in HS solution.
Results and Discussion Size distribution of HA with different pHbufferingagents:Figure 1 shows the representative fractogram of SHA at pH 6.5 with different pHbuffering agents, and without any buffersbut with just NaCl as a reference. It was found that TRIS made an increase of modal value of size.In the case of other HS with TRIS, we could also confirm this trend. Because TRIS molecules are positively charged at pH 6.5, the charge potential of HS could be partly screened out. The decrease of their charge potential may affect an interaction between HS samples and the accumulation wall in theflow channeland their retention time. In contrast, the shape of fractograms and modal value with MOPS and MES are similar to those without buffer. On the basis of this result, weregarded the MOPS asthe least disruptive buffer among them and utilized only MOPSin subsequent experiments. Size distribution of HA at different pH: Change of the pH between 6.5 and 8.5 in the presence ofMOPS exerted little influence on the size distribution of SHA (Fig. 2).This tendency was observed in the case of other HS samples. Recovery ratio tended to decrease with decreasing pH. This may be attributed to some
Materials and Methods Three kinds of the IHSS standard sample (Suwanee River humic acid (SHA), Elliot humic acid (EHA) and Suwanne River fulvic acid (SFA)) and a commercial HA purchased from Aldrich(AHA) were utilized.They were dissolved in NaOH solution at pH ~ 10 and then diluted with dionized water to make 50 mg/L HS solutions. The pH of these solutions was adjusted with NaOH/HCl solution in the presence of three types of pH-bufferingagent(2-(N-morpholino) ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS) and 2-amino-2-hydroxymethyl-propane-1,3diol (TRIS)) to test the effect them on the size distribution of HS. Each pH buffer solution was adjusted to be 5 mM. To stabilisze the ionic strengh, appropriate amount of NaCl was added in MOPS and MES solution. Aside from these samples, we preparedAHAsolution with different concentrations of
198
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
legend correspond to recovery ratio of the sample and modal value of hydrodynamic diameter, respectively.
attractive interaction between HS samples and the accumulation wall because of the loss of their negative charge by protonation of their acidic functional groups. Size-dependent Cation bindingby HS: Figure 3 depicts the fractograms of AHA and the amount of Eu3+ bound when 0.01, 0.1 and 1 M was added in the AHA solution, respectively.It was observed that the peak of the fractogram of AHA was different from the peak of Eu3+ boundin all cases. Thisgap means that Eu3+preferentially bound to larger fraction of AHA. Namely, Eu3+have a high affinity for larger fraction rather than the abundant smaller fraction. It was found that the addition of trivalent cation had little impact on the size distribution of AHA at least 1 M.Humicaggregation induced by multivalent cation can be avoided. It is known that the HS aggregation is related to the amount of cation bound and its electric potential [5]. In the present case, dispersion state can be kept because of low loading ofmetal ion. In the case of UO22+, we observed similar trend.
3+
Eu = 0.01 M
3+
Eu = 0.1 M
3+
Eu = 1 M
Figure 3.Size-dependent Eu3+binding by Aldrich humic
Figure 1.Representative fractogram of Suwanee River humic acid with different pH-buffering agents, and without any buffers but with just NaCl as a reference. The values in legend correspond to recovery ratio of the sample and modal value of hydrodynamic diameter, respectively.
acid and size distribution of AHA with different additive concentrations of Eu3+(0.01, 0.1 and 1 M) in the presence of MOPS buffer at pH 7.5.
REFERENCES
(1) Tipping, E.,Cation binding by humic substances, Cambridge University Press, Cambridge, 2002. (2) McCarthy, J.F.; Zachara, J.M., Environ. Sci. Technol. 1989, 23 (5), 496-502. (3) Schimpf, M.E.;Petteys, M.P.,Col. Surf. A, 1997, 120, 87-100. (4) Siripinyanond, A.; Worapanyanond, S.;Shiowatana, J., Environ. Sci. Technol. 2005, 39, 3295—3301. (5) Weng, L.; Temminghoff, E.J.M.; van Riemsdijk, W.H., Eur. J. Soil Sci.2002, 53, 575-587.
Figure 2.Representative fractogram of Suwanee River humic acid with MOPS at different pH. The values in
199
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Use of Ultrafiltration and Diffusive Gradients in Thin Films to Evaluate the Influence of the Dissolved Organic Matter in the Arsenic Speciation E. S. J. Gontijo (a) *, A. S. C. Monteiro (b), M. L. Fernandes (c), D. A. dos Reis (c), M. T. Domingues (a), C. H. Watanabe (a), P. S. Tonello(a), H. M. P. Roeser(c), K. Friese (d), A. H. Rosa (a)
(a) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Av. Três de Março, 511, Alto da Boa Vista, 18087-180, Sorocaba, SP – Brazil (b) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Rua prof. Francisco Degni, 55, Quitandinha, 14800-060, Araraquara, SP – Brazil (c) Universidade Federal de Ouro Preto (UFOP), Morro do Cruzeiro, S/N, 35400-000, Ouro Preto, MG – Brazil (d) Helmholtz Zentrum für Umweltforschung UFZ, Brückstraße 3a, 39114, Magdeburg, Germany * Corresponding author e-mail: [email protected] Keywords: metal speciation, arsenic, dissolved organic carbon, ultrafiltration, DGT Abstract The speciation of As was studied in waters sampled in a tropical region in Brazil. The metals Al and Fe were also investigated because they may affect the complexation of As by dissolved organic matter (DOM). The dissolved fraction was determined using a 0.45 µm filter. An ultrafiltration system was used to determine the fraction <1KDa, which was considered as “free” fraction (or inorganic complexes). DGT devices were deployed at some points in order to determine labile and inert fractions of the metals analysed. Dissolved organic carbon was below 6 mg L-1 for all samples. The percentage of the fraction complexed with DOM was smaller for As if compared with Fe and Al. About 50% of dissolved As in the waters studied was occurred as inert fraction. The dissolved form of Fe and Al had higher percentages in the inert fraction indicating the formation of more stable complexes with DOM. Introduction The chemical forms of an element in aquatic environments are related to their mobility and toxicity. In this way, the total concentration of a metal in the water do not reveal its real level of contamination to organisms (1). Therefore, the dissolved metal fraction of a metal is more associated to bioavailability than the total concentration. However, even this dissolved fraction just provides incomplete information about toxicity of an element (2). The dissolved organic matter (DOM), for instance, may form complexes with metals changing its lability, bioavailability, mobility and toxicity. Consequently, it is important to study specific fractions of metals/metalloids and its interactions with dissolved organic carbon (DOC) for a better understanding of their role in the environment (3). Several techniques have been used in studies related to the speciation of metals. Ultrafiltration (UF) with 1 KDa membrane, for example, is a procedure where a metal in the filtrate is assumed to be a “free” metal (or inorganic complexes) (3). A technique with the advantage of in situ measurements used to determine labile metals in water is the diffusive gradients in thin films (DGT) (1). The Quadrilátero Ferrífero (QF) is a tropical area located in southeast of Brazil that is widely known for its mineral deposits. The exploitation of these deposits is associated with problems related to trace metals. Arsenic (As), for instance, is a metalloid that occurs naturally associated with sulphide-rich gold (Au) bearing rocks in the QF (4). However the Au exploitation contributed to an increase in the
concentration of this metalloid, which is known to appear in higher levels in water, soils and sediments of the area (5). In the environment, the mobility of As is potentially affected by DOM and metals like iron (Fe) and aluminium (Al) (6). The aim of this research was study the As, Al and Fe speciation by fractionating waters sampled in the QF using 1 KDa ultrafiltration membranes and DGT devices. In addition, the influence of the dissolved organic matter in the complexation of metals and metalloids was evaluated. Experimental Samples of water were collected at 10 points in southeast of QF in the rainy season. The samples for Al and Fe analysis (around 2 L) were stored in plastic bottles. The samples for total organic carbon (TOC) and DOC (previously filtrated) analyses were stored in 60 mL amber-glass bottles. At 5 points, DGT devices were deployed for around 1 week to determine in situ the labile and inert fractions of the elements analysed in the samples. For the analyses of Al and Fe, a chelex-100 gel was used. For As analysis a ferrihydrite gel was used (7). The temperature was measured in situ before and after the experiment. In the laboratory, part of the samples for metal analysis was filtered in 0.45 µm filters in order to determine the dissolved fraction of the metals/metalloid. Part of the permeate was filtered in 1 KDa membranes (in a UF system) to determine the “free” fraction of the elements analysed. All fractions were acidified with HNO3 after the filtration. The Chelex DGT devices were eluted with HNO3 (1 M) and the ferrihydrite DGT devices were
200
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Concentration (mg L-1)
6
TOC
5
DOC
4 3
1 1
2
3
4
5 6 7 Sampling points
8
9
% As Inert
Inert As (µg L-1)
% As Labile
<0.09
0.174
-
-
-
6
<0.09
0.377
-
-
-
7
0.800
0.479
59.9
0.321
40.1
9
1.060
0.531
50.1
0.529
49.9
The study of the speciation helps to understand the behaviour of metal/metalloids in the environment, especially considering the bioavailability to organisms and DOM.
10
Figure 1. TOC and DOC measured in all samples collected
in QF. The point 4 showed higher concentrations of DOC (4.48 ± 0.04 mg L-1). The DOC of the points 3 and 8 was not determined.
REFERENCES
% As Complexed to DOM
As complexed to DOM (µg L-1)
"Free" As (µg L-1)
Dissolved As (µg L-1)
Particulate As (µg L-1)
Total As (µg L-1)
Fractionation of Al, Fe and As [Table 1] shows the amounts of As determined in the total, dissolved, particulate, and “free” fractions and complexed to DOM. The points 1 and 10 showed higher concentrations of total As. Most of As available in the samples is present in its “free” form. Point 9 had higher percentage of As complexed to DOM. The results showed that greater amounts of Fe and Al were complexed to DOC if compared with As.
Point
4
Table 2. Fractions labile and inert As determined from deployment of DGT devices in 5 points of QF. These fractions for point 1 were not determined for As.
2
0
Labile As As (µg L-1)
Point
Results and Discussion TOC and DOC of the samples: [Figure 1] shows the results of TOC and DOC for all samples collected in QF. All results were below 6 mg L-1.
Dissolved As (µg L-1)
Inert and labile Al, Fe and As [Table 2] shows the labile and inert fraction of As in waters of the samples collected at some points of QF. It was observed that about 40 to 50% of As present in the dissolved fraction at the points 7 and 9 was inert and therefore formed very stable complexes with organic matter. At the points 4 and 6 the labile fraction was above the dissolved fraction. This result could be explained by the fact that the dissolved metal was measured just in the beginning of deployment of DGT. For Fe and Al the inert fraction had higher percentages indicating the formation of more stable complexes.
eluted with diluted HCl (1:4). Al and Fe of the total (no filtered), dissolved and ultrafiltrated fractions and from the DGT devices were measured using an ICPOES (Agilent Technologies 700 Series). For As measurements a graphite furnace atomic absorption spectrometer (Varian AA240Z) was used. Acid digestion was performed before analyses for the total and dissolved fractions. Diffusion coefficients to DGT calculations were taken from literature (7, 8). The TOC and DOC analyses were carried out in a TOC Analyser (Analytik Jena multi N/C 3100).
1
24.06
3.68
20.38
20.12
0.26
1.28
2
0.33
0.33
<0.09
<0.14
-
-
3
0.12
0.12
<0.09
<0.14
-
-
4
0.45
0.45
<0.09
<0.14
-
-
5
0.3
0.3
<0.09
<0.14
-
-
6
0.75
0.75
<0.09
<0.14
-
-
7
1.58
0.78
0.80
0.73
0.07
8.75
8
1.11
0.9
0.21
0.2
0.01
4.76
9
2.26
1.2
1.06
0.45
0.61
57.55
10
55.9
8.79
47.11
40.59
6.52
13.84
(1) Forsberg, J.; Dahlqvist, R.; Gelting-Nystrom, J.; Ingri, J. Environ. Sci.Technol. 2006, 40, 3901-3905. (2) Goveia, D.; Lobo, F.A.; Burba, P.; Fraceto, L.F.; Dias Filho, N.L.; Rosa, A.H. Anal. and Bioanal. Chem. 2010, 397, 851-860. (3) Tonello, P.S.; Rosa, A.H.; Abreu Jr, C.H.; Menegário, A.A. Anal. Chim. Acta. 2007, 598, 162-168. (4) Varcjao, E.V.V.; Bellato, C.R.; Fontes, M.P.F.; Mello, J.W.V. Environ. Monit. Assess. 2011, 172, 631-642. (5) Borba, R.P.; Figueiredo, B.R.; Matschullat, J. Environ. Geol., 2003, 44, 39-52. (6) Redman, A.D.; Macalady, D.L.; Ahmann, D. Environ. Sci.Technol., 2002, 36, 2889-2896. (7) Panther, J.G.; Stillwell, K.P.; Powell, K.J.; Downard, A.J. Anal. Chim. Acta, 2008, 622, 133-142. (8) Zhang, H., DGT - for measurements in waters, soils and sediments (Technical Documentation), 2003. 58 p.
Acknowledgments: This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors gratefully acknowledge the Universidade Federal de Ouro Preto (UFOP) for the support in the field trips in QF.
Table 1. As in the fractions total, particulate (>0.45 µm), dissolved (<0.45 µm), “free” (<1KDa) and complexed to DOM (obtained by difference between dissolved and “free”). The point 9 showed higher amounts of As complexed to DOM.
201
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Immobilisation of 15N derived from 15N-enriched black nitrogen into soil organic matter M. Lopéz-Martín*, J. M. De la Rosa, H. Knicker Instituto de Recursos Naturales y Agrobiología, CSIC. P.O. Box 1052, E-41080 Sevilla, Spain *[email protected] Keywords: NMR spectroscopy, 15N enrichment, charcoal, N stabilization, vegetation fires, incubation Abstract. A medium-term pot experiment of 16 months was designed to study the incorporation of 15N from burnt and unburnt plant residues into the stable soil organic matter fraction. Our results show that after 2 weeks all soils were enriched in 15N, demonstrating that Black Nitrogen (BN) was relatively quickly incorporated into the soil. Although 15N from the mineral fertilizer was identified in the soil at the beginning of the experiment, our study indicated that it was not efficiently immobilized over a longer time periodframe. In contrast 15N from the added pyrogenic organic matter (PyOM) remained incorporated into the humic material until the end of the experiment. Preliminary 15N NMR studies confirmed that some of the heteroaromatic N from BN was transformed into amide N most likely of microbial origin as which it remained stabilized until the end of the experiment. Our experiments lead to the conclusion that in the post fire environment BN plays an important role as a N-fertilizer for the newly recovering plant cover. Our results confirmed that BN is less biochemically recalcitrant than commonly assumed. Therefore N-rich PyOM may act as slow-release N source for the growing plant. Introduction In particular, Mediterranean ecosystems are often affected by wildfires, which can cause a series of changes in the soil properties such as pH, texture and color. During burning, PyOM is formed by incomplete combustion of plant debris and humic material. During this process biomolecules such as carbohydrates, lipids and proteins are cyclized by deshydratation yielding in a highly aromatic material. However, the degree of those alterations clearly depends upon the strength of the charring. Comparably, the chemical composition of charcoal strongly depends upon the nature of the fuel [1]. Whereas cellulose-rich wood results in PyOM rich in benzofurans, N-rich sources form charcoals containing considerable amounts of so called black nitrogen (BN) [2]. Thus, vegetation fires modify the quality and quantity of the soil organic nitrogen (SON) not only by reducing its labile fraction and increasing the amount of more recalcitrant forms, but also by the input of BN. Commonly, it is assumed that charred material is biochemically more stable than fire unaffected humic material. However, recent studies indicated that charcoal is biochemically less recalcitrant than commonly assumed [3,4]. In laboratory incubation studies it was even indicated that BN can be quickly used for plant growth [5]. With this in mind, the goal of the present study was to obtain more insights into the humification and stability of BN. Therefore, a pot experiment was designed were plants were grown on soils amended with different forms of 15N which were representing typical N sources in fire-affected soils. They comprised 15Nenriched plant residues, 15N-PyOM from grass or K15NO3. Additional experiments were performed in which these amendments were mixed with N-sources without any 15N enrichment to obtain insights if the presence of mineral fertilizer affects the humification of organic N residues. The incorporation of the isotopic label into SOM was followed by isotopic ratio mass spectrometry over a time span of 16 months.
Nuclear magnetic resonance (NMR) spectroscopy was applied to follow the humification of grass-PyOM. Material and methodos For the incubation, pots were prepared where Lolium perenne was grown on soils from a fire-affected (FA) and fire-unaffected FU Cambisol from the Sierra de Aznalcóllar (Southem Spain) sampled seven years after a severe fire. The pots were amended by topping the soils with either K15NO3 (15Ni ) (4.41 mg 15N), 15Nenriched plant residues (15N-OM) (10.69 mg 15N), plant residues without 15N-enrichment (OM) + 15Ni (4.45 mg 15N), 15N-OM + KNO3 (5.4 mg 15N) without isotopic enrichment (14Ni), 15N-enriched burnt plant residues 15N-PyOM (15.50 mg 15N), 15N-PyOM + 14N (7.8 mg 15N), and PyOM + 15N (4.43 mg 15N). In addition a control series was prepared without any soil amendment. The PyOM was produced by charring grass residues at 350°C in the presence of oxygen for 8 minutes. The pots were watered with deionized water. After 2 weeks, 1, 5, 8, 12 and 16 months pots were sampled in duplicates after the plant leaves were cut for the last time. The litter layer and the root residues were dried and stored for further analysis. The fate of the 15Nlabel was monitored in the dried bulk samples by isotopic ratio mass spectrometry (IRMS). Selected samples were subjected to solid-state NMR analysis to obtain further information about the humification process. Results Above-ground plant biomass production. In our experiments, comparable above ground plant productions were obtained for the fire and fireunaffected soils. With respect to the control pots and the experiment with only Ni, the addition of organic N clearly increased plant growth during the first month of the incubation. Here, higher plant yields were obtained for the plots amended with fresh plant residues than with PyOM. Although the above ground
202
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
biomass production was still slightly higher from the amended soils than in the control pots at the end of the incubation after 16 months.
Considering that vegetation fires combust the litter which serves as the source for future labile organic N, burning can greatly reduce the amount of plantavailable N. Inorganic N which may accumulate in the soil after a fire is highly susceptible to leaching if not used fast enough by a newly growing plant cover. On the other hand, N bound in PyOM accumulating on the topsoil can act as an important slow-release fertilizer for the recovery of the vegetation. As such BN may have a more important ecological role for the post-fire environment than previously assumed.
Incorporation of 15N from fresh and charred plant residues into SOM. Although the litter layer (organic amendment) was still observable after 2 weeks of incubation, the soils of all treatments were already enriched with 15N indicating that aside from 15Ni, 15Nenriched organic residues were translocated into the soil (Figure 1) most tentatively with the infiltration of water during watering. An impact of the fire history of the soil was not observed. Compared to the soils with 15 N-OM, more 15Ni was recovered in the samples with 15 N-PyOM + 14Ni. This is possibly because the N of the fresh plant litter had a higher bioavailability than that of PyOM and was used for the growth of the new grass. Isotopic ratio determination of the leaves of the new plants confirmed this assumption. However, this amount decreased to levels which were only slightly higher than natural abundance until the end of the experiment. This indicates that the added 15Ni was not efficiently immobilized over a longer time frame. A different picture was revealed from the experiments with 15N-PyOM. Here the continuing incorporation of the organic N source increased the 15N-levels in the soil until month 5. Thereafter, the isotopic label in the soils decreased again, either due to leaching of PyOM residues or because 15Ni which had been already incorporated into the SOM was mobilized and became available for the growing plants. However, up to 3% of the 15Ni added with 15N-PyOM remained incorporated into humic material at the end of the experiment after 16 months. Preliminary 15N NMR studies confirmed that some of the heteroaromatic N from BN was transformed into amide N most likely of microbial origin.
% of
15
N add
6
Fire affected 3
8 months 12 months 1
% of 15Nadd
2
4 2 0
1 month
5 months 4
5
7 51 month
2 weeks 7
16 months 0
Fire unaffected
2 weeks
6
5 months 3 8 months 12 months 1 16 months
Figure 1: Content of 15N in the humic material of a fireaffected and fire unaffected Cambisol from the Sierra de Aznalcóllar, Southern Spain after amendment with various N sources and planting with grass seeds during a 16 months pot experiment.
References
Discussion This study shows that soil amendments with PyOM, increase plant biomass production at a short term scale but has no major beneficial effect with respect to plant production after several months. Our study demonstrated a quick transport of 15N- PyOM into the mineral soil and a fast incorporated into the SOM. However, the fact that only a part of the immobilized 15 Ni survives until the end of the experiment confirms that BN can be degraded and its N is released from the aromatic charcoal network to become bioavailable and usable for the build-up of new plant material. It supports recent observation that at least in topsoils the mean residence time of PyOM is only slightly higher than fire-unaffected humified SOM [6]. This is in line with the results of the solid-state 13C NMR spectroscopic analysis revealing that in the spectra of the soils used in the present study, the clear pattern of PyOM is lost in the sample taken 7 years after the last intense fire (unpublished data). It may be argued that most of the plant-available N in PyOM derived from peptide structures that were only weekly altered by the thermal treatment. However, solid-state 15 N NMR spectra assured that those compounds were not present to a higher extent in the used grass char but were newly formed during the charring process.
(1) Knicker, H.; Hilscher, A.; González-Vila, F.J.; Almendros, G. Geochem. 2008, 39, 935-939. (2) Knicker, H. Org. Geochem. 2010, 41, 947-950. (3) Hamer, U.; Marschner, B.; Brodowski, S.; Amelung, W. Geochem. 2004, 35, 823-830. (4) Hilscher, A.; Knicker, H. Org. Geochem. 2011, 42, 42-54. (5) De la Rosa, J.M.; Knicker, H. Soil Biol. Biochem. 2011, 43, 2368-2373. (6) Knicker, H.; González-Vila, F.J.; GonzálezVázquez, R. Soil Biol. Biochem. 2013, 56, 31-39.
Acknowledgment: The authors would like to thank the Ministerio de economía y competividad (MINECO) (Spain) (CGL2009-10557) and the European Regional Development Fund (FEDER) for their financial support. The People Programme (Marie Curie Actions) of the European Union´s Seventh Framework Programme FP7/2007-2013 is acknowledged for funding the Project PCIG12-GA2012-333784 (Biocharisma). Thank MINECO and CSIC for funding of JAE-Doc contract of J.M. de la Rosa.
203
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Biochar induced alterations of soil properties and its organic matter; discerning how it improves crop production J.M. de la Rosa*, M. Paneque, M. Velasco-Molina, F.J. González-Vila, A. Z. Miller, H. Knicker Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS-CSIC). Reina Mercedes, Av. 10. 41012, Seville (Spain) * Corresponding author e-mail: [email protected] Keywords: pyrolysis; field experiment; sunflower production; characterisation; Mediterranean climate Abstract In this study, four pyrolysis biochars (B1:wood, B2: paper-sludge, B3: sewage-sludge, B5: chips-wood certified) and one kiln-biochar (B4: grapevine wood) were characterized by determining different chemical and physical properties such as, elemental composition (C, H, N,O), pH, ash content, water holding capacity (WHC), specific surface area, as well as a suite of analytical techniques which included field emission scanning electron microscopy (FESEM-EDS), Fourier-transform infrared spectroscopy (FT-IR) and 13C solid-state nuclear magnetic resonance (NMR) spectroscopy. Those biochar characteristics were related to the soil properties, germination rates and to the plant biomass production during a field experiment of 7 months in which a calcareous Cambisol from SW Spain was amended with 0, 10 and 100 t ha-1 of the 5 biochars (n=6). Elemental and spectroscopy analyses suggested a generally high aromaticity for all the biochars. For the wood-derived biochars (B1, B4 and B5), IR and NMR spectra showed differences in specific functional groups present at biochars (for instance presence of lignin structures, carboxylic groups, etc.). Structural and compositional differences evidenced on the surface of biochars by FESEM-EDS were helpful in explaining their diverse physical characteristics. The field experiment unveiled that all biochar amendment increased not only the aromaticity of the soil organic matter (SOM) but also the germination rate and plant production. account that biochar application is irreversible and potential alterations of the SOM are expected to last on a long term scale. In order to avoid unwanted negative impacts, a good knowledge of the properties of the applied biochars together with their effect on soil functionality is needed. Therefore we studied five biochars produced from different feedstock are related their chemical and physical properties to alterations of the soil organic matter and their potential to increase the fertility of a Cambisol from SW Andalusia, Spain.
Introduction Considering the increasing amount of organic agricultural and urban wastes, there is an urgent need for finding a sustainable strategy which allows managing and reducing these kinds of residues. In this sense, biochar has been proposed as a novel tool to achieve this goal at the same time that provides other agricultural and environmental benefits. Biochars contain large amounts of carbon. Thus, when applied to land, they have the potential to significantly, increase soil organic matter (SOM) contents, which is in critical decline in many regions of the world. Specifically, biochar may offer a new strategy for restoring carbon to depleted soils and since biochar is considered to be more stable than SOM concomitantly sequestering significant amounts of CO2. The latter is in accordance with the European´s commitment to lower its GHG emissions to 20% below their 1990 level by the year 2020. Concerning agriculture, researches have pointed to biochar as a new ecological amendment which may enhance soil quality and plant growth [1]. However, the effectiveness of biochar in enhancing plant fertility is not only a function of soil type, climate, and type of crop [2] but also of the biochar properties. The inherent chemical variability of biochars due to different feedstock and production conditions implies a high variability of their effect on soil properties. In addition it has to be taken into
Experimental The used biochars were produced from wood (B1), paper-sludge (B2), sewage sludge (B3) chip woods (B4) and grapevine wood (B5). They were provided by the COST action TD1107 and the companies “Swiss Biochar” and “Bodegas Torres”. They were were characterized for their elemental composition (C, H, N, O), pH, ash content, water holding capacity (WHC) and specific surface area (SSA-N2). For a more detailed analysis, a suite of analytical techniques such as Fourier-transform infrared spectroscopy (FT-IR), 13C solid-state nuclear magnetic resonance (NMR) spectroscopy and field emission scanning electron microscopy (FE-SEM) was used.
204
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Those biochar characteristics were related to the germination rates and to the plant biomass production of Helianthus annuus planted during field experiment . This experiment started in February 2014 and is performed on a calcic Cambisol (WRB 2007)[3] located at the experimental station “La Hampa” of the Instituto de Recursos Naturales y Agrobiología de Sevilla. About 10 and 100 t ha-1 of the five biochars were amended. Soil properties, SOM composition of the top 5 cm and soil microbial biomass were analysed during the growing time. After 6 months of growth, above-ground biomass will be harvested. Plant height, chlorophyll content and sunflower seeds production will be recorded and weights of fresh biomass and harvested seeds will be determined.
and the EC of this alkaline soil but caused an improvement of the WHC, water retention and soil porosity. Those changes are most tentatively responsible for the observed faster germination and augmented growth of the plants (Figure 1).
Results and Discussion
Acknowledgments:
REFERENCES (1) (2) (3)
Sohi, S.P.; Lopez-Capel, E.; Krull, E.; Bol, R. 2009. CSIRO Land and Water Science Report 05/09. Blackwell, P.; Riethmuller, G.; Collins, M. 2009. Ed by Lehmann, J.; Earthscan, J.S. London, UK. IUSS Working Group WRB, 2007. World Reference Base for Soil Resources, first update 2007. World Soil Resources Reports 103. FAO, Rome.
The Marie Curie Actions of the European Union's Seventh Framework People Programme (REA grant agreement nº PCIG12-GA-2012-333784-Biocharisma project), the Spanish Ministry of Economy and Competitiveness (MINECO GCL2012-37041 and 2011BR0097) and the European Regional Development Fund (FEDER) are thanked for the financial support of the present study. Ph.D. J.M. De la Rosa was the recipient of a fellowship from the CSIC JAE-Doc program co-financed by the European Social Fund (JAE-DOC-056). The European Biochar Network (Biochar as option for sustainable resource management-COST action TD1107) and Bodegas Torres (Spain) are thanked for providing the biochar samples.
Figure 1. Image of the field experiment with biochar
amended soils and representative sunflowers for 100 t ha-1 of biochar amendment after 81 days of growth.
Biochar 1, 2, 4 and 5 showed comparable elemental composition (C, H, N) high pH (10.3-10.4), and WHC values (178-266%). In contrast, B3 showed a low pH (6.7) and WHC (27% as well as a low C (18%), but a hight N (2%) content. The solid-state NMR spectra confirmed high aromaticity which was already suggested by the H/C and O/C atomic ratios suggested a generally high aromaticity for all the biochars. Although B1 could not be analyzed by NMR spectroscopy due to its strong reflection of the emission signal, most tentatively caused by its graphite-like structure its high aromaticity was confirmed by FT-IR spectroscopy. For the wood-derived biochars (B1 and B4), this technique resulted in typical signals assignable to lignin. The FESEM-EDS distinguished not only compositional but structural differences of the studied biochars. Macropores were evidenced on the surface of B1 (wood biochar), collapsed structures in B2 (paper sludge), high amount of mineral deposits (rich in Al, Si, Ca and Fe) and organic phases in B3(sewage sludge) and vessel structures for B4 (vineyard wood). First results of the ongoing field experiment showed an increase of the aromaticity of the SOM in the topsoil of biochar amended soils. However, addition of biochar did not alter negatively the (pH
205
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Chemical composition and origin source of soil organic matter under four vegetation in Doñana National Park N. T. Jiménez-Morillo(a)*, F. J. González-Vila (a), O. A. Leal (b) and J. A. González-Pérez (a)* (a) IRNAS-CSIC. Avda. Reina Mercedes, 10, Sevilla, E-41012 (Spain). (b) Federal Univ. Rio Grande do Sul, Bento Gonçalves Avenue 9500, 91501-970, Porto Alegre (Brazil). * Corresponding author e-mail: [email protected]; [email protected]
Keywords: Py-GC/MS, isotopic ratio mass spectrometry, EA/TC-IRMS, Py-GC-(FID)-C\TC-IRMS Abstract:
This study deals with the chemical characterization of soil organic matter (SOM) under different ground covers from a Mediterranean climate (Andalusia, South Spain), for it using techniques of analytical pyrolysis, with this techniques, we could find that organic matter consists mainly of seven chemical families, as they are, alkanes / alkenes, fatty acids, aromatic, lignin, steranes, sugars and peptides. Also we had been used different approaches for the isotopic signature study of stable light elements in bulk (low-complexity) samples. Light element isotope ratios (δ 15N, δ13C, δ18O and δD) were measured in the whole soil samples (EA/TC-IRMS) and δ13C and δD values were also estimated in a number of specific compounds -previously identified by Py-GC/MS released after pyrolysis (Py-GC-(FID)-C\TC-IRMS). Introduction Among the most suitable techniques for the direct study of complex organic matrices such is soil organic matter (SOM) is analytical pyrolysis. The technique consists of a thermolytic degradation of macromolecules into small fragments that may be separated and identified by gas chromatography–mass spectrometry (Py-GC/MS). Pyrolysis of SOM generates a wide range of products that can be related to their origin (e.g., methoxyphenols from lignin, anhydrosugars and furan derivatives from polysaccharides, and N-containing molecules from proteins; González-Vila et al., 2001; Leinweber & Schulten, 1995). In addition, recently the technique has been effectively hyphenated with other detection devices like isotope ratio mass spectrometry (IRMS) that will provide additional information relevant for the monitoring of biogeochemical processes including tracing the origin and dynamics of SOM pools. In this study we use different approaches for the isotopic signature study of stable light elements in bulk (low-complexity) arenosol samples collected under different vegetation covers from a Mediterranean climate (Andalusia, South Spain). Light element isotope ratios (δ15N, δ13C, δ18O and δD) were measured in the whole soil samples (EA/TC-IRMS) and δ13C and δD values were also estimated in a number of specific compounds -previously identified by Py-GC/MS- released after pyrolysis (Py-GC-(FID)C\TC-IRMS).
samples were sieved to fine earth (< 2 mm) to discard coarse elements and litter. Pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) was performed for SOM characterization using a double-shot pyrolyzer (Frontier Laboratories, model 2020i) attached to a GC/MS system Agilent 6890N. Compound assignment was achieved via single-ion monitoring for various homologous series, via low-resolution mass spectrometry, and comparison with published and stored (NIST and Wiley libraries) data. Bulk isotopic signature of light elements (δ15N, 13 δ C, δ18O and δD) was analyzed using a Flash 2000 HT (N, C, S, H and O) elemental analyzer coupled to a Delta V Advantage IRMS (Thermo Scientific) (EA/TC-IRMS). The direct study of specific compounds isotopic signature of light elements (δ13C and δD) was done by coupling a pyrolysis unit (double-shot pyrolyzer “Frontier Laboratories, model EGA/Py-3030D”) – to a gas chromatograph fitted with a flame ionization detector (GC/FID) and coupled to the Delta V Advantage IRMS (Thermo Scientific GC-Isolink System) (Py-GC-(FID)-C\TC-IRMS). Isotopic ratios are reported as parts per thousand (‰) deviations from appropriate standards recognized by the international atomic energy agency (IAEA) (Valkiers et al., 2007).
Experimental The Soil samples were collected in a circular area (radius 5m) under frequent vegetation covers found in sandy soils from the Doñana National Park (SW Spain): cork oak (Quercus suber, QS), eagle fern (Pteridium aquilinum, PA), pine (Pinus pinea, PP) and rockrose (Halimium halimifolium, HH). Dry soil
Pyrolysis characterization: The organic matter in each soil sample presented rich pyrolisates with a large number of chemical compounds. These could be best classified in seven groups according to its chemical nature or probable biogenic origin (Fig 1)
Results and Discussion
206
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
pyrolysis is depicted in Fig. 3 and Table 1. These results are actually in the process of analysis and discussion. The results presented during the meeting will be discussed in relation to possible different sources contributing to SOM as well as to specific micro-climatic conditions that may be affecting SOM isotopic signature at a local scale.
Figure 1. Distribution (relative abundance) of main chemical families identified in SOM pyrolysates (AlK: Alkane/ Alkene; Ar: Aromatic; FA: Fatty Acid; L: Lignin; P: Peptides; St: Steranes and Su: Sugars) under the different studied vegetation types (QS: Quercus suber; PA: Pteridium aquilinum; PP: Pinus pinea and HH: Halimium halimifolium).
EA/TC-IRMS: Bulk soil isotopic signatures of light elements in each sample are shown in Fig 2. The δ13C signature is clearly in the range of C3 plant (-26 to -30 ‰) (O´Leary 1981) and the different plant canopies (tree, shrubs or ferns) caused only slight variations in δ13C (STD=0.42). Nitrogen isotope signature (δ15N) is also in line with that commonly found in plant or land organisms as described in Létolle (1980). Cross plots of δ15N vs. δ18O may provide information about nitrate (NO3-) sources and N cycling (Kendall, 1998), in our case, it was compatible with a predominant nitrate source from atmospheric deposition (δ15N range: -5 to 5 ‰; δ18O range: 20 to 70 ‰). No conclusive results could be obtained from the δD isotopic signature probably due to overlapping of the δD signals from the organic and the mineral fractions. For a more accurate δD analysis additional steps allowing their separation would be necessary (Ruppenthal et al., 2013, and references therein). 60
δ (‰ )
40
QS
PA
PP
HH
Figure 3. Example of compound specific (δ13C and δD) analysis (Py-GC-(FID)-C\TC-IRMS) is soil under Quercus suber (QS). Numbers on traces corresponds to compounds of known structure as determined by conventional analytical pyrolysis (TIC QS: Py-GC/MS) and listed in Table 1. Table 1. Isotopic signature of light elements in selected peaks released directly from pyrolysis of soil under Quercus suber (QS) (Py-GC-C\TC-IRMS). Nº 1 2 3 4 5 6 7 8 9 10 11 12 13
δ13C
δD
δ18O
-40 -60 -80 -100 -120
δ13C (‰) -27.58 -27.51 -27.52 -26.56 -26.66 -26.80 -27.47 -27.99 -27.47 -27.24 -28.42 -28.52 -26.87 -27.98±1.48
REFERENCES
0
δ15N
δD (‰) -65.93 -68.81 -73.51 -75.91 -65.06 -47.84 -64.41 -62.41 -78.90 -63.61 -77.81 -55.44 -43.34 -45.68±17.07
(1) González-Vila F.J.; Tinoco P; Almendros G; Martin F. J. Agric. Food Chem. 2001, Res 49: 1128–1131.. (2) Kendall C; Silva S.R.; Stober Q.J.; Meyer P. Am. Geophys. Union Trans. 1998, Res 79: S88 (3) Leinweber P and Schulten HR. J Anal Appl Pyro. 1995, Res 32: 91–110. (4) Letolle R. 1980. In: Fritz P & Fontes JC (Ed) Handbook of Environmental Isotope Geochemistry (pp 407-433). Elsevier, Amsterdam. (5) O'Leary M.H. Phytochemistry. 1981, Res. 20 (4), 553567. (6) Ruppenthal, M.; Oelmann, Y.; Wilcke,W. Envir Sci Tech. 2013, 47: 949-957. (7) Valkiers S, Varlam M, Ruβe K, Berglund M, Taylor P, Wang J, Milton M, De Bièvre P. Int J Mass Spectrom. 2007, Res 263(2–3): 195-203.
20
-20
Name Family Trimetil benceno AROMATIC 1-Undecene AlKENE Phenol AROMATIC Guaiacol LIGNIN Phenol, 4-methylAROMATIC Phenol, 2-methoxy-4-methylLIGNIN Phenol, 4-ethyl-2-methoxyLIGNIN 2-Methoxy-4-vinylphenol LIGNIN Syringol LIGNIN 2H-Pyran-2,4(3H)-dione, 3-acetyl-6-methylSUGARS Cyclopentanone, 2-(1-methylpropyl)SUGARS Vinylsyringol LIGNIN Levoglucosan SUGARS Bulk isotopic signature of QS samples (Mean±STD)
Stable Isotopes
Figure 2. Bulk isotopic signature of light elements under the different studied vegetation types (Quercus suber QS, Pteridium aquilinum PA, Pinus pinea PP and Halimium halimifolium HH).
Acknowledgments: “Ministerio de Economía y Competitividad” through project GEOFIRE (ref. CGL2012-268 38655-C04-01) and a research contract to Nicasio T. Jiménez-Morillo (ref. BES-2013-062573).
Py-GC-(FID)-C\TC-IRMS: An example of the compound specific IRMS analysis of selected peaks released directly from
207
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Role of Humic Acids on DNA Hypomethylation Caused Fipronil Stress in Vicia faba Seedlings M. Turan (a) *, E. Arslan(b) , G. Agar(b), M.S. Taspinar, M. Gulluce(b), H. Ogutcu (c), F. Sahin(a) (a)
Yeditepe University, Department of Genetics and Bioengineering. Istanbul, Turkey. (b) Ataturk University, Faculty of Science, Department of Biology, Erzurum, Turkey. (c) Ahi Evran University, Faculty of Science, Department of Biology, Kırsehir, Turkey. * Corresponding author e-mail: [email protected] Keywords: CRED-RA, DNA methylation, fipronil, humic acids Abstract The aim of the present study was to evidence the possible antagonistic effect of humic acids against fipronil induced DNA methylation changes by using CRED-RAs (Coupled Restriction Enzyme Digestion-Random Amplification) in Vicia faba seedlings. The results showed that five concentrations of fipronil (0.5, 1, 2, 3 and 4 ppm) caused DNA methylation changes. In additional when different five concentrations of HA (%2, %4, %6, %8 and %10) were added together with 0.5, 1, 2, 3 and 4 ppm of fipronil, DNA methylation changes decreased. Results suggested that HA have an antagonistic effect against fipronil stress induced DNA methylation changes.
were obtained from the Department of Field Crops, Faculty of Agriculture, Ataturk University (Turkey). Vicia faba seeds were surface-sterilized with 0.5 % NaOCl (sodium hypochlorite) solution for 10 min and then washed with sterile water three times. Seeds were placed on two layers of filter paper moistened with 25 ml distilled water. 25 seeds were used in each petri dish. The dishes were kept at 25 ± 1°C under dark condition until primary roots were grown at 0.5-1 cm length. After, the Vicia faba seedlings were exposed to different concentrations of fibronil (0.5, 1, 2, 3, 4 ppm) and humic acid (0%, 2%, 4%, 6%, 8% and 10%) for 96 h. Treated seedlings were grown in pots of a peat/soil mix (5 plants/pot) at 25±1°C with a 16 h photoperiod of 60 µmol photons/m2s provided by white fluorescent lamp, at a relative humidity of 70– 75%. Each treatment was replicated three times. Bulk leaves were randomly collected from ten plants for each treatment after emergence of leaf 3 (leaves numbered from base) and were stored at -80 °C. Total DNA was isolated by CTAB method (7). CRED-RAs Digestion and PCR: Genomic DNA sample from each treatment were separately digested with Hpa II and Msp I endonucleases (which cut the sequence 5´-C/CGG-3´with different sensitivity to cytosine methylation; Msp I cuts if the inner C is methylated, whereas Hpa II cannot cleave in the presence of methyl groups). After checking digestion on agarose gel, were prepared a PCR reaction cocktail. CRED-RA PCR reaction was contained 25 ng digested DNA, 400 µM dNTP, 10 pmol primer, 2,5 mM MgCl2, 1 U Taq DNA polymerase and 1X PCR buffer (10X) in a total volume of 20 µL. DNA amplification was carried out in a thermocycler programmed as follows: 1 cycle of 5 min at 95°C, 42 cycles of (1 min at 94°C, 1 min at 36°C and 2 min at 72°C ), 1 cycles of 15 min at 72°C. 16 CRED-RA primers were tested
Introduction Pesticides can be classified based on their chemical structure. Fipronil is a member of the phenyl pyrazole class of pesticides, which is principally chemical with a herbicidal effect. Actively marketed in many industrialized and developing countries, its worldwide use is increasing. Fipronil is highly toxic for crustaceans, insects and zooplankton. Fipronil is recommended for insects control in various crops such as sugarcane, soybeans, corn, eucalyptus and others. However, beyond the target insects, fipronil has demonstrated high toxicity on insects no target. In additional, the genotoxic effect of fipronil on humans has been investigated by using different test systems including micronucleus, comet assay and many studies have shown that fipronil has genotoxic, cytotoxic and toxic potential (1, 2). There is not yet a study examining the epigenetic effect of fipronil on the animals. On the other hand, only report is available role of fipronil on epigenetic modifications in plant (3). Recently, several studies have demonstrated that humic substances (HSs) provide a protective effect against herbicides and metals cytotoxicity in several animal and plant species. Some researchers have been reported that HAs (humic acids) can be the anticlastogenic and can expose antitoxic and antimutagenic activity (4). In additional HAs have been showed that caused decreasing number of genetic anomalies in seedlings of Vicia faba, treated with various herbicides (5). There is one study about the epigenetic potential of HAs in plant (6) but there is no information that protective role of HAs against fipronil which induces epigenetic in plants. Experimental Sample Collection: Fipronil was obtained from Sigma Chemical Company, USA and Vicia faba seeds
208
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
with bulked DNA of control treatment (0 ppm fipronil+ % 0 humic acid). Only 8 primers amplified polymorphic amplicons and used in CRED-RA PCR reactions. These primers for CRED-RA analyses are; GTCCACACGG (OPB–8), CTGCTGGGAC (OPB– 10), ACCAGGTTGG (OPH-14), TCTCAGCTGG (OPH-16), CACTCTCCTC (OPH–17), GAATCGGCCA (OPH-18), AGAGCCGTCA (OPY7), AGGCCCGATG (OPW-6). Electrophoresis: The PCR products (20 μl) were mixed with 6x gel loading buffer (3 μl) and subjected to agarose (1.5% w/v) gel electrophoresis in 0.5x TBE (Tris-Borate- EDTA) buffer at 70 V for 150 min. Amplification products separated by gel electrophoresis were stained in ethidium bromide solution (2 μl Etbr/100ml of 1x TBE buffer) for 40 min. The amplified DNA products were detected using the Bio Doc Image Analysis System and analysed using the Uvi-soft analysis package (Cambridge Electronic Design Ltd, Cambridge, UK). Data analysis: The average number of CRED-RA pattern polymorphisms (%) were calculated for each dose to realize CRED–RA analysis. To calculate the number of polymorphisms (%), the following formula was used 100x a/n where a is the average number of polymorphic bands detected in each treated sample, and n is the number of total bands in the control. Polymorphisms in CRED-RA profiles included disappearance of a normal band and appearance of a new band compared with the control. The average was calculated for each experimental group. To compare the sensitivity of each parameter, changes in these values were calculated as a percentage of their control (set to 100%).
against toxic elements such as cadmium, zinc, mitomycin C, mercury and maleic hydrazide mercury in different animals and plants (5). There is one study about effect of HA on epigenetic modifications in plant. On the other hand, no report is available protective role of HA against fipronil which induces epigenetic in plants. Also, some studies have showed that HSs have transcriptional interact with biochemical constituents and signaling pathways (8). The DNA methylation changes induced by HSs may relate to affect the transcription and translation processes of specific genes, to improve the plant resistance under stress conditions. This result suggests that some components of humic acids could be an alternative for amelioration effect against chemical mutagens in plant. REFERENCES
(1) Lourenco, C.T.; Carvalho, S.M.; Malaspina, O.; Nocelli, R.C.F. Bull. Environ. Contam. Toxicol. 2012, 89, 921–924. (2) Jacob, C.R.O.; Soares, H.M.S.; Carvalho, M.; Nocelli, R.C.F.; Malaspina, O. Bull. Environ. Contam. Toxicol. 2013, 90, 69–72. (3) Gulluce, M.; Ercisli, S.; Agar, G.; Arslan, E.; Turan, M.; Sahin, F. International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2014). Feb. 6-7, 2014. Bali (Indonesia). 67-68. (4) Marova, I.; Kucerik, J.; Duronova, K.; Mikulcova, A.; Vlckova, Z. Environ. Chem. Lett. 2011, 9, 229–233. (5) Ferrara, G.; Loffredo, E.; Senesi, N.; Marcos, R. Mutat. Res. 2006, 603, 27–32. (6) Yildirim, N.; Agar, G.; Taspinar, M.S.; Turan, M.; Arslan, E. Acta Agr. Scand. 2014B-S P. http://dx.doi.org/10.1080/09064710.2014.891650. (7) Li, G.; Quiros, C.F. Theor. Appl. Genet. 2001, 103, 455–461. (8) Menzela, S.; Bouchnaka, R.; Menzela, R.; Steinberg, C.E.W. Aquat. Toxicol. 2011, 105, 640– 642.
Results and Discussion In total, 16 oligonucleotide primers with % 60-70 GC content were used for CRED-RA analyzing and only eight gave specific and stable results. Compared with the PCR products obtained from the control DNA, fipronil treatments resulted in apparent changes in CRED-RA patterns. These changes are characterized by variation loss of normal bands or appearance of new bands. Increasing concentration of fipronil caused DNA hypomethylation. The methylation value was 8.5 in control. However, the highest methylation value was 7.4 and the lowest was 5.2 in fipronil applications. In additional, value was progressively increased when fipronil and HAs applications combined with together. In this case, the highest methylation value was 9.2 and the lowest was 8. When plants are exposed to environmental stress such as biotic and abiotic, they can be antistress protective action, biochemical, physiology and molecular levels, induced DNA methylation and histon modification. Especially these epigenetically changes may be suggest a mechanism for plants adaptation under stress. DNA hypomethylation effect of fipronil may relate to affect the transcription and translation processes of specific genes, to improve the plant resistance under stress conditions. Our results showed that HAs treatment amelioration effect against DNA hypomethylation caused fipronil. Previous researches have reported the protective effect of HAs
209
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The comparison of granular blended humic-phosphate fertiliser and mono ammonium phosphate on solubility of phosphorus and on phosphorus availability A. Kargosha (a) *, M. Rose (a), T. Cavagnaro (b), A. Patti (a)
(a) Address a. School of chemistry,Monash University,Wellington Rd, Clayton, VIC 3800. Melbourne. Australia (b) Address b. School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, SA, 5064.Australia * Corresponding author e-mail: [email protected] Keywords: Solubility,blended humic-phosphate, phosphorus, availability, mono ammonium phosphate, frtiliser Abstract: A study was conducted to evaluate the effect of humic- derived substances on phosphorus solubility and availability.Re-granulate humate-phophate fertilisers with 5 different humates concentration were used. The study was run in two phases.in phase (I) the solubility of monoammonium phosphate was compared with blended humicmonoammonium phosphate fertiliser. The results showed phosphorus solubility increases by increasing the humic concentration in new fertilisers. The availability of phosphorus in calcareous soil from two different phosphorus fertilisers was studied in phase (II). Results have showed that the pure phosphate fertiliser without humics released more phosphorus to the soil, and then the lower rate of humate fertiliser released more phosphorus comparing with the higher rate humic fertiliser. coating DAP with resin reduces N losses (Gracia et al., 1995). However, low-rate of HA (1.7kg/ha) accompanying MAP fertilization caused no significant difference in P uptake compared with MAP without HA (Clain et al.2007).Thus, despite evidence showing the beneficial effects of HA on P availability and uptake, there is still uncertainty about how to obtain consistent improved responses under various environmental conditions.
Introduction Humic substances (humics) are 60% to 80% of soil organic matter. They are huge molecule with different structure and composition, which are specified by their aromatic, ring type structure with mostly dark colour. Because of their complexity are known to be resistance against microbial attack. They extracted from soil using a strong base (NaOH or KOH). The humics are considered as a soil conditioners. Their role in soil nutrition improvement has been proved by studies. For example, it is showed that the brown coal (as one the organic matter) alkaline extraction humic derived has positive effects on soil nutrients efficiency and plant uptake as well as stimulation of crop growth (Fong et al., 2006).It also has been showed that BC derived products have significant effect on soil health by improving plant growth, enhancing soil microbial activity and carbon capture. Imbufe et al (2004) have showed that humic substances from brown coal increase soil electrical conductivity (EC) by providing reactive sites for cation exchange as well as nutrients transport to plant improvement and pH buffering (Imbufe et al., 2004). Humic substances can improve P efficiency by enhancing P water-soluble in calcareous soils (Parvage et al., 2012). Addition of humic acids to soil beside P fertilizer significantly increased the amount of water soluble phosphate, strongly retarded the formation of occluded phosphate and increased P uptake and yield by 25% (Wang et al., 1995). Also, coating phosphorus fertilizers with humic derived alkaline extracts such as K humates leads to P efficiency increase. Diez and et al (1991) showed that diammonium phoshpate (DAP) coated with humates enables phosphorus fixation to be controlled in calcareous soils, being sufficiently stable to persist in the available fraction for the time it takes the crop to grow. A glass house study showed by
Methods and materials S90 (powder form) as a commercial Victoria brown coal potassium- humtae derived product was blended with Monoammonium phosphate (MAP) considered as phosphate fertiliser, based on different phosphorus: carbon ratio. Phase (I): four blended humate-MAP fertilisers selected for this stage. The p:c ratio of the humate fertilisers were: control (no humate), 4.98, 2, 1 and 0.20. A certain amount of fertilisers that contained 10 mg phosphorus were added to 50ml distilled water and leave for 20 days. Samples were collected in 1, 3, 8, 15 and 20 days after incubation start. Samples were filtered with 0.45um filters and analysed for available phosphorus by Murphy and Riley method (1962). Phase (II): in this phase of study the new blended humate-MAP fertilisers compared with MAP in regard to available phosphorus in soil. Two p: c ratios 2 and 0.2 were considered for humate-phosphate fertilisers. Alkaline soil was chosen for this step. A certain amount of granular fertilisers were placed in the middle of petri dishes and after reaching soil to the field capacity moisture they sealed and left for two weeks. After incubation period each petri dish divided to 4 radiuses by concentric plastic rings. The available phosphorus extracted with resin exchange phosphorus
210
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
References (1) Clain A. Jones, Jeffrey S. Jacobsen & Aaron Mugaas (2007): Effect of Low‐RateCommercial Humic Acid on Phosphorus Availability,Micronutrient Uptake, and Spring Wheat Yield,Communications in Soil Science and Plant Analysis, 38:7-8, 921933Paciolla, M.D. and White A.B. Environ. Sci. Technol. 1999, 33, 1814-1818. (2) Amer F, Bouldin DR, Black CA and Duke FR (1955) Characterization of soil phosphorus by anion exchange resin adsorption and p32 equilibration. Plant and Soil 6: 391-408 (3) Murphy J and Riley JP (1962) A modified single solution method for the determination o1 phosphate in natural waters. Anal Chim Acta 27:31-36 (4) Parvage, M.M., Ulen, B., Eriksson, J., Strock, J. and Kirchmann, H., 2012.Phosphorus availability in soils amended with wheat residue char. Journal of Bio Ferti Soils, 49: 245-250 (5) Diez, J.A., Cartagena, M.C. & Vallejo, A. (1991). Controlling phosphorus fixation in calcareous soils by using coated diammonium phosphate. Fertilizer research. 31: 269-274. (6)Urrutia O, Guardado I, Erro J, Mandado M, GarcíaMina JM (2012). Theoretical chemical haracterization of phosphate-metal-humic complexes and relationships with their effects on both phosphorus soil fixation and phosphorus availability for plants.J Sci Food Agric. 2013 Jan;93(2):293-303.
method (Amer etal., 1955) followed by Murphy and Riley (1962) method for phosphorus analysis.
Mg P/50 ml DI water
Results and Discussion 1. Phase (I): phosphorus solubility The blended humate-MAP fertilisers have released more available P to the solution. By increasing the ratio of humate in blended fertiliser, more P was released in water. This was probably due to presence of ammonium cation (NH4) + in MAP granules that promotes P release because of binding to humate particles that have negative charge. In both fertilisers, there was a decrease of soluble P in 3 days after starting incubation. It is likely that the physical binding between k-humate and phosphate fertilisers at the time of manufacturing, has caused to release more P in T, then after two days chemical reaction happened between phosphate ions and humate resulting in some P participation (Fig 1).
Days after start Figure 1. Comparison of available phosphorus between MAP and humate-MAP
Mg P/L
2. Phase (II): phosphorus availability in soil Obviously the available phosphorus decreased by distance. The amount of resin exchange phosphorus (REP) in radius 4 (r4) far from granules was the lowest. The results showed that the MAP fertilizer with no humate, released the highest available phosphorus. This was likely due to Ca+2 ions exist in alkaline soil. The negative charged humate prefer to bind to Ca+2 instead of NH4+ of MAP (Fig 2). The highest ratio of p: c that contains less humate in blended fertilisers in blended humate-phosphate fertilizer also showed more available phosphorus compare to lowest ratio of p: c (0.20).
Figure 2. Comparison of available phosphorus between MAP and humate-MAP K0: no humate , K2: p:c = 2 , K4: p:c= 0.2
211
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The biological activity modulation of engineered nanomaterials in soils under the humic substances’ influence M. Gladkova (a), V. Terekhova(a,b)
(a) Lomonosov Moscow State University, Soil Science Faculty, Moscow, Russian Federation (b) Institute of Ecology and Evolution RAS, Moscow, Russian Federation e-mail: [email protected] Keywords: nanomaterials, humic substances, modulation, bioassay, water extracts, soils Abstract Organisms’ test-responses of three trophic levels (producers, consumers and reducers) on nanomaterials of different nature: carbon-containing (nanodiamonds) and metal-containing (nanodioxide titanium and nanomagnetite) and humate’s response reactions to it in water, water extracts from natural and artificial soils were analyzed. The complex nature of "dose-effect" of studied nanomaterials was marked. Unidirectional influence of humic substances on nanomaterials toxicity manifestation wasn’t revealed. The neutralization of nanoparticles toxicity by humate, which confirms the universality of detoxication properties of humates was marked.
Introduction Widespread engineered nanomaterials and their accumulation in environments gives grounds to consider them as a special kind of pollutants. Currently the most effective areas of humic substances’ (HS) application are known. Their use as detoxicants of organic and inorganic pollutants is one of the most important (1,2,5). Several researchers noticed some compounds’ toxic effect increase in the presence of HS simultaneously (4). Nanomaterials’ biological activity in soils and HS influence on nanomaterials remain poorly understood despite of considerable attention given to the nanomaterials’ study in environments. The objective of this research is to study engineering carbonand metal-containing nanomaterials’ toxicity change under humic substances’ influence.
analyzed. In another set of experiments the nanoparticles’ toxicity and humate’s response reactions to it in water extracts of podzolic soil (Chashnikovo, Moscow region, A horizon) and artificial soil – model soil, prepared in accordance with ISO 11268-1, were investigated. Results and Discussion Bioassay showed that soil contaminated with nanomaterials exhibit inhibiting and stimulating biological activity. Biotic response level fluctuations in nanoparticles’ presence in water and in soil sample extracts were noticed. Depending on the type of medium and nanomaterials humate’s detoxication effect on test-cultures varies. Nanomaterials’ bioassay in water on test-cultures of different trophical levels with and without HS was carry out: •Nanodioxide titanium inhibited producers’ testfunctions (higher plants – root length) in all range of concentrations (0.5-500 mg/l). At the same time humate in all concentrations, except 50 mg/l, relieves inhibition, stimulating root growth and seed germination. Nano-TiO2 has a stimulating effect on infusorium and bacteria test-cultures and the HS presence increased twice more stimulating effect compared to higher plants . •Nanomagnetite unlike nanodioxide titanium stimulated the development of higher plants, at all concentrations, except 50 and 100 mg/l, which showed an inhibitory effect. HS effect on the nano-Fe3O4 bioactivity at different test-cultures appeared ambiguous: at high concentrations (100 and 500 mg/l) inhibition of higher plants’ roots and bacterial luminescence stimulation was observed, at low concentrations - on the contrary, inhibition of bacterial luminescence, and stimulation of the plant roots and infusoriums’ survival. •Adding humate to nanodiamonds (particle size 15100 nm) inhibited toxic effects. This effects are more evident in concentration 500 mg/l. Thus, research has shown that the toxic effect of nanomaterials in water was nearly removed in the
Experimental In this work humate «Pow-Humus» (Le-PhK, Khumate, originated from leonardite, manufactured by German firm «Humintech»); carbon-containing nanomaterials - nanodiamonds produced by industrial detonation synthesis of high explosives (DNDs, different size free particles in aqueous suspensions up to 15, 30 and 100 nm, «SNK», Snezhinsk, Chelyabinsk region, Russia), metal-containing – nanodioxide titanium (nano-TiO2, <25 nm, «SigmaAldrich», U.S.) and magnetite (nano-Fe3O4, 30 nm, MAI, Russia) were investigated. Nanomaterials’ concentration varied in range 5-500 mg/l; humate’s concentration was 5 mg/l in water. The research is based on standard environmental soil control methods recommended for industrial and state issues. The bioassay of standardized test-cultures represented by different trophical levels such as producers (higher plants Brassica juncea L.), consumers (infusorium Paramecium caudatum Ehrenberg), reducers (bacterial biosensor - genetically modified strain of Escherichia coli) was carried out. In one set of experiments organisms’ test-responses on nanomaterials in water (0.5-500 mg/l) and humic preparation’s response reactions to them were
212
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
presence of humate Le-PhK (5 mg/l). In some cases, HS combined with nanomaterials increases toxic effects in concentrations 50 and 500 mg/l. Bioassays of nanomaterials with and without HS on test-cultures of different trophical levels in soil extracts from natural and artificial soils found: •Nanodioxide titanium in the extract of podzolic soil is almost neutral for higher plants. Humate addition caused stimulating effect in all concentrations (0.5-500 mg/l) from 8 to 35%. Phytotesting on model soil extract showed stimulatory effect in all concentrations. Humate stimulates further growth of Brassica juncea roots for 3-14 %. Bioassay on infusoriums showed that nano-TiO2 impact has mostly inhibitory effect except 0.5 mg/l, in which stimulation was showed. Adding HS eliminates this inhibition with the exception of 0.5 mg/l, in which it certainly inhibited survival of infusoriums not only in the extract of podzolic soil, but also in artificial soil. Nano-TiO2 significantly increased the bacteria luminescence in both mediums, and the addition of humate further enhanced this effect. •Nanomagnetite phytotesting mainly shows little stimulatory effect in podzolic soil extract, excepting 500 mg/l. Adding humate doesn`t affect nanomagnetite’s nature of impact. Nano-Fe3O4 impact is neutral in the artificial soil medium. Adding humate at low concentrations depresses higher plants roots development. Natural soil extract with 100 mg/l nanomagnetite concentration showed acute toxic effect on infusoriums survival, but humate completely eliminated this effect. However, in the range of lower concentrations (5-10 mg/l) nanomagnetite toxicity increases. Nano-Fe3O4 reduces infusoriums survival in the range 100-500 mg/l in the model soil medium, HS further enhances toxicity at 500 mg/l. Bacteria luminescence inhibited in the whole range of concentrations (0.5-100 mg/l) in podzolic soil media, and in the range of 100-500 mg/l in artificial soil medium. Humate exhibits inhibitory effect in both mediums. Nanomagnetite’s exposure stepwise nature has been established. Equal inhibitory activity is typical for 0.5 mg/l concentrations and 10 times bigger concentrations (ex. 5 mg/l). Equal stimulating activity is typical for 1 mg/l concentrations and 10 times bigger concentrations (ex. 10 mg/l). Such dependence is difficult to explain by the basics of different soil matrixes. This can be attributed to different mechanisms of impact in each concentration range. Unidirectional influence of humic substances on nanomaterials toxicity manifestation wasn’t revealed. The complex nature of "dose-effect" of studied nanomaterials can be witnessed. Determined by a number of peculiarities: concentrations of nanomaterials diffed by an order or two have a similar effect; the bioactivity sign changes from concentration to the concentration "stimulation-inhibition"; Average zone concentration effect in some cases is lower than in small concentration, it was also noticed in other researches (6). We may also conclude that the bioactivity of engineering nanomaterials entering environments (water or soil, enriched with natural organic matter)
can be modified by the presence of humic substances. Generalizing humate (5 mg/l) impact data positive effect is clearly evident in conjunction 1) with nanomagnetite on infusoriums in water (10 mg/l), bacteria (500 mg/l); extraction from podzolic soil on infusoriums (0.5 and 100 mg/l), from artificial soil (100 mg/l); 2) with nanodioxide titanium on infusorium (1 mg/l), bacteria (10 mg/l); extraction from podzolic soil on higher plants (10 mg/l), infusorium (1 and 50 mg/l). This confirms the universality of detoxication properties of humates (7) and expressed in neutralizing nanoparticles toxic effect. The obtained bioassay data of the three nanomaterial types (nano-TiO2, nano-Fe3O4 and DNDs-U) showed that toxicity depend on the physical nature of the nanoparticles (metal or carboncontaining), size and ability to form aggregates. Marked increase in the toxic effect of nanodiamonds with decreasing particle size of 100 nm 30 nm 15 nm is consistent with the known data for nanomaterials of different nature (3). REFERENCES
(1) Kaniskin, M.A., Izosimov, A.A., Terekhova, V.A., Yakimenko, O.S., Pukalchik, M.A. Theoretical and Applied Ecology 2011, 1, 86-93. (2) Perminova, I.V. Chemistry and Life 2008. 1, 20-30. (3) Radilov, A.S., Glushkov, A.V., Dulov, S.A. Nanotechnology&Science 2009, July, 1, 86-89. (4) Servos, M.R., Muir, D.C.J. Environ. Toxicol. Chem. 1989. 8, 141-150. (5) Tan, K.H. CRC Press. 2003, 386. (6) Terekhova, V., Gladkova, M. Eurasian Soil Science 2013. Vol. 46, 12, 1203–1210. (7) Yakimenko, O., Terekhova, V. Eurasian Soil Science 2011. Vol. 44, 11, 1222–1230.
213
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The effect of Humic acid on the Acute Toxicity of Benzo[a]pyrene to Algae, Pseudokirchneriella subcapitata. Y. Yanagi (a) *, Y. Okuyama(b), Y. Ochi(a) , N. Fujitake(c) , T. Kobayashi(d) (a)
Faculty of Agriculture, Yamaguchi University, Yoshida 1677-1, Yamaguchi, Yamgaguchi 7538515, Japan. Faculty of Environmental Horticulture, Minamikyushu University, Tateno 3764-1, Miyakonojo, Miyazaki 8850035, Japan. (c) Graduate school of Agriculture, Kobe University, Rokkodai 1-1, Kobe, Hyogo 6578501, Japan. (d) College of Bioresource Sceinces, Nihon University, Kameino1866, Fujisawa, Kanagawa 252-0880, Japan. * Corresponding author e-mail: [email protected] (b)
Keywords: Humic substances, Benzo[a]pyrene, Growth inhibition, Green algae, Polycyclic aromatic hydrocarbon, Toxicity Abstract: Partition coefficients (Koc) for the binding capacity of benzo[a]pyrene to humic acids from different soil in Japan were determined using fluorescence quenching technique. The highest Koc values showed in Andisol humic acid and there was no significant difference in the values of the other two humic acids (Inceptisol and Histosol). The magnitude of the Koc values seems to be related to atomic H/C ratios of HAs. In addition, the effects of three humic acids on the acute toxicity of benzo[a]pyrene to the green algae Pseudokirchneriella subcapitata were examined. The observed growth inhibition of P. subcapitata by BaP decreased along with an increase in the concentration of all HAs. The extent of a decrease in growth inhibition rate in the presence of humic acids showed similar trend to Koc values. Therefore, adsorption of benzo[a]pyrene with humic acids might be participated in the detoxification of benzo[a]pyrene. Introduction Polycyclic aromatic hydrocarbons (PAHs) are the most widespread of environmental contaminants. PAHs have a high potential for bioaccumulation owing to their lipophilic nature and acute toxicity and possess mutagenic, teratogenic, or carcinogenic effects. They are produced by incomplete combustion of organic matter such as fossil fuels, garbage, wood, and food. Humic substances (HS) can adsorb to PAHs and play an important role in the flocculation and the accumulation of PAHs. Therefore, these substances affect to the transport, fate, and bioavailability of PAHs in soil, sediment, and water. A large number of studies have examined the sorption characteristics of PAHs to HS and demonstrated that the binding capacity of HS for PAHs relate to the structure of HS such as aromaticity and atomic H/C ratio (1). In addition, several studies also showed the mitigating activity of the accumulation and the toxicity of PAHs by HS (2, 3, 4). However, relatively limited researches have been conducted on benzo[a]pyrene (BaP), an extraordinarily hydrophobic PAH, sorption to HS and the mitigating activity of HS to acute toxicity for BaP. The aim of the present study was to determine the binding capacity of humic acids (HAs) for BaP and to evaluate the detoxification effect of HAs to the acute toxicity of BaP. HAs derived from three different soils were used to measure partition coefficients (Koc) and to the BaP acute toxicity test using green algae.
Experimental The HAs used in this study was prepared from A horizon soil samples of Inceprisol (HO), Histosol (IJ), and Andisol (SG) in Japan according to the IHSS method. The elemental composition of HAs was determined by dry combustion method using elemental analyzer. The HAs was dissolved in the small amount of 0.1 mol L-1 NaOH, diluted with MQ water, and adjusted to pH 7.5. The partition coefficients between BaP and HAs were determined by fluorescence quenching technique (5). BaP dissolved in acetone (final conc. 1.0 µg L-1) was added to the vial and the acetone was evaporated. Five millilitre of carbonate buffer (pH7.5) was then added to the vial and shaken for 24 h. Five millilitre of HA solution, in concentrations ranging from 0 to 2.6 mg C L-1 (pH7.5), was added to the vial and shaken for 24 h. The quenching of the fluorescence of BaP was measured by spectrofluorometer. The excitation and emission wavelength were 380 nm and 405 nm, respectively. The fluorescence intensity of BaP was corrected for the influence of background HA and inner filter effect. The corrected fluorescence intensity of BaP with (F) /without (F0) HA was used in the Stern-Volmer equation. Koc values were calculated from the slopes of the Stern-Volmer plots. The green algae, Pseudokirchneriella subcapitata NIES-35, was used the acute toxicity test. Ten milliliter of synthetic nutrient medium containing HAs, in concentrations ranging from 0 to 1.1 mg C L-1, was added to the flask. BaP dissolved in ethylene glycol monomethyl ether was added to this flask to achieve a final concentration of 5 µg L-1 and shaken for 24 h. P. subcapitata (1 × 105 cells) were inoculated to each vial
214
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
and incubated on a shaker (130 rpm, 25˚C, 16 h light and 8 h dark). After 96 h incubation, the number of algal cells was counted. The medium without BaP was used as a control. The growth inhibition was expressed as a percentage of the control. Results and Discussion Partition Coefficients: Stern-Volmer plots for fluorescence quenching of BaP with three HAs are shown in Figure 1. SG showed highest affinity and Koc value was 35.2 × 106 L kg C-1. There was no significant difference in Koc between HO and IJ (3.1 and 2.5 × 106 L kg C-1, respectively). These values were distributed within the range of Koc values for BaP sorption reported by Turner and Rawling (6). The element content and atomic ratio were shown in Table 1. SG had lowest C %, H %, and atomic H/C ratio than HO and IJ had. Several researchers reported that the atomic H/C ratio of HS correlated negatively with the Koc values of PAHs. Our result agreed with these observations.
25
Figure 2. Growth inhibition of P. subcapitata by BaP with HO (Inceptisol HA; ), IJ (Histosol HA; ), and SG (Andisol HA; ). Bars represent ± SD (n = 3)
inhibition decreasing effect among three HAs. HO and IJ showed lower effect on growth inhibition and no significant difference in these HAs. The extent of a decrease in growth inhibition rate in the presence of HAs showed similar trend to Koc values. Therefore, adsorption of BaP with HAs might be participated in the detoxification of BaP. REFERENCES (1) Perminova, I.V.; Grechishcheva, N.Y.; Petrosyan, V.S. Environ. Sci. Technol. 1999, 33, 3781-3787. (2) McCarthy, J.F.; Jimenez, B.D. Environ. Toxic. Chem. 1985, 4, 511-521. (3) Johnsen, S.; Kukkonen, J.; Grande, M. Sci. Total Envirom. 1989, 81/82, 691-702. (4) Perminova, I.V.; Grechishcheva, N.Y.; Kovalevskii, D.V.; Petrosyan, V.S.; Matorin, D.N. Environ. Sci. Technol. 2001, 35, 3841-3848. (5) Gauthier, T.D; Shane, E.C; Guerin, W.F. Environ. Sci. Technol. 1986, 20, 1162-1166. (6) Turner, A.; Rawling, M.C. Water Res. 2002, 36. 2011-2019. (7) Haitzer, M.; Abbt-braun, G; Traunspurger, W.; Steinberg, C.E.W. Environ. Toxicol. Chem. 1999, 18, 27822788. (8) Steinberg, C.E.W.; Haitzer, M.; Brüggemann, R.; Perminova, I.V.; Yashchenko, N.Y.; Petrosyan, V.S. Internat. Rev. Hydrobiol. 2000, 2-3, 253-266.
Figure 1. Stern-Volmer plots for fluorescence quenching of BaP with HO (Inceptisol HA; ), IJ (Histosol HA; ), and SG (Andisol HA; ). BaP concentration was 1.0 µg L-1 . Table 1. Elemental composition and atomic ratio of HAs used in this study. HA HO IJ
Element content (weight %) C H N O 54.5 5.90 4.77 34.8 49.6 4.23 2.10 44.0
Atomic H/C 1.30 1.02
ratio O/H 0.48 0.65
Acknowledgments: This research was partially funded by the Japan Society for the Promotion of Science through a Grantin-Aid for Young Scientists (B) (No. 24710014).
Detoxifying Effect of HAs: The relationships of growth inhibition rate of P. subcapitata by BaP vs. concentration of HAs are shown in Figure 2. The observed growth inhibition of P. subcapitata by BaP decreased along with an increase in the concentration of all HAs. These findings agree with the previous reports on BaP bioconcentration and other PAHs toxicity (2, 3, 4, 7). SG showed the highest growth
215
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Fractionation and characterization of dissolved organic matter from Sorocabinha and Itapanhaú rivers (SP/Brasil) A.S.C. Monteiro(a)*, E.S.J. Gontijo(b), C.H. Watanabe(b), J.P. Pinheiro(c), A.H. Rosa(b) (a)
Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Rua prof. Francisco Degni, 55, Quitandinha, 14800-060, Araraquara, SP – Brasil (b) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Av. Três de Março, 511, Alto da Boa Vista, 18087-180, Sorocaba, SP – Brasil (c) IBB/CBME, DQF/FCT, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal * Corresponding author e-mail: [email protected] Keywords: DOM, AHS, tangential ultrafiltration, UV/VIS, trace metals, Stripping chronopotentiometry Abstract The fractionation and characterization of the dissolved organic matter (DOM) of Sorocabinha e Itapanhaú Rivers were performed in order to evaluate its structural composition and the ability to interact with trace metals such as Cd, Pb e Zn. The Aquatic humic substances (AHS) extracted were fractionated using tangential ultrafiltration yielding five fractions. UV-VIS spectrometry (E254/E456, E280/E350, SUVA254) and elemental analysis were applied for qualitative characterization of the DOM. The total, dissolved and free fraction (<1KDa) trace metals were analysed in the natural waters and after filtration in 0.45 µm. The results indicates that the material is mostly fulvic in nature and that the most abundant molar mass fraction is <5 KDa while the most aromatic is 5
Introduction Dissolved organic matter (DOM) is a key player in the chemistry of the aquatic environment. In natural waters it is mostly a mixture of degradation products of animal and vegetal origin, being operationally divided in two main categories: chemically welldefined compounds or non-humic substances (ex: proteins, amino acids, lipids and carbohydrates) and aquatic humic substances (AHS). The DOM, especially the AHS fraction is chemically heterogeneous and polydisperse in size, being their structural and chemical composition highly variable with the origin and age of the material (1). The diversity of functional groups bestow a polyfunctional and polyelectrolytic nature to AHS (and DOM) and therefore defines its interaction with metal ions that spans a broad range of free energy of complex formation, hence controlling their bioavailability and mobility (2). Thus characterization of the DOM, especially of the AHS, is useful to understand these phenomena. UV/VIS spectroscopy is useful to characterize, differentiate and classify DOM using ratios between absorbance at characteristic wavelengths. When the DOM is fractionated by molar mass we observe differences in the characteristic absorbance ratios of the fractions, thus allowing a characterization of the DOM polydispersity and chemical nature (3). Further information on the metal ion binding can be obtained by quantifying total, dissolved metal and free metal by ICP/OES and/or Stripping Chronopotentiometry (SCP) directly in the natural water and in the filtrated of 1KDa. It is also important to quantify the metal ions in the different colloidal fractions, resulting from successive tangential ultrafiltration steps.
Experimental Samples of surface water were collected in Itapanhaú (It) and Sorocabinha (So) rivers in the State of São Paulo, southeast of Brasil. The samples were stored in plastic containers to analyse the trace metals (Cd, Pb and Zn), dissolved organic carbon DOC and to perform UV-VIS spectrometry measurements. Aquatic humic substances (AHS) were extracted using DAX-8 resins based in methodology proposed by Thurman and Malcolm, and recommended by the IHSS (4). The AHS extracted was fractionated using ultrafiltration systems obtaining five fractions [>100 KDa (F1), 30
216
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The Zn concentration is within the usual natural levels and is mostly complexed by the dissolved and particulated fraction (total-dissolved) being the free zinc below the detection limit. The high Pb concentration in the Sorocabinha river is likely due to the lead mines operating in the region (8), and is predominantly complexed in the particulated fraction.
Results and Discussion
H (%)
N (%)
S (%)
O (%)
H/C
So
44.5
4,5
0,05
50,9
1,2
0,8
It
42.6
4.2
1.4
51.8
1.1
0.9
Sample-Metal
Total Metals (µg L-1)
Dissolved Metal (µg L-1)
Free Metal (µg L-1)
So -Cd
<0.2
<0.09
< 0.14
So- Pb
32.45
<2.57
<3.86
So- Zn
19.00
8.30
<2.62
It- Cd
<0.2
<0.09
<0.14
It-Pb
< 2.6
<2.57
<3.86
O/C
C (%)
Sample
Elemental composition: The atomic rations H/C and O/C of both AHS are similar (Table 1). The higher H/C ratio indicates a lower humification degree, thus indicating a higher aliphatic content in agreement with the predominantly fulvic nature of the aquatic materials (6).
Table 1. Elemental composition of the AHS extracted from the Sorocabinha (So) and Itapanhaú (It).
Fractionation and UV/VIS characterization The COD obtained was 52.50 mg.L-1 and 16.20 mg.L-1 for (So) and (It) respectively. Figure 1 shows that the majority of the AHS appears in the lower molar mass fractions (56.5% <5 KDa (F5) and 13.7% between 5 KDa and 10 KDa(F4)).
It- Zn 12.08 6.24 <2.62 Tabela 3. Total dissolved and free Cd, Pb and Zn determined by ICP/OES in Sorocabinha (So) and Itapanhaú (It).
The fractionation and characterization of the DOM indicates that the material is mostly fulvic in nature and the most abundant molar mass fraction is <5 KDa while the most aromatic is 5
(1) Chen, J.; Gu, B.; LeBoeuf, E.J.; Pan, H.; Dai, S. Chemosphere. 2002, 48: 59–68. (2) J. Buffle, Complexation reactions in aquatic systems, Ellis Horwood, Chichester, 1988. (3) Baker, A.; Spencer, Robert, G.M. Environ. Sci.Technol.2004, 333: 217-232. (4) Thurman, E.M.; Malcolm, R.L. Environ. Sci. Technol. 1981, 15:463-466. (5) Tonello, P.S.; Rosa, A.H.; Abreu Jr, C.H.; Menegário, A.A. Anal. Chim. Acta. 2007, 598, 162-168. (6) De Oliveira, L. C.; Sargentini, T.; Rosa, A. H.; Rocha, J. C.; Simoes, M. L.; Martin-Neto, L.; Da Silva, W. T. L.; Serudo, R. L. J. Braz. Chem. Soc. 2007, 18: 860-868. (7) Svetlana M. I.; Yu, O.D.;Sergey A. L.;Yuriy V. A.; Vladimir V. D.; Yuliya A. Z.; Liudmila S. S.; Viersa, J; Pokrovsky O.S.; Geochemistry, 2014, 66: 14-24. (8) De Figueiredo, B.; Borba, R.; Angélica, R. Arsenic Occurrence In Brazil And Human Exposure. Environ. Geochem. Ealth, 2007,29: 109-118.
Figure 1. %C (w/w) in the different fractions of the AHS extracted from the Sorocabinha (So).
The ratio E254 /E436 (Table 2) indicates that the origin of the DOM is dominantly aloctonous (4< E254 /E436<11). Both the SUVA and the ratio E280/E350 show that the F4 fraction (5
E254/E436
E280/E350
SUVA 254
So
15.28
3.16
6.21
AHS
12.43
2.34
5.77
F>100
5.49
2.09
6.75
30
12.87
3.33
3.69
10
8.32
2.36
6.45
5
6.31
24.33
7.27
Acknowledgments: The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support. AHR and JPP thank CNPq Brasil PVE scholarship 400572/2013-3. JPP thanks Fundação para a Ciência e a Tecnologia (FCT) for funding support in project FCTANR/AAG-MAA/0065/2012) and Project PestOE/EQB/LA0023/2013.
F<5 10.95 2.45 6.78 Tabela 2. Characteristic UV/VIS ratios E254/E436, E280/E350, SUVA 254.
Concerning the trace metals present in the sample Table 3 shows that Cd is absent in both rivers and Pb is below detection limit in the Itapanhaú.
217
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The effect of effluent organic matter (EfOM) on Cu bioavailability to Daphnia magna J. Yoo, J. Jung*
Division of Environmental Science and Ecological Engineering, Korea University, Seoul, Republic of Korea * Corresponding author e-mail: [email protected] Keywords: Effluent organic matter, Copper, Bioavailability, Fractionation, Oxidative stress Abstract Effluent discharges of treated wastewater represent an important source of organic matter to aquatic ecosystems. Rapid development of industry has led to large amounts of effluent organic matters (EfOMs) being released into water bodies, which greatly influence the composition and property of dissolved organic matters. It is well known that natural organic matters (NOMs) affected the bioavailability of trace hazardous compounds to aquatic organisms. However, it is unknown how EfOMs contribute to ecosystem processes in streams receiving wastewater effluent discharges. In this study, effluents of liquid crystal display (LCD) manufacturing plants (LCD-EfOM) and Suwannee River NOM (SR-NOM) were isolated into hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) fractions by DAX-8 and XAD-4 resins (Amberlite, USA), and characterized by specific ultraviolet absorbance (SUVA) analysis. In addition, acute toxicity and oxidative stress responses (activities of superoxide dismutase, catalase and malondialdehyde) of each fractionated samples with copper (Cu) were conducted using Daphnia magna. As a result, we observed the SUVA value of the SR-NOM to be large than LCD-EfOM. Such differences indicate that the SR-NOM may have more aromatic character than the LCD-EfOM. In addition, organic matters (OMs) were alleviated acute toxicity of Cu to daphnids, and the reduction efficiency of SR-NOM were higher than LCD-EfOM. Lower levels of antioxidant enzymes were expressed in HPO samples than isolated TPI and HPI samples. These findings suggest that physicochemical properties of OMs have affect to Cu bioavailability, which needs to be further studied with toxic mechanisms between Cu and OMs. Introduction Effluent discharges of treated wastewater represent an important source of organic matter to aquatic ecosystems. Wastewater derived DOM was shown heterogeneity and complexity and was more aliphatic than the DOM in natural waters (1). Although the importance of DOM from natural sources in stream system is well established, little work has examined anthropogenic sources of organic matter such as in wastewater effluents; thus, it is unknown how effluent organic matter (EfOM) contributes to ecosystem processes in streams receiving effluent discharges. The presence of DOM affects the availability of trace hazardous chemicals by serving as a sorptive phase that can be transported through aquatic environments (2, 3). In particular, some of the functional groups on EfOM have a high affinity for trace metals, and EfOM may therefore strongly affect the mobility of trace metals in aquatic systems. In this study, EfOMs from liquid crystal display (LCD) wastewater treatment effluents and Suwannee River NOM were isolated into hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) fractions, and characterized by specific ultraviolet absorbance (SUVA) analysis. In addition, acute toxicity and oxidative stress responses to Daphnia magna were evaluated to characterize toxicological properties of isolated OM. Biochemical endpoints can be used as early and sensitive reporters relative to acute toxicity because they are typically the initial stress response in organisms and can be detected before death (4). In particular, enzymatic antioxidants including
superoxide dismutase (SOD), catalase (CAT), and malondealdehyde (MDA) have been widely used to characterize defense mechanisms and evaluate toxicity induced by oxidative stressors (5, 6). Therefore this study aimed to evaluate lethal (acute toxicity) and sub-lethal (oxidative stress) toxicity in isolated EfOM and NOM samples with copper using D. magna, and to identify the toxic mechanisms between copper and isolated organic matters. Experimental Effluent samples was collected from liquid crystal display (LCD) wastewater treatment in Gyeonggi-do, Korea. The samples were transported to the lab, filtered using Watman GF/C glass fiber filters (0.45µm), acidified to pH 2 using HCl, and stored in the refrigerator at 3 oC. Suwannee River standard NOM (SR-NOM) sample was also examined in this work. The Samples were isolated into hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) fractions using the Amberlite resins (DAX-8 & XAD4 resins, Amberlite, USA) and cation exchange resins were adopted from previous studies (2, 7, 8). Toxicity tests with D. magna were conducted in copper-spiked test media with 0.03 mg Cu/ L (for acute toxicity test), 0.01 mg Cu/ L (for oxidative stress test) and 5 mg /L DOC concentrations of each isolated samples. These samples were added to reconstituted water containing 2 mM CaCl2, 0.5 mM MgSO4, 0.77 mM NaHCO3, and 0.078 mM KCl. To buffer the pH of the test solutions, 750 mg L21 3-N-morpholino-propanesulphonic acid (MOPS) was added and pH was adjusted to 6.5 with NaOH and HCL.
218
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Acute toxicity tests were carried out according to OECD 202 standard procedures (6) with D. magna neonates (≤ 24 h old). Each sample were conducted for the toxicity test using 10 neonates and 50mL of test solution in triplicates, respectively. Immobility (%) (defined as no response to gentle agitation for 15 s) was recorded after 24h and 48 h. Oxidative stress responses were analyzed according to the study by Barata et al. (9) using D. magna independently cultured for 5 d. Twenty juveniles were exposed to 250 mL isolated OM samples without feeding at 20 ± 2 ◦C in photoperiods of a 16 h light:8h dark for 48 h. The animals were then homogenized in phosphate buffer (100 mM) at pH 7.4. After centrifuging the homogenates at 10,000 × g for 10 min, antioxidant enzyme activity and lipid peroxidation in the supernatants were analyzed. Enzymatic measurements were conducted in a microplate spectrophotometer (BioTek Inc., Winooski, VT, USA) using commercial assay kits, including SOD (WST) kits from Dojindo Laboratory (Tokyo, Japan), CAT (K773-100) kits from BioVision Inc. (San Diego, CA, USA) and MDA (NWK-MDA01) kits from Northwest Life Science Specialties (Vancouver, WA, USA). All tests were carried out following the manufacturer’s instructions.
Immobilization (%)
100
24h (%)
48h (%)
80 60 40 20 0
ISO
Raw
HPO
TPI
HPI
Raw
LCD EfOM
HPO
TPI
HPI
SRNOM
Figure 1. Acute toxicity (immobilization) of the isolated OM samples with Cu (0.03mg L-1) to Daphnia magna in 5 mg L-1 DOC.
A
0.05 0.04
SOD (mU/mg)
CAT (mU/mg)
0.03 0.02 0.01 0
Raw HPO TPI Contol ISO
B
10 8 6
HPI
LCD EfOM
Raw HPO TPI
HPI
SRNOM
MDA (µM/mg)
4 2 0
Raw HPO TPI Contol ISO
LCD EfOM
HPI
Raw HPO
TPI
HPI
SRNOM
Figure 2. Antioxidant enzyme activity of (a) superoxide
Results and Discussion The percent of effluent organic matter isolated in the hydrophobic (HPO) fraction of the LCD WWTP effluent was less than typically found for SR-NOM isolates. EfOMs isolated from LCD WWTPs effluent had lower SUVA value than these of SR-NOM. SUVA value of the HPO fractions for each LCDEfOM and SR-NOM samples to be larger than their corresponding TPI, HPI fraction. Such differences suggest that the HPO fractions may have more aromatic character than the TPI fractions. The SUVA has been frequently employed to estimate aromatic content of organic compounds because the absorbance of energy at 254 nm corresponds to π - π * transitions typical of aromatic rings (10). [Figure 1.] As a result of acute toxicity test, most hydrophobic fractions of LCD-EfOM and SR-NOM showed less toxicity to D. magna. Al-Reasi et al. (11) also reported that more aromatic DOMs are more protective against Cu toxicity to D. magna. [Figure 2A, 2B]. In addition, lower levels of antioxidant enzymes were expressed in HPO samples than isolated TPI and HPI samples, likely due to the alleviation effect of OMs. Heavy metals can generate reactive oxygen species, such as hydroxyl radical, superoxide anion radical and hydrogen peroxide, which result in oxidative stress to aquatic organisms (9, 12). These findings suggest that EfOMs have different physicochemical and toxicological properties compared with those of terrestrial SR-NOM. These different properties of OMs have affect to Cu bioavailability, which needs to be further studied with studied with toxic mechanisms between Cu and OMs.
dismutase (SOD), catalase (CAT), and (b) malondialdehyde (MDA) to Daphnia magna after exposure to the isolated OM samples with Cu (0.01mg L-1) in 5 mg L-1 DOC.
REFERENCES
(1) Czerwionka, C.; Makinia, J.; Kaszubowska, M; Majtcz, J. and Angowski, M. Water Sci Technol. 2012, 65, 1583-1590. (2) Croue, J. -P.; Benedetti, M. F.; Violleau. D. and Leenheer., J. A. Environ Sci Technol, 2003, 37, 328-336. (3) Pernet-coudrier, B.; Blouzot, L.; Varrault, G.; Tusseau-vuillemin. M. –H.; Verger, A. and Mouchel, J. –M. Chemosphere. 2008, 73, 593-599. (4) Kim, S.; Kim, W.; Chounlamany, V.; Seo, J.; Yoo, J.; Jo, H and Jung J. J. Hazard. Mater. 2012, 227.228, 327-333 (5) Kim, K. T.; Klaine, S. J.; Cho, J.; Kim, S. H. and Kim, S. D. Sci.Total Environ. 2010, 408, 2268–2272. (6) OECD 202, Organization for Economic Cooperation and Development, Paris, 2004. (7) Aiken, G. R.; McKnight, D. M.; Thorn, K. A. and Thurman, E. M. Org Geochem, 1992, 18, 567-573 (8) Ma, H.; Allen, H. E. and Yin, Y. Water Res, 2001, 35(4), 985-996. (9) Barata, C.; Varo I.; Navarro J.C.; Arun S. and Porte C. Comp Biochem Phys C, 2005, 140, 175-186. (10) Abbt-Braun, G. and Frimmel, F. H. Environ Int. 1999, 25, 161-180. (11) Al-Reasi, H. A.; Smith D. S. and Wood, C. M. Ecotoxicology, 2012, 21, 524-537. (12) Xu, H.; Song, P.; Gu, W. and Yang, Z. Ecotoxicol. Environ. Saf. 2011, 74, 1685–1692.
Acknowledgments: This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (2012R1A1A2041989).
219
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The Effect of Humic Acid on Yield and Fe Uptake of Soybean
Ayhan HORUZa*, Cengiz ÖZCANb, Mumin DİZMANc, Ahmet KORKMAZa, Güney AKINOĞLUa a
Ondokuz Mayıs Üniversity, Agricultural Faculty, Department of Soil Science, Samsun, Turkey b Directorate of Food Agriculture and Animal Breeding, Nevşehir, Turkey c Yeditepe Üniversity, Department of Genetics and Bioengineering, Istanbul, Turkey *Corresponding autor e mail: [email protected] Keywords: Humic acid, Iron, Soybean, Yield
Abstract: The objective of this research was to examine the effect of humic acid (HA) on yield and iron uptake of soybean (Glycine max L.). The experiment was conducted in randomized plot design with the treatments of 0, 0.5, 1.0 and 2.0 kg Fe/da (FeSO4.7H2O) dose applications. Humic acid was added as a dose of 10 kg HA/da in each treatment. In the study, it was found that iron application with HA increased yield, leaf and Fe content in grain significantly (P<0.01). The highest yield amount and 100 grain weight of soybean were 378.07 kg/da and 20.64 g in 1.0 kg Fe/da+HA application, respectively. The highest Fe content in leaf and grain were 365.40 ppm and 165.17 ppm in 0.5 kg Fe/da+HA application, respectively. Consequently, the application of 0.5 and 1.0 kg Fe/da with 10 kg HA to soyabean was recommended.
Introduction The effects of humic substances on plant growth depend on the source and concentration, as well as on the molecular fraction weight of humus. Lower molecular size fraction easily reaches the plasma lemma of plant cells, determining a positive effect on plant growth, as well as a later effect at the level of plasma membrane, that is, the nutrient uptake (1). The stimulatory effects of humic substances have been directly correlated with enhanced uptake of macronutrients, such as nitrogen, phosphorus and sulfur, and micronutrients, such as Fe, Zn, Cu and Mn (2). These substances also affect the solubility of many nutrient elements by building complex forms or chelating agents of humic matter with metallic cations (3). Humic acid caused increases the uptake of nitrogen, phosphorus, K+, Ca2+, Cu2+, Mn2+, Zn2+ and Fe3+. Humic substances increased root length in Helianthus annuus L. (4), in maize roots, and uptake of micronutrients such as Zn2+, Fe3+, Mn2+ and Cu2+. Humic acid added to micronutrient fertilizers increased the uptake of Cu, Zn, Fe and Mn in tropical kuzdu (Pueraria phaseoloides;). Humic acid has an essential role in agricultural processes. It increases cation exchange capacity and enhances soil fertility, converting the mineral elements into forms available to plants (5). Fe deficiency as chlorosis is a widespread problem for soybean grown on alkaline, calcareous soils, and yield limiting problem for soybean production in the some parts of Turkey (6). While Fe is the primary plant nutrient, relatively small amounts of Fe is required to support the process of growth and quality of plants (7). The objective of this research was to examine the effect of humic acid (HA) on yield and iron uptake of soybean (Glycine max L.) in a field experiment.
with the treatments of 0, 0.5, 1.0 and 2.0 kg Fe/da (FeSO4.7H2O) doses. Seeds were sown on May 3rd, 2005 in four 6m rows per plot (70 cm row spacing). Humic acid was added as a dose of 10 kg HA/da in each treatment with three replications. Soybean plants were harvested about 140 days after the sowing and washed three times with tap water and then with deionized water before oven dried at 65°C for 72 hr. The grain yield at 15% moisture was recorded, and each of plant part (leaf and grain) oven dried were ground in a steel mill, and submitted to 550°C digestion and Fe determined by AAS (Atomic Absorbance Spectrophotometer-AA400). Table 1. Some soil physical and chemical properties. Soil property Values pH, saturated mud 8,05 EC, dS/cm 0,07 Lime , % 9,36 OM, % 2,28 CEC, me/100 g 37,44 Sand, % 35,51 Mil, % 37,67 Clay, % 26,82 Texture Loam Available nutrients P, ppm 14,69 K, ppm 117,83 Ca, me 100/g 26,72 Mg, me 100/ g 7,83 Zn, ppm 0,97 Fe, ppm 6,70 Mn, ppm 18,20 Cu, ppm 2,14 Results and Discussion Effect of humic acid plus Fe applications on soybean yield components: Effects of humic acid (HA) applications with different Fe doses on yield components of soybean are given in Table 2. According to the variance analyses results, humic acid plus Fe applications significantly increased (P<0.01) grain yield and 100 grain weight of soybean compared with the control (only humic acid). While the maximum soybean yield was obtained from HA+1 kg/da Fe application, higher than HA+1 kg/da Fe doses caused decreases in yield (Figure 1).
Experimental A field experiment was carried out in Çarşamba district located on the eastern part of Samsun in Turkey. The main characteristics of soil are reported in Table 1. Soybean (Glicine max L.) cv. Savoy was used as a test plant. Inoculation of seeds with appropriate strain of Rhizobium japanicum L. was carried out. The experiment was conducted in a randomized plot design
220
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Table 2. Effect of humic acid and Fe fertilization on yield components Fe dose Yield 100 grain weight Leaf Grain kg da-1 kg da-1 g Fe content, mg kg-1 0 200,13c 14,95c 328,96b 125,23b 0.5 281,37b 19,73b 365,40a 165,16a 1 378,07a 20,64a 317,23c 118,26b 2 363,36a 19,63b 279,07d 128,07b
Leaf Grain Fe uptake, g da-1 65,84b 25,06b 102,81a 46,47a 119,94a 44,71a 100,49a 46,12a
*
There is not a significant difference between the values showed with the same letters in the same column at 1% level.
Leaf
400
Grain
350 300 250 200 150
Leaf
Grain
350
Fe content, ppm
Yield, kg/da
400
300 250 200 150 100
0
0,5
1
1,5
50
2
0
Fe, kg/da
Figure 1. The effect of Fe applications on soybean yield
0,5
Fe, kg/da
1
2
Figure 2. The effect of Fe fertilization on leaf and y = -86,239x + 257,15x + 193,1 grain Fe contents R² = 0,9697 2
y = -86,239x 2 + 257,15x + 193,1 R² = 0,9697
Increasing Fe application doses increased grain yield between 200,30 and 378,07 kg/da, and 100 grain weight between 14,95 and 20,64 g. The highest increments in soybean yield and 100 grain weight were 88,91% and Grain Lea f 38,06%, respectively (Table 3).
140
Leaf
Grain
Fe uptake, g/da
120
Table 3. Effect of humic acid and Fe fertilization on increment of yield components 100 grain Yield Leaf Grain Fe dose weight -1 kg da Increment, % 0 0.5 40,59 31,97 11,07 31,89 1 88,91 31,30 -3,57 -5,56 2 79,93 38,06 -15,17 2,27
100
Grain
Lea f
80 60 40 20 0 0
0,5
Fe, kg/da
1
2
Figure 3. The effect of Fe fertilization on leaf and grain y = -86,239x + 257,15x + 193,1 Fe uptake. R² = 0,9697 2
REFERENCES
(1) Nardi S, Pizzeghello D, Muscolo A, Vianello A. Physiological Effects of humic substances in plant growth. Soil Grain Lea f Biol. Biochem. 2002, 34(11), 1527-1536. (2) Chen Y., Clapp C.E., Magen H., Cline V.W. Stimulation of Plant Growth by Humic Substances: Effects on Iron Availability. In: Ghabbour, E.A. and Davies, G. (eds.), Understanding Humic Substances: Advanced Methods, properties and Applications. Royal Society of Chemistry, Cambridge, UK.. 1999, 255-263. (3) Lobartini J.C. Orioli G.A. and Tan K.H. Characteristics of soil humic acid fractions separated by ultrafiltration. Communications in Soil Science and Plant Analysis, 1997, 28, 787-796. (4) Kolsarici O, Kaya M.D., Day S., Ipek A., Uranbey S. Effects of humic acid doses on emergence and seedling growth of sunflower (Helianthus annuus L.). Akd. Uni. J. Agric. Faculty, 2005, 18(2), 151-155 (5) Stevenson, F.J. Humus Chemistry: Genesis, composition, reactions, 2nd edition, John Wiley and Sons, Inc, New York. 1994 (6) Caliskan S, Ozkaya I, Caliskan M E and Arslan M. The effects of nitrogen and iron fertilization on growth, yield and fertilizer use efficiency of soybean in a Mediterranean-type soil. Field Crops Res. 2008, 108, 126-132. (7) Fageria N.K. Adequate and toxic levels of zinc for rice, common bean, corn, soybean and wheat production in cerrado soil. Rev. Bras. Eng. Agri. Ambien. 2000, 4, 390-395.
Effect of humic acid plus Fe application on Fe content and uptake of soybean: Iron application with humic acid (HA) to the soybean significantly increased (P<0.01) leaf and grain Fe contents according to the control (Table 2). Fe content changed between 279,07 and 365,40 ppm in leaf, and between 118,26 and 165,16 ppm in grain (Figure 2). Also, the maximum increments in Fe contents of soybean leaf and grain have been found as 11,07% and 31,89% in HA + 0,5 kg Fe da-1 dose, respectively (Table 2). Similarly, HA+Fe application to the soybean significantly increased (P<0.01) leaf and grain Fe uptakes according to the control (Table 2). The highest Fe uptake was found as 119,94 g/da in leaf and 46,74 g/da in grain (Figure 3). It is known that Fe can be complexed with organic ligands such as humic acid. Due to fact that experimental soil had moderately lime content (9.36%), Fe uptake (65.84 g/da) by the soybean plants grown in the control plots was lower than that in the HA+Fe applications. According to the results of this study, humic acid (10 kg/da) plus Fe treatments increased the yields, and Fe uptake of soybean plant. The highest value for yield components were obtained from 1 kg Fe/da soil fertilizations and the highest Fe content and Fe uptake were obtained from 0,5 kg Fe/da fertilization.
Acknowledgments: We are thanks to Black Sea Agricultural Research Institute, Samsun for providing the full support of this study in field stage.
221
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
A Meta-analysis of Plant Growth Response to Humic Substances M. T. Rose(a), Karen Little(a), Alicia Brown(a), W. R. Jackson(a), T. R. Cavagnaro(b) and A. F. Patti(a), (a)
School of Chemistry, Monash University, Clayton, Victoria, 3800, Australia School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB1 Glen Osmond, SA, 5064, Australia (b)
* Corresponding author e-mail: [email protected] Keywords: humic substances, plant growth, meta-analysis, lignite, brown coal Abstract Lignite (or brown coal) is a rich source of humic substances. Commercially available humic containing preparations often use lignite as the humic acid source. These lignite-derived products, including humic acids and organo-mineral soil conditioners, are also being marketed as one solution to soil degradation through their effects on plant growth. In this paper, we present results from a literature meta-analysis of reports involving humic substance preparations from various sources including lignite to improve soil fertility and plant growth. Our findings suggest that complex interactions between humic substances, soil types, environmental conditions and plant species mean that a ‘one-size fits all’ product or solution is unlikely. Changes to soil characteristics brought about by LDPs in particular are more apparent over longer time periods than a single cropping season. We identified a set of continuous and categorical predictors that were hypothesized to influence the responsiveness of plants to HS applications, measured as the ratio of the shoot (SDW) or root dry weight (RDW) of treated plants to non-treated control plants These groups fell under four broad areas: environmental conditions; plant type; HS properties; and the method of HS application. The actual predictors are shown in the table below.
Introduction Commercially-available humic products in agriculture have been promoted as an option to improve crop and pasture productivity, as well as providing beneficial effects in the soil. The mechanisms for these benefits are not well understood and numerous explanations have been put forward. The scientific literature is lacking in reporting studies where humic substances (HS) preparations have been applied and crops have been grown to completion, thus evaluating the economic and other benefits claimed from the use of HS preparations. The mechanisms of action reported include; a direct effect of humic acids on the plant, thereby influencing plant metabolism1; improved plant nutrient uptake due to the complexing ability of HS with soil nutrients2 and indirect effects through stimulation of soil microbial communities3. The aim of this paper is to report on a detailed literature analysis5 that evaluated the magnitude and likelihood of plant growth responses to humic substances. The meta-analysis ranked the factors that contributed to positive growth promotion. These factors included; source of the humic substances, environmental conditions, type of plant being treated and the manner in which the humic substances were applied.
Table 1 Predictors of Plant Response
3. Statistical analysis A mixed-effect meta-analysis model was used to test the significance of predictors one-at-a-time, using the R package ‘metafor’4. All data were weighted according to their inverse variance
Methodology 1. Literature Search A search of the databases Scopus and ISI Web of Science was conducted. A combination of search terms including ‘humic’ AND ‘plant’ AND ‘effect’ AND ‘growth OR yield’; designed to provide an unbiased selection of potential studies, rather than act as an exhaustive search for all studies in this area was used. From a total of 390 papers, 81 were retained for the analysis.
Results and Discussion 1. Aggregated plant growth responses to HS The random effects model predicted that HS application significantly (Fig. 2) increases both SDW and RDW by 19±3% and 20±4%, respectively SDW response was not significantly influenced by the growth media or the application site, but was significantly affected by the source of HS, stressful
2. Data classification
222
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
growing conditions, the type of plant being treated and the rate of HS applied Only peat-derived HS did not significantly affect shoot biomass accumulation (4±12%) (Fig. 1A). Brown coal derived HS increased SDW response (12±4%), but was less effective than HS extracted from green waste compost (29±8%), manure compost (28±8%) and soil (25±8%). A similar trend was observed for RDW (Fig. 1B)
3. Optimisation of application rate of commercial HS Commercial HS are most often sourced from brown coals (BC) The SDW response of plants to BC-derived HS is nonlinear and is better described by two quadratic functions rather than a single quadratic or higher polynomial An initial sharp peak in growth response is observed between 5-40 mg kg with a maximum around 20 mg kg followed by a more gradual growth increase from 40-200 mg kg. There is a greater opportunity to maximise plant growth promotion by applying BC-derived HS in the lower range (5-40 mg) of the initial peak, which would also be economically rational. Conclusions The growth response of plants to HS is generally positive, but is influenced by a number of environmental and management factors. These included: source of the HS which had a strong impact on whether plant growth was significantly improved; plant type and stress conditions also influenced the response to HS, but to a lesser extent. Interactions between each of these factors and the HS application rate moderate the plant growth response, emphasizing the complexity of obtaining predictable responses. The majority of papers reporting experiments on HS lack information about the organic structure and chemical composition of the HS amendments. Data about the soil, such as the nature of native organic matter, pH, EC, texture, and mineral nutrient concentrations are also lacking. More applied research is needed but requires farmer involvement in scientifically designed trials in local conditions.
Fig.1 Estimated SDW (A) and RDW (B) response (weighted mean ± 95% confidence level) of plants to HS application for significant predictors. Ratios > 1 indicate growth promotion; < 1 indicate growth suppression. The number of data points in each group is given in parentheses
Plants were significantly more likely to increase shoot growth in response to HS application under highly stressful conditions (28 ±6%) than non-stressful conditions (18±3%) RDW increases were observed more frequently in plants grown in soil as compared with hydroponics 2. The importance of predictor interactions in explaining growth response to HS The BRT analysis revealed an interaction between HS source and application rate as the most important in explaining the variation in both shoot and root growth response to HS use. Increasing rates of green waste compost HS application to both monocots and dicots was positively related to SDW over untreated control plants, but the application of soil-derived HS stimulated SDW more effectively at lower application rates. Increasing rates of lignite HS were negatively related to root growth under conditions of stress, but did not consistently affect growth under non-stress conditions. The opposite occurred with soil-derived HS, with a positive root growth response to increased application rates under high stress conditions.
References 1. Nardi, S., et al., Physiological effects of humic substances on higher plants. Soil Biology and Biochemistry, 2002. 34(11): 1527-1536 2. Chen, Y., et al., Mechanisms of plant growth stimulation by humic substances: the role of organoiron complexes. Soil Science and Plant Nutrition, 2004. 50(7): 1089-1095. 3. L. P. Canellas and F. L. Olivares, Physiological responses to humic substances as plant growth promoter, Chem.Biol. Tech. Agr.,2014, 1, 3-11 4. Veichtbauer, W., Conducting meta-analysis in R with the metafor package. J. Stat. Softw., 2010, 36,145. 5. Rose, M.T., et al., Chapter Two - A Meta-Analysis and Review of Plant-Growth Response to Humic Substances: Practical Implications for Agriculture, in Advances in Agronomy, L.S. Donald, Editor., Academic Press. 2014, 124: 37-89. Acknowledgements: The support of Brown Coal Innovation Australia (BCIA) is gratefully acknowledged. KRL was a recipient of a BCIA research scholarship and an Australian Postgraduate Award scholarship provided by Monash University. TRC gratefully acknowledges the Australian Research Council for supporting his research through the Future Fellowship program (FT120100463).
Fig. 4. SDW response to different application rates of brown coal-derived HS. Dashed lines show quadratic fits to rates less than 50 mg kg-1 (short dash) and rates greater than 50 mg kg-1 (long-dash).
223
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Organic Matter in High Mountain Volcanic Soils J.A. González-Pérez(a)*, N. Rodríguez-Eugenio(b), F.J. González-Vila(a), C.D. Arbelo(b), A. Rodríguez-Rodríguez(b) (a) IRNAS-CSIC. Avda. Reina Mercedes, 10. Sevilla, E-41012 (Spain). (b) Univ. La Laguna, Avda. Astrofísico Fco. Sánchez s/n, La Laguna, Tenerife, E-38204 (Spain). * Corresponding author e-mail: [email protected] Keywords: Andosols, Analytical pyrolysis, Allophanes, Organo-mineral complexes. Abstract. Volcanic soils accumulate soil organic matter (SOM) usually through the formation of organo-mineral complexes. In terms of residence time, SOM quality depends on whether it is free or associated to the mineral fraction forming metal (non-allophanic andosols) or allophane complexes (allophanic andosols). This work deals with carbon sequestration mechanisms in volcanic ash soils from the Canary Islands. Physical and chemical soil properties were analysed and SOM studied by analytical pyrolysis (Py-GC/MS) in bulk and in two density fractions. Our findings points to different mechanisms for SOM protection 1) In allophanic soils is mainly associated to noncrystalline organo-mineral complexes, is rich in polysaccharides and N-compounds probably from chitin. 2) In nonallophanic soils a selective preservation mechanism is observed, SOM is formed of relatively unaltered plant components related to a protective effect of amorphous minerals favouring organo-metal complexes/coatings. ferrihydrite or halloysite. In addition two density soil fractions (δlight < 1.9 g ml-3 < δheavy) were separated using a tungsten salt (3Na2WO49WO3) solution as described in Rumpel et al. (2012). The molecular features of SOM were studied in detail by direct analytical pyrolysis (Py-GC/MS) at 500º C in bulk soil and the two density fractions. In addition and in search of molecular markers also different parts of the predominant plants and soil’s decaying litter were also analysed. The pyrolysis and chromatographic conditions used can be found in González-Pérez et al. (2012).
Introduction Soils formed on volcanic materials are rich in noncrystalline minerals as allophane, imogolite and other aluminium silicate clay minerals, as well as in sesquioxides (aluminium and iron oxides and hydroxides). These soils accumulate high quantities of soil organic matter (SOM) usually attributed to the formation of organic-mineral complexes. In acidic environments (pH < 5) the formation of nonallophanic (metal-humus complexes) soils predominate over allophanic soils which formation is favoured at higher pH conditions (pH > 5). In terms of residence time, SOM quality depends on whether it is free, no associated to the soil matrix, or is associated to the mineral fraction forming metal (non-allophanic andosols) or allophane complexes (allophanic andosols). Nonetheless, SOM protecting mechanisms that act in these soils remain uncertain, and it is no clear whether organic-mineral/metal complexes are the main protection mechanism in these soils (Hernández & Almendros, 2012 and references therein). The aim of this work is to enlighten which protection mechanisms are acting preferentially in high mountain volcanic soils from the Canary Islands.
Results and Discussion The study of non-crystalline minerals (ratio Alo + ½ Feo) in relation with the SOM fraction associated to organo-mineral complezes (Cp) allow us to diferenciate four soil types according to the andic properties: non-allophanic, allophanic, intermediate and mineral soils. All studied soils show low pH, and a relatively scarce SOM content, except nonallophanic soils (Fig. 1). Soil density fractionation shows that SOM is preferentially associated to the light fraction (C-LF). However this does not necessarily is a free or weakly associated to soil particles C pool, being best related with C in organic-mineral complexes (Cp) (Fig. 2). It is known that density fractionation does not discriminate well between light volcanic materials from free SOM, especially in incipient/young andosols rich in vitreous materials (vitric andosols). Direct analytical pyrolysis (Py-GC/MS) of both light (LF) and heavy (HF) soil fractions allow us to relate andic properties with SOM organic molecular features (Fig 3). In general the molecular features of C-LF is in line with a plant, fungal and general biogenic origin whereas the analysis of C-HF fraction provide more specific information about possible Csequestering mechanisms.
Experimental The samples studied were taken from 19 diagnostic A horizons, chosen to be representative of the different parent materials, vegetation and climatic conditions in the area (Teide National Park). Physical and chemical properties possibly controlling SOM accumulation were analysed in whole soils as described in Blakemore et al. (1981) and included the extraction of active metal forms (acid-oxalate extraction) Alo, Feo and Sio, and organometallic complexes (pyrophosphate extraction) Cp, Alp, Fep and Sip. This allows an estimation of the organic carbon complexaton forms present in the soil i.e. in the form of Al-humus, Fe-humus or associated to allophane,
224
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The study of the structural features of SOM in this diverse and almost pristine collection of high mountain ash soils allow the identification and confirms the existence of two different main mechanisms involved in SOM preservation and stabilization. Those mechanisms have been also appointed previously by Nierop et al. (2009), and that seems to be strongly influenced by the type of andosol: 1) In allophanic andosols (i.e. soils 44, 45, 111, 150) SOM is predominantly associated to noncrystalline minerals forming organo-mineral complexes. The structure of this OM as seen by PyGC/MS shows a high relative proportion of polysaccharide and protein derived N-compounds with a probable secondary (fungal) origin that may adsorb to the short range order minerals. This has been previously observed in andosols (Buurman et al., 2007; González-Pérez et al., 2007) and recently directly in allophane-type nanoparticles (CalabiFloody et al., 2011). 2) In non-allophanic andosols (i.e. soils 36, 18, 56, 130, 106) SOM binds mainly to metals forming organo-metal complexes (Shoji et al., 1993). In this situation a selective preservation of relatively unaltered plant components seems to prevail. The structure of this OM as seen by Py-GC/MS is dominated by aliphatic compounds (saturated and unsaturated hydrocarbons) retaining the plant signature as well as a clear lignin component with abundance of methoxyphenols. This SOM molecular assemblage may be related to a sequestration mechanisms implying a protective role of amorphous minerals with, either the formation of stable microaggregates where SOM is encapsulated over the long term, the formation of mineral coatings (Matus et al., 2008) or of complex tri-dimensional nets of alkyl material and metal colloids, that combined with toxic free cations in an acidic environment, may restrict microbial attack (Hernández et al., 2012).
Figure 1. Non-crystalline minerals (ratio Alo + ½ Feo) vs. organic carbon associated with organo-mineral complexes (Cp) of high mountain ash soils.
Figure 2. SOM pools in high mountain ash soils. C-LF: associated to the light fraction, C-HF: associated to the heavy fraction, Cp: associated to organo-mineral complexes.
REFERENCES
(1) Hernández, Z.; Almendros, G. Soil Biol. Biochem. 2012, 44, 130-142. (2) Blakemore, L.C.; Searle, P.L.; Daly, B.K. New Zealand Soil Bureau 1981. Sci. Rep. 80. (3) Rumpel, C.; Rodríguez-Rodríguez, A; GonzálezPérez, J.A.; Arbelo, C.; Chabbi, A.; Nunan, N.; GonzálezVila, F.J. Biol. Fertil. Soils 2012 48, 401-411. (4) González-Pérez, J.A., Chabbi, A.; de la Rosa Arranz, J.M.; Rumpel, C.; González-Vila, F.J. Org. Geochem. 2012. 53, 119-130. (5) Nierop, K.G.L.; Kaal, J.; Jansen, B.; Naafs, D.F.W. Geophysical. Res. Abst. 2009 11, EGU2009-1227. (6) Buurman, P.; Peterse, F.; Martin, G.A. Eur. J. Soil Sci. 2007 58, 1330-1347. (7) González-Pérez, J.A.; Arbelo, C.D.; González-Vila, F.J.; Rodríguez-Rodríguez, A.; Almendros, G.; Armas, C.M.; Polviilo, O. J. Anal. Appl. Pyrolysis 2007 80, 369-382. (8) Calabi-Floody, M.; Bendall, J.S., Jara, A.A.; Welland, M.E.; Theng, B.K.; Rumpel, C.; Mora, M.D.L.L. Geoderma 2011 161, 159-167. (9) Matus, F.; Garrido, E.; Sepúlveda, N.; Cárcamo, I.; Panichini, M.; Zagal, E. Geoderma 2008 148, 180-188.
Figure 3. Multivariate (PCA) analysis of SOM pools in high mountain ash soils. C-LF: up and C-HF: down. The analysis is based in the relative abundance of identified pyrolysis released compounds (Py-GC/MS).
225
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Chemical and Spectroscopic Characterization of Humic Substances Isolated from South-Bohemian Peat V. Enev(a) *, F. Novák(b), M. Klučáková(a) (a)
Centre for Materials Research, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic (b) Biology Centre AS CR v.v.i., Institute of soil Biology, Na sádkách 7, 370 05 České Budějovice, Czech Republic * Corresponding author e-mail: [email protected] Keywords: humic substances, UV/Vis, FTIR spectroscopy, fluorescence spectroscopy, absorption and fluorescence coefficients Abstract The aim of this work was study chemical composition and structure of different HS. Object of our study were two samples HS which were isolated from South-Bohemian peat from the quarry Branná near Třeboň, Czech Republic. Isolation of HS was performed according to the procedure recommended by the IHSS. All samples of HS were characterized by elemental analysis (EA), ultraviolet-visible spectroscopy (UV/Vis), infrared spectroscopy (FTIR) and steady-state fluorescence spectroscopy. Absorption coefficients (E ET/E Bz, E250/E365 and E 465/E665) of HS were calculated from the absorbance values. Fluorescence coefficients (Milori index and HIX) of HS were calculated from the area of the emission spectra. Biological/autochthonous index (BIX) and fluorescence index (FI) of HS were calculated from the ratio of different emission intensities. Excitation spectra were recorded over the range of 300–500 nm at a fixed emission wavelength of 520 nm. Total luminescence spectra (TLS) were obtained in the form of excitation/emission matrix (EEM) by scanning the wavelength emission over the range of 300– 600 nm, also the excitation wavelength was in 5 nm steps from 240 to 550 nm. The following fluorescence coefficients were obtained: [a] fluorescence index (FI) (4); [b] Milori index (5); [c] biological/autochthonous index (BIX) (6); [d] Zsolnay index (HIX) (7). The fluorescence intensity (IF) values (in CPS/MicroAmp.) of samples were corrected using method of Lakowicz (8). The correction method of Lakowicz uses:
Introduction Humic substances (HS) are a major component of natural organic matter (NOM) and are the dominant products of plant and animal degradation by microbial activity. HS, the main organic constituents of soil and sediments are widely distributed over the earth’s surface, occurring in almost all terrestrial and aquatic environments. Humic substances are complex mixtures of high to low molecular weight species, so they are polydisperse systems with a specific distribution of molecular weights. The object of our study was to investigate the chemical properties and humification degree of HS. For this purpose, elemental analysis (EA), UV/Vis spectroscopy, FTIR spectroscopy and steady-state fluorescence spectroscopy were used (1, 3).
(1) where F corr and F obs are the corrected and uncorrected fluorescence intensities and Aex and Aem are the absorbance values at the current excitation and emission wavelengths.
Experimental The objects of our study were two different samples of HS. HA and FA were isolated from SouthBohemian peat from the quarry Branná near Třeboň, Czech Republic, by conventional procedure recommended by the IHSS. Absorption coefficients (E ET/EBz, E250/E 365 and E465/E 665) of HS were calculated from the absorbance of HS in UV/Vis spectral range (2). The FTIR spectra of HS were recorded over the range of 4000–400 cm−1. A Nicolet iS50 FTIR spectrophotometer operating with a peak resolution of 4 cm−1, and 128 scans were performed on each acquisition. Fluorescence spectra were recorded in aqueous solutions of 10 mg·L−1 HS after overnight equilibration at room temperature, using FluoroLog luminescence spectrophotometer. The pH-value of the samples was adjusted to seven using a standard phosphate buffer. Emission spectra were recorded over the range of 380–600 nm at a constant excitation wavelength of 360 nm.
Results and Discussion Elemental analysis and UV/Vis spectroscopy: The values of the different absorption indexes calculated from the UV/Vis spectra of peat HS and elemental composition are presented in [Table 1, 2]. The higher value of E ET/E Bz ratio of HA may be indicative of the presence of O-containing functional groups (hydroxyl, carbonyl, carboxyl, ester and ether groups). Thus, lower E ET/E Bz ratio of FA will be associated with scarce substitution on the aromatic ring or with the substitution with aliphatic functional groups. The lower value of E 2/E 3 ratio of peat HA may be indicative of the presence of structures with higher molecular weight, aromaticity and humification degree. The lower value of humification index for HA confirmed the presence of HS with higher molecular
226
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
weight and humification degree. The higher value of E4/E 6 ratio of FA may be indicative of the presence simple aromatic structures with higher degree of substitution with oxygen containing functional groups.
network, and other unsaturated bond systems capable of a great degree of conjugation in large molecular size and extensively humified “macromolecules”. The fluorescence EEM spectrum of peat FA were located by two fluorescence maxima at an excitation/emission wavelength pair of 250/430 nm (fulvic-like) and 305/430 nm (humic-like) which are typical for terrestrial origin. On the contrary, the prevalence of fluorescence bands and peaks with high relative intensity at short wavelengths, such as those measured for the peaks of FA, is associated with the presence of simple structural components of wide molecular heterogeneity and small molecular weight, small degree of aromatic condensation, small level of conjugated fluorophores, and small humification degree.
Table 1. Elemental composition of HS (weight %) sample HA FA
C 54.10 52.53
H 4.39 4.26
N 2.18 1.43
O 38.85 41.00
S 0.48 0.79
Table 2. Absorption indexes (EET/EBz, E2/E3 and E4/E6) of HS sample HA FA
EET/EBz 0.53 0.51
E2/E3 3.06 4.14
E4/E 8.50 11.00
FTIR spectroscopy: All spectra feature common and distinctive absorption bands, with some differences in their relative intensity. The main characteristics of these spectra are the following: about 3400–3300 cm−1 (O– H stretching and, secondarily, N–H stretching of various functional groups); about 2935–2925 cm−1 (asymmetric C–H stretching or of CH2 groups); about 1720–1710 cm−1 (C=O stretching of COOH), whose higher relative intensity was determined for FA; 1620– 1600 cm−1 (aromatic C=C skeletal vibrations, C=O of strongly H-bonded conjugated ketones, whose higher intensity was determined for HA; about ≈1510 cm−1 (preferentially ascribed to simple aromatic C=C vibrations, N–H deformation and, C=N stretching of amides); about 1420 cm−1 (O–H deformation and C–O stretching of phenolic OH); about ≈1380 cm−1 (C–H deformation of CH2 and CH3 groups, and/or asymmetric stretching of COO− groups); about 1270– 1260 cm−1 (C=O stretching of aryl esters), whose higher intensity was detected for HA; about 1220 cm−1 (C–O stretching of aryl ethers and phenols); 1130– 1080 cm−1 (C–O stretching of secondary alcohols and/or ethers); and, finally, about 1045–1041 cm−1 (C– O stretching of polysaccharides or polysaccharide-like substances, and/or Si–O of silicate impurities).
Table 3. Position of excitation-emission wavelength pair of the main peaks in the EEM spectra and values of fluorescence intensity these fluorescence peaks of HS sample HA FA
1st maximum (peak A) EEWP IF (nm) (CPS/M.A.) 270/490 0.86×10 250/430 2.47×10
2nd maximum (peak C) EEWP IF (nm) (CPS/M.A.) 455/510 0.16×107 305/430 1.44×107
The values of the different indexes calculated from the emission spectra (FI, BIX, Milori index and HIX) are presented in [Table 4]. The values of FI and BIX of peat HS are typical for terrestrial origin and autochthonous sources. The higher values of Milori index and HIX of HA may be indicative of greater humification degree. Also, values of FI, BIX, Milori index and HIX of HS are in agreement with previous results. Table 4. Fluorescence coefficients (FI, BIX, Milori index and index HIX) of HS
Steady-state fluorescence spectroscopy: Emission fluorescence spectrum of HA possess unique broad band, with a maximum at 488 nm and a shoulder at 458 nm. The emission spectrum of FA featured one intense peak at 467 nm. The long wavelength of the main fluorescence peak of HA indicate the presence of condensed aromatic ring and other unsaturated bond systems, a high degree of conjugation, and electron-withdrawing groups such carbonyl and carboxyl groups. The short wavelength of the fluorescence peak of sample FA suggest the presence of simple structural components of wide molecular heterogeneity and small molecular size, low degree of aromatic polycondensation, low level of conjugated fluorophores, and low humification degree. The values of the fluorescence intensity and excitation-emission wavelength pair of the main peaks in the EEM spectra of HS are presented in [Table 3]. The fluorescence EEM spectrum of HA was characterized by two unique fluorophores centered at an excitation/emission wavelength pair (EEWP) of 270/490 nm (peak A) and ≈445/510 nm (peak C). The long wavelength and less fluorescence intensity of the major peak of HA may be ascribed to the presence of an extended, linearly-condensed aromatic ring
sample
FI
BIX
HA FA
0.73 1.01
0.44 0.40
Milori 2.98×10 2.11×10
HIX 11.59 7.52
References
(1) c1994. Humus chemistry: genesis, composition, reactions. 2nd Ed. New York: John Wiley and Sons. (2) Fuentes, M.; González-Gaitano, G.; García-Mina, J.; Tranter, G.; Tranter, G.; Gonçalves, M. Org. Geochem. 2006, 37, 1949–1959. (3) Valencia, S.; Marín, J.; Restrepo, G.; Frimmel, F. H. Sci. of the Tot. Environ. 2013, 442, 207–214. (4) McKnight, D.; Boyer, E.; Westerhoff, P.; Doran, P.; Kulbe, T.; Andersen, D. Lim. and Oceano. 2001, 46, 38– 48 (5) Milori, D.; Martin-Neto, L.; Bayer, C.; Mielniczuk, J.; Vagnato, V. Soil Sci. 2002, 167, 739–749. (6) Huguet, A.; Vacher, L.; Relexans, S.; Saubusse, S.; Froidefond, J. M.; Parlanti, E. Org. Geochem. 2009, 40, 706–719. (7) Zsolnay, Á.; Baigar, E.; Jimenez, M.; Steinweg, B.; Saccomandi, F. Chemosphere. 1999, 38, 45–50. (8) Lakowicz, J. 2006. Principles of fluorescence spectroscopy. 3rd ed. New York: Springer.
Acknowledgments: Materials Research Centre at FCH BUTSustainability and Development, REG LO1211, with financial support from National Programme for Sustainability I (Ministry of Education, Youth and Sports)
227
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Aggregation of Soil Humic Acid Fractions with Respect to Their Functional Group Contents U. Jovanović (a) *, Dj. Čokeša (a), A. Savić(a), M. Marković (a), S. Radmanović(b) (a)
University of Belgrade - Vinča Institute of Nuclear Sciences, Chemical Dynamics Laboratory, P.O. Box 522, 11001 Belgrade, Serbia (b) University of Belgrade - Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia * Corresponding authors e-mail: [email protected] Keywords: Soil Humic Acid, Aggregation, Functional Groups, Fractionation Abstr act Soil humic acid (HA) was fractionated with respect to its functional group contents and the aggregation of unfractionated HA and obtained fractions were studied. The fractionation procedure applied resulted in three fractions having different functional group contents. The fractions revealed pronounced differences in aggregate sizes. The conclusion is: the higher the functional group content, the less pronounced the HA aggregation process. again with deionized water and as HA was not completely eluted, 10% NaCl solution was pumped through the column eluting fraction 3 (F3). To precipitate all the HA fractions obtained, pH was adjusted to <1. Precipitates were filtrated and washed with 0.1M HCl until Na+ concentration in filtrates was <0.1 ppm (determined by AES), and finaly washed off from filters using deionized water. Samples were dried at 37 0C. After negative reaction to Cl- was confirmed by AgNO3 solution, HA suspensions (0.2 g/L) were prepared using 0.1M NaCl. Acid-base titrations of HA functional groups were performed according to (2). Functional group content was not determined in F2 due to small amount of sample obtained. To perform size measurements, the HA suspensions were first adjusted to pH>8, then acidified to pH 3 and equilibrated for 6 days. The size measurements were performed by Dynamic Light Scattering (DLS) using a Zeta–sizer Nano ZS with 633 nm He-Ne laser (Malvern, UK).
Introduction Humic acids (HAs), similar to other fractions of humic substances (HSs), have a large number of reactive functional groups influencing the HA aggregation in solutions. Regardless of the origin of humic acid (aqueous or soil), this aggregation process is dependent on environmental conditions and strongly influences the mobility of soluble ionic and molecular pollutants. There are a lot of literature data on functional group contents of various HAs (1, 2). In some HAs, functional group contents were determined after fractionation (3). But the relation between the HA aggregation and the functional group content in various HA fractions, although very important, was not studied until now. Experimental Unfractionated HA (UF) was extracted from Rendzic Leptosol (Calcaric) (according to WRB-2006 (IUSS Working Group WRB, 2006)), which is the mostly widespread soil in Serbia. RLHA was obtained from the soil sample originating from Stari Slankamen, Serbia, developed on sandy marl, at 187 m.a.s.l., area under grassland. The soil sample taken from the A horizon (0-20 cm depth) had the characteristics as follows: grayish yellow brown (10YR5/2) color (dry soil), sandy loam texture, 14,78% CaCO3, pH in water 7.69, 4.31% total organic C, 33.64% humic acids, 32.24% fulvic acids, and 34.12% humin. RLHA sample was isolated using a modified IHSS method (HA gel was dried at 35C, powdered, and sieved using a 0.05 mm sieve). RLHA elemental composition was determined to be as follows: C 52.04%, H 5.18%, O 37.96%, N 4.82%, C/N 10.80, H:C 0.100, and ash 1.63%. HA was fractionated as follows (1): 0.1% HA water suspension was initially adjusted to pH 7 and pumped through a column packed with secondary amine weak base resin (Amberlyst® A21 free base) at a rate of 4 ml/min. The effluent collected is termed as fraction 1 (F1). After rinsing with deionized water, the resin was eluted with 1M NaOH solution until discoloration and the fraction 2 (F2) obtained. The resin was washed
Results and Discussion Results are presented in Table 1 and Figure 1. Fractionation procedure applied resulted in three fractions. Carboxyl and phenolic content of unfractionated HA [Table 1] are in accordance with the values of IHSS soil HA samples (2). Both carboxyl and phenolic contents are higher in F3 compared to UF, while functional group contens in F1 are lower than in UF [Table 1]. Particle size distribution in the HA fractions obtained is obviously different in comparison with UF [Figure 1]. Both UF and F1 contain small (diameter approx. 60 nm), medium (diameter approx. 200-500 nm) and large (diameter approx. 2500-5500 nm) particles. In UF the number of small particles is higher than in F1 containing mostly of medium and large particles. F2 is almost free of small and medium particles, but with two groups of large particles. F3 contains only very small particles (diameter approx. 12 nm). From the presented results the following can be concluded: the higher the functional group content, the
228
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
less pronounced the HA aggregation process. Namely, the HA sample with the highest content of functional groups (F3) revealed the smallest particle size. On the contrary, in sample F1, having smaller number of functional groups, the highest content of large particles was determined. Table 1. Functional group contents of HA fractions
sample UF F1 F3
carboxyl content (meq/gC) 7,16 5,35 8.82
phenolic content (meq/gC) 2,66 1,60 3,01
25
Volume (%)
20 15 10 5 0 1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
25
Volume (%)
20 15 10 5 0 25
Volume (%)
20 15 10 5 0 25
Volume (%)
20 15 10 5 0 Size (d.nm)
Figur e 1. Particle size for a) unfractionated HA, b) F1, c) F2, d) F3.
REFERENCES
(1) Ritchie, J.D. and Perdue, E.M. Geochimica et Cosmochimica Acta. 2003, 67, 85-96. (2) Janoš, P.; Kříženecká, S.; Madronová, L. Reactive & Functional Polymers 2008, 68, 242-247. (3) Lin, C.-F.; Liu, S.-H; Hao, O. Wat. Res. 2001, 35, 2395-2402.
229
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Study of Copper Binding By Humic Acids from an Acidic Lake Using Fluorescence Spectroscopy JM. Cabrera (a) *, M. Diaz(a), FS. García Einschlag(b)
Address a: INIBIOMA, Quintral 1250, San Carlos de Bariloche (8400), Río Negro, Argentina. Address b: INIFTA, Calle 64 Diag. 113, La Plata (1900), Buenos Aires, Argentina. * Corresponding author e-mail: [email protected] (a)
(b)
Keywords: Humic Acids – Acidic Lake – Copper Complexation Abstract Apparent binding constants (KS) and site concentration (LC) were studied for humic acid (HA) – copper complexes using fluorescence spectroscopy. Humics were extracted from the sediments of Lake Caviahue (pH=2,8). Since downstream, water is used for human consumption, understanding heavy metal sedimentary chemistry is of key importance. Previous studies showed high affinity of HA for Cu +2. To study this system, experiments were conducted at pH=3, within the sedimentary metal concentration range (μM to mM). Matrix analysis showed three fluorescent components, although component C2 was found to be of minor importance. Non-linear regression was used to calculate the above mentioned parameters associated to each component. Excitation and emission maxima (λexc/λem) were: C1=465/505 nm and C3=360/445 nm. KS values (~2,0 ± 0,8 104 M-1) did not differ significantly between components, whereas LC values ranged from 4,8 ± 0,5 10-5 M for C1 to 7,7 ± 0,8 10-5 M for C3.
about 1–6 10-3 M. Since downstream water is used for human consumption, it is important to assess the affinity of humic materials for transition metals. In the present work we used copper as a model cation, in order to foresee changes in water column concentrations.
Introduction Humic acid (HA) metal complexing capacity by different fluorophoric components, like quinones, catechols, phtalates, phenol, amines and salicilates can be studied by excitation-emission fluorescence matrices. Given that fluorescence quenching (or enhancing) may arise when metals are bound to the fluorophore (1), apparent binding constants can be calculated (2). Lake Caviahue is located 1600 m.a.s.l. in the Copahue-Caviahue Provintial Park (37° 53' S; 71° 02' W), Neuquén, Argentina. It has unique characteristics, like very low pH (2,2 – 3), high electric conductivity (1600 μS cm–1), high concentractions of S, Fe, Al, Cl, P, and trace elements among others. It has two mayor influxes, the Río Dulce (pH=6) and the Río Agrio (RA) (pH=1,9). The RA is born on the Copahue Volcano caldera with a pH of 0,8, a temperature of 82ºC, 2 g L-1 of dissolved iron and a conductivity of 560 mS cm–1 (3). Caviahue is a naturally glaciated occurring lake with an average depth of 60 mts and a total volume of 0,47 km3. Sediments, which are >85% andesitic, have very high organic matter content (5– 10%), high P concentrations (1 mg g -1 as NaOH extractable P) and heavy metals like copper (up to 60 μg g-1 of sediment). As the electric potential is –120 mV (in average) and pH is between 3 and 4, the metal is found as Cu+2, associated with the pyrite and the organic matter fractions (Cabrera, unpublished data). Iron should be oversaturated (10-1–10-2 M) for pyrite to form under these conditions, so humics become of key importance as Fe+2 concentrations in pore water are
Experimental HA were extracted from the lake according to the protocols of IHSS. EEM were taken at 20°C, with 100 ppm [HA]. [Cu +2] ranged from 10 μM to 1,2 mM. pH was adjusted to 3 (to HA and metal stock) and ionic strength to 10-3 with HCl and NaOH 0,1M. EEM were decomposed with the KINESIM software which combines regression analysis with matrix methods for analysis of bi-linear data. For a given EEM, the number of contributing factors (fluorescent components, (FC)), in the experimental matrices, was estimated by Singular Value Decomposition. Then, the MCR-ALS method was applied to retrieve both the excitation and emission spectra of each component. For the comparison between EEM obtained with different Cu2+ concentrations the matrix augmentation strategy was used. Results and Discussion Singular value decomposition showed that the EEM obtained in the absence of copper could be deconvoluted into three fluorescent factors or FC (fig. 1C, 1D and 1E). Despite the number of fluorophoric species may be higher, by adding up the contributions
230
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
of three linearly independent factors, the original fluorescence matrix could be reproduced within the experimental error (figs. 1A, 1B and 1F).
Ni+2, Zn+2 and Cd+2 (8). Emis s io n S pe c tra o f C1
Emis s io n S pe ctra o f C3 60000
Re lative Emis
Re lative Emis
60000
C1-Fre e C1-Bo und
45000
30000
15000
C3-Fre e C3-Bo und
45000
30000
15000
0
0 30 0
35 0
4 00
4 50
5 00
55 0
30 0
60 0
350
40 0
Co nc entratio n Pro file s of C1
50 0
55 0
60 0
Co nc entratio n Pro file s of C3 1.2
Re lative Co nc
1.2
Re lative Co nc
4 50
WL / Emis
WL / Emis
1.0 0.8 0.6
C1-Free C1-Bo un d
0.4
1.0 0.8 0.6
C1-Free C1-Bo un d
0.4
0.2
0.2
0.0
0.0
-0.2
-0 .2 0
2 00
4 00
6 00
80 0
10 0 0
Cu / M
12 0 0
1 40 0
0
2 00
4 00
60 0
80 0
1 00 0
1200
14 0 0
Cu / M
Figure 2. Component C1 and C3 Component 1 fluor -
escence behaviour for the emission maximum and speciation scheme.
Conclusions Caviahues sedimentary HA showed high affinity for Cu+2. Pyrite is present as the only conspicuous metallic sulfide and it is far from its most favoured conditions. In addition, pore water concentration of iron and other metals is 103 times higher than that found in slightly acidic or alkaline environments. Therefore we suggest that humics play an important rol regarding the copper content in this anoxic sediment, and probably other metals also capable to interact with organic ligands. The copper content of pyrite is also not negligible, should changes in REDOX contitions occur, (e.g. by mixing due to seismic activity) the solubilized copper will be fully complexed by HA given the values found KS and the fact that HAs are in excess when compared to extractable metal concontent (<1% of the sediment).
Figure 1. A: EEFM of native HA. B: Sum of the modeled
components #1+2+3. C: Calculated residues. D: C1. E: C2. F: C3.
It should be taken into account that the decomposition does not simulate non linear dispersive resonance phenomena such as 1st order Raman or 2nd order Rayleigh. Moreover, component 2 (C2) is the least important of the three, thus only C1 and C3 were used for K S estimation. The excitation and emission maxima for these components are as follows (λexc/λem) C1=465/505 nm y C3=360/445 nm. For the matrices recorded in the presence of Cu 2+, it was observed that emission maxima associated to C1 and C3 shifted gradually towards higher energy wavelengths as copper concentration was increased (table 1, at higher [Cu +2]), same results were reported by (4) and (5) for other metals.
REFERENCES
(1) Philpot, W. D., & Vodacek, A. Rem. Sens. Environ. 1989, 29, 51–65.
(2) Ryan, D. K., & Weber, J. H. Anal. Chem., 1982, 54(6), 986–990.
Table 1: fitting values and emission maxima. FC Ks (M-1) CL (M) C1
(2,4 ± 0,8) 104
(4,8 ± 0,7) 10–5
C3
(1,6 ± 0,5) 104
(7,7 ± 0,8) 10–5
λem HA (nm)
λem HA – Cu+2 (nm)
505
480
C1
(3) Pedrozo, F. L., Temporetti, P. F., Beamud, G., & Diaz, M. M. J Volcanol. Geoth. Res. 2008, 178(2), 205–212.
(4) Senesi, N. In: Adriano Domy, C. (Ed.), CRC Press, Boca Raton, USA, 1992, pp. 425–491. (5) Provenzano, M. R., D'Orazio, V., Jerzykiewicz, M., & Senesi, N. Chemosphere. 2004, 55(6), 885-892.
445 415 C3 Speciation schemes (fig. 2) and table 1 showed similar KS for the two kind of binding sites. Although KS for C1 almost doubles C3, difference is within the standard deviation. In contrast, for C3, ligand concentration (CL) was ~62% higher than for C1. Values obtained here are similar to those found by other authors (e.g. 2 10-5 – 7,9 10-5). In addition, conditional constants were in agreement with values published by (6) and (7) for Cu+2 ions and also similar to those reported for other transition metals such as
(6) Luster, J., Lloyd, T., Sposito, G., & Fry, I. V. Environ. Sci. technol. 1996, 30, 1565–1574. (7) Plaza, C., Brunetti, G., Senesi, N., & Polo, A. Anal bioanal chem. 2006, 386(7–8), 2133–2140.
(8) Terbouche, A., Ramdane–Terbouche, C. A., Hauchard, D., & Djebbar, S. J. Environ. Sci. 2011, 23, 1095– 1103. Acknowledgments: This study received financial support from ANPCyT (2008-1105), UNComahue (Program 04/B166) and CONICET (PIP 11220090100013). I want to especially thank IHSS for the travel award.
231
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characterization of tetramethylammonium ion binding to fulvic acid molecules by the use of 1H DOSY method. Hiroki KODAMA(a), Tohru MIYAJIMA(b), Kotaro NAKATA(c)
(a) Analytical Research Center for Experimental Science, Saga University, Japan, (b) Faculty of Science and Engineering, Saga University, Japan, (c) Nuclear Fuel Cycle Backend Research Center, Abiko Research Laboratory, Central Research Institute of Electric Power Industry, Japan * Corresponding author e-mail: [email protected] Keywords: DOSY,
hydrophobicity
tetraalkylammonium, fulvic acid, cationic probe, polyelectrolyte,
Abstract Binding of cationic organic molecule, tetramethylammonium ions(TMA+) to dissociated fulvic acid molecules(FA-) of various origins has benn evaluated by using 1H DOSY method. Chemical shift() together with diffusion coefficient (D) value have been determined as a function of , the degree of neutralization of FA molecules. Specific interaction between TMA+ and moieties of FA- molecules has been revealed, which is dependent of the chemical nature of the FA- samples examined. The results were compared with a PAA-/TMA+ system, where TMA+ ions bind to PAA- mainly due to territorial binding mode. 1H DOSY method can characterize the counterion binding to FA molecules. dm-3 protonated samples dropwisely. Samples of polyacrylic acid (PAA) Dand fulvic acid; JHSS standard(DFA), Inogashira fulvic acid; JHSS standard (IFA) as well as Kuta fulvic acid (KFA) have been examined. NMR measurements have been performed with 5mm sample tubes, non-spinning on Varian VNMRJ 400 at ca. 23℃. Diffusion encoding was carried out with a BPPSTE method(Bopolar pulse stimulated echo).
Introduction In spite of the importance of binding nature of cationic species to fulvic acid(FA) molecule in their migration processes in various environments, qualification and quantification of the FA binding still remain unclarified. The FA binding characterization is complicated because several fundamental forces, such as electrostatic, hydrophobic-hydrophilic, as well as specific interaction such as -interaction and metalcoordination, are known to operate concurrently in aqueous solutions. It is well known that polyelectrolytic nature is common to negatively charged FA molecules, though the magnitude is not so pronounced as polyacrylic acid(PAA) of highly dissociated state(1,2). In the present work, the binding property of tetramethylammonium ions(TMA+) to weakly acidic polyanions, such as FA- and PAA- has been investigated by a 1H DOSY method(3,4); the binding characteristics have been compared in order to clarify the nature of FA binding. The chemical shift change as well as the variation in the diffusion coefficient upon neutralization of acidic forms of the polyions with TMA+OH- have been monitored. Two representative binding modes, territorial binding and site binding, are considered operative concurrently. If only territorial binding due to polyelectrolytic nature is operated then the variation in the diffusion coefficients(D) of TMA+ ions is expected, though the chemical shift() change of TMA+ molecules cannot be anticipated. On the contrary, if site binding of TMA+ ions to the molecule backbones is operative then the change in both D and is anticipated.
Results and discussion In Fig. 1 is shown a representative 1H DOSY spectrum obtained with 5000ppm Inogashira FA sample dissolved in D2O neutralized with tetramethylammonium hydroxide(TMA+OH-). The signal appearing at 3 ppm() and in the range of 8 to 10x10-10m2s-1(D) can be assigned to the carbon to nitrogen of TMA+ ions. The and D values obtained are plotted against in Fig. 2 and 3, respectively. In order to examine the ion binding characteristics, the TMA+ binding systems of different FAsamples(Dando, Inogashira, and Kuta) as well as PAAhave been measured. The D value of free TMA+ itself was 10.00; when TMA+OH- was added to H+PAA- solution the D value of TMA+ ions drastically decreased with indicating electrostatic binding of TMA+ to a PAA- domain, whereas the corresponding value was maintained at about 3.02 ppm, being equal to the value of free TMA+ ions. These results show that the biding mode of TMA+ to PAA- molecules is just due to a terrestrial mode, which has been reported earlier by Lindman (5). On the contrary, the binding mode of FA- molecules to TMA+ is much different from the hydrophilic PAAmolecules. The chemical shifts remained constant at about 3.17ppm, which is quite different from the free TMA+ ions (3.02 ppm). Also, the D values remained
Experimental 1H DOSY experiments were carried out by adding aliquot of TMA+OH- solutions to 50x10-3 monomol
232
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
(4) A. J. Simpson, ‘Determining the molecular weight, aggregation, structures and interactions with soil organic matter using diffusion ordered diffusion ordered NMR spectroscpy’, Magn. Reson. Chem., 40,572-582 (2002) (5) P. Stilbs, B. Lindman, ‘FT NMR Self-Diffusion for the study of counterion binding in polyelectrolyte solutions.’, J. Magn. Reson., 48, 132-137 (1982)
almost constant (6.90) in whole range investigated. These phenomena indicate that TMA+ ions are bound to hydrophobic FA- molecules mainly in a site-binding mode though the electrostatic i. e., non-specific binding mode(terrestrial binding) operates at the same time. When the electrostatic potential formed around PAAmolecule is increased so strong at , the D value of TMA+ decreased to 5.5, on the contrary, the D value remains constant at 7 for all FA- samples, which indicates the electrostatic potential formed around the FA molecules is much weaker than PAA- molecules of fully dissociated state. Small differences observed among the FA- samples(Figs. 2 and 3) may correspond to the differences in the chemical structures, mainly hydrophilic-hydrophobic and character of the molecules. Since it has been shown by the present work that monitoring the chemical shifts and the diffusion coefficients of countercations of FA- molecules give new and straightforward insights into ion binding to humic substances molecules, systematic binding study of series of tetraalkylammonium ions to various FAsamples are under investigation in our laboratory. Not only weakly acidic polyions, such as PAA- or PMA-, but also strongly acidic polyions, such as PVS- and PSSare taken as examples of polytions and are referenced to FA- samples of various origins.
Acknowledgments:
This study was done under contracts awarded from METI (Min- istry of Economy, Trade and Industry).
REFERENCES (1) G.S. Manning, J. Chem. Soc., 51, 924 (1969) (2) T. Miyajima, M. Mori, “Complexation equilibria of humic substances in aqueous solution”, Bunseki kagaku, 45, 5, 369-399 (1996) (3) K. F. Morris, B. J. Cutak, A. M. Dixon, C. K. Larive, ‘Analysis of diffusion coefficient distributions in humic acid and fulvic acid by means of diffusion ordered NMR spectroscpy’, Anal. Chem., 71, 53155321 (1999)
233
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Structural characterisation of the aromatic moieties of HS by novel NMR methodology N. G. A. Bell(a), J.W.T. Blackburn(a), A. Michalchuk(a), L. Murray(a), M. C. Graham(b), D. Uhrín(a) * (a) EastChem School of Chemistry, University of Edinburgh, Scotland, UK (b) School of Geosciences, University of Edinburgh, Scotland, UK * Corresponding author e-mail: [email protected]
Keywords: Nuclear Magnetic Resonance, NMR, 13C methylation, peat fulvic acid. Abstract Humic substances - chromatographically ‘inseparable’ mixtures of thousands of compounds - yield very complex NMR spectra. This prevents the use of standard NMR methods from elucidating structural features of individual compounds. To achieve this, the complexity of NMR spectra needs to be reduced through spectroscopic ‘separation’. Tagging of HS molecules with isotopically labelled nuclei provides the means towards this goal. Once in place, the tags serve as filters, reducing the number of observed signals and at the same time, as spies, report on their immediate chemical neighbourhood. We illustrate how n-dimensional NMR experiments in combination with isotope labelling can provide structural information. The methodology developed so far using a model mixture (Bell, N.G.A et al. Chem. Commun. 2014, 50, 1694-1697) aims at characterising aromatic HS moieties carrying OH and COOH groups. Here we show, for the first time, the initial results obtained on a HS sample, Scottish peat fulvic acid. information about the nature of their COOH and OH groups (4) Herein we present the first NMR experiments designed specifically for the structural elucidation of methylated HS. We demonstrate their usage on a model mixture as well a sample of a Scottish peat fulvic acid (FA). . Experimental A mixture of 2-hydroxybenzoic acid, 3hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid and 4hydroxybenzioc acid (all 0.10 mmol) was methylated using a procedure outlined in (5). For NMR experiments the nine methylated compounds (~1.4 mM each) in 550 μl of CDCl3 were prepared in a sealed NMR sample tube. The FA sample was prepared according the IHSS protocol from a peat sample taken from a lowland raised bog located at Red Moss in Balerno, Scotland. A sample (61 mg) was methylated using a modified version of the procedure outline in (5). For NMR investigations the methylated FA (26.5 mg) was dissolved in 550 μl of CDCl3 and sealed in a NMR sample tube. The NMR spectra were acquired on an 800 MHz Avance III (Bruker) NMR spectrometer equipped with a z-gradient triple-resonance TCI cryoprobe. The details regarding the 3D INEPT-INADEQUATEHSQC experiment are given in (5). The 3D HMQCHMBC spectrum was acquired using t1, t2 and t3 acquisition times of 7.9, 8.7 and 240 ms, respectively. Spectral widths of 80, 16 and 8 ppm were used. Four scans were acquired into each of 256 F 1, 56 F 2 and 3072 F3 complex data points. The relaxation time was 1.3 s resulting in the total experimental times of 1 day and 5 hours. The 1JCH (145 Hz) and 3JCCH3 (6 Hz) Hz were used to calculate the evolution delays.
Introduction Humic substances (HS) - one of the most complex mixtures of organic compounds on Earth, contain compounds of different sizes and structural characteristics. These mixtures pose a great challenge to any analytical technique. It is therefore no surprise that despite their obvious importance in many biogeochemical processes, the composition of HS has only been characterised in terms of molecular types and the occurrence of major functional groups. However, in order to understand HS functional roles in the environment we need to have a better understanding of how these functional groups decorate the carbon skeletons of HS compounds. The most promising tools for this task are high resolution NMR and mass spectrometry (MS) (1). Recent advances in these techniques have led to their increased applications in characterisation of HS. E.g. the use of FT-ICR-MS has allowed the molecular formula determination of thousands of ionisable HS compounds (2), or even the structural elucidation of a handful of compounds using MS-MS experiments (3). On other hand, NMR is somewhat lagging behind when it comes to characterising individual molecules. Standard NMR techniques cannot easily deal with the sheer number of compounds making up HS. Thus some form of spectroscopic ‘separation’ has to be done should we stand a chance of obtaining at least partial structural information. One way of achieving this is through chemical modification of HS. Introducing an NMR spy, which removes the vast majority of resonances and focuses only on its surroundings, is a very promising approach. The methodology presented here aims at characterising aromatic moieties of HS carrying OH and COOH groups and relies on introducing 13Cenriched -O13CH3 and -COO13CH3 groups into HS. HS have been methylated in the past and the inspection of -O13CH3 resonances yielded some rudimentary
234
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
chemical shift indicated by a dashed line in Fig. 3c. This plane indicates the spectral simplification achievable from this experiment.
Results and Discussion Testing the methodology on a model mixture. Using a model mixture of 9 13C methylated compounds a set of NMR experiments was designed utilising spin-spin interactions (Fig. 1) involving 13CH3 groups.
a)
c)
b)
d)
Figure 1. Spin-spin interactions of 13C methyl groups available to probe the surrounding chemical environment.
By directing the polarisation transfer through the 13CH3 groups simplified spectra are obtained showing only the resonances of the nuclei in close vicinity of the methyl spy. This methodology is illustrated using a 3D INEPT-INADEQUATE-HSQC experiment (5). This experiment provides chemical shifts of the methyl 1H and 13C atoms, the quaternary aromatic or carboxylic acid carbons as well as 1H and 13C chemical shifts of the atoms in the ortho position to the methoxy groups as highlighted in Fig. 2 on a model mixture of 9 compounds.
Figure 3. 1H,13C correlated spectra of methylated FA (a) 2D 1 H, 13C HSQC showing carboxylic esters, aromatic methoxy, aliphatic methoxy and strained methoxy groups; (b) 2D 1H, 13 C HMBC showing correlations of protons from aromatic methoxy, aromatic carboxylic ester and aliphatic carboxylic ester groups; (c) A 2D HMQC projection of the 3D HMQCHMBC; (c) A 2D HMBC plane of the 3D HMQC-HMBC taken at the 13CH3O chemical shift indicated by a dashed line in Figure 3c.
The two examples presented here illustrate how novel multidimensional NMR experiments can be used to obtain chemical shifts of nuclei in the vicinity of 13C enriched Me groups. These chemical shifts are analysed and used to propose structural fragments of aromatic moieties of HS. The power of this approach lies in the fact that chemical shifts of multiple nuclei from the same molecule can be identified. This is not possible using standard NMR techniques applied to complex mixtures such as HS.
Figure 2. The chemical shifts of the nuclei highlighted, provided by the 3D INEPT-INADEQUATE-HSQC experiment.
REFERENCES
The chemical shifts of the nuclei highlighted in Fig. 2 permitted the unambiguous structural determination of the 9 aromatic compounds.
(1) Hertkorn, N.; Ruechar, C.; Meringer, M.; Gugisch, R.; Frommberger, M.; Perdue, E.M.; Witt, M.; SchmittKopplin, P. Anal. Bioanal. Chem. 2007, 389, 1311-1327. (2) Stenson, A.C.; Marshall, A.G.; Cooper, W.T. Anal Chem. 2003, 75, 1275-1284 (3) Witt, M.; Fuchsar, J.; Koch, B.P. Anal. Chem. 2009, 81, 2688-2694. (4) Thorn, K.A.; Steelink C; Wershaw, R.L. Org. Geochem. 1987, 3, 123-137. (5) Bell, N.G.A; Murray, L.; Graham, M.C.; Uhrin, D. Chem. Commun. 2014, 50, 1694-1697.
3D HMQC-HMBC spectrum of the methylated FA. This experiment correlates 1H and 13C chemical shifts of the methyl groups with the carbonyl carbons of esters and the carbons carrying the methoxy group. Fig. 3 illustrates how this method delivers chemical shifts of nuclei close to the Me groups. Fig. 3a shows the 2D 1H, 13C HSQC spectrum of the 13C-methylated FA identifying different OMe types. Fig. 3b shows a 2D 1H, 13C HMBC spectrum mapping the nonaliphatic carbons coupled to OMe protons. Fig. 3c shows an HMQC projection of the 3D HMQC-HMBC spectrum illustrating the fact that this method only selects correlations to non-aliphatic carbons. Finally, Fig. 3d shows an HMBC plane taken at the 13CH3O
Acknowledgments: This work was supported by the NERC grant NE/L00044X/1 to MCG and DU. NGAB would like to acknowledge the support of the University of Edinburgh Principal’s Career Development Scholarship and NERC. We thank J. Bella for maintaining the NMR spectrometers.
235
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
H/D exchange of skeletal protons to develop targeted synthesis of humic-like substances A. Zherebker (a) *, Y. Kostyukevich(b), A.S. Kononikhin(b), A.I. Konstantinov(a), E.N. Nikolaev (b), I.V. Perminova(a) (a) Department of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia. (b) Emanuel Institute of Biochemical Physics of RAS, Moscow, Russia. * Corresponding author e-mail: [email protected] Keywords: humic, mass spectrometry, high resolution, isotopic exchange Abstract. Natural humic materials possess enormous structural heterogeneity. To overcome this problem, the humiclike products of phenolic copolymerization are frequently used as their synthetic analogues. However a direct analysis of molecular compositions of the synthetic products shows heterogeneity that not allows us to obtain complete structural information. Thus, we present another approach to obtain structural information of natural humic substances. Labeling of humic substances by skeletal H/D exchange enable to determine kind and quantity of protons in HS. Such information will allow to develop targeted synthesis of low dispersed structure analogues of HS. We anticipate our results to be a starting point for systematic studies on skeletal isotopic exchange in humic substances and on molecular adequacy of the humic-like materials. commercial 7 Tesla LTQ FT Ultra mass spectrometer equipped with Ion Max Electrospray Ion source (Thermo Electron Corp., Bremen, Germany) located at the facilities of the Institute of Biochemical Physics of RAS (Moscow, Russia). The samples were dissolved in methanol at concentrations of 1 g∙L-1. V; MS spectra (m/z 300-600) were acquired in the FTICR with resolution R = 400 000 at m/z 400. The average FTICR mass spectrum was a sum of 400 consecutive scans. The LTQ FT tuning mix was used for external mass calibration. The FTICR MS data were processed using the lab-made “Transhumus” software designed by A. Grigoriev, which is based on previously described total mass difference statistics algorithm (4). 1 H NMR spectra were acquired using a Bruker Avance 400 NMR spectrometer operating at 400 MHz proton frequency. The 1H NMR spectra were acquired in a 5 mm tube using 90 excitation pulses (90(1H) = 9 µs relaxation delay, 150 scans). 10 mg of HS were dissolved in DMSO-d6 for 1H NMR analysis. As a reference for proton assignments, a signal of residual protons of DMSO-d6 located at 2.5 ppm was used. Fourier transformation, phase correction and integration were performed using ACD-labs software Version 10 (Advanced Chemistry Development, Canada). Chemical shifts were expressed relative to tetramethylsilane.
Introduction Humic substances (HS) are natural organic compounds that are ubiquitous throughout the environment. They are characterized with nonstoichiometric elemental compositions and extreme structural heterogeneity, which provides for their recalcitrant character and multiple life-sustaining functions (1). However, this molecular heterogeneity hinders greatly systematic organize data of analysis of humic materials. One approach to solve this problem is synthesis of well-defined structure analogues of HS and testing adequacy of results obtained by highresolution method, such as ultrahigh resolution Fourier Transform mass spectrometry, which emerged at the end of the 20th century as an indispensable tool for exploring complex systems (2). However works on the study of the adequacy of the compounds obtained in terms of the molecular ensemble are missing. Another approach is using FTICR MS to observe selective isotopic labeling of the mobile protons in natural HS that gives structural information about quantity of mobile hydrogen atoms in every identified formulas (3). The objectives of this study were: (1) to synthesize aromatic humic-like products; (2) to use high resolution FT ICR MS for molecular level analysis of the synthesized reaction products; (3) to show capability of labeling of natural HS by catalyzed H/D exchange of skeletal protons to obtain information enable to develop new models of HS.
Results and Discussion Synthesis of HS-like substances: oxidative polymerization with hydroquinone (HQ) was conducted under alkaline conditions using potassium persulfate as a radical initiator. The obtained copolymerization product was fractionated into humicacid (MHQ-HA) and fulvic-acid (MHQ-FA) like fractions by precipitating acid-insoluble HA-like fraction and extracting acid-soluble FA-like fraction on Amberlite XAD8 resins. Humic-like products were
Experimental Humic materials used in this study were Arctic creek near the river Kolyma DOM –Y3. H/D exchange was conducted in sealed tube at 120 ˚C by adding D2O and NaOD as catalyst. Synthesis of humic-like substances was conducted via the oxidative polymerization of 3-(4-hydroxy-3-methoxyphenyl)-3oxopropionic acid (M) with hydroquinone (HQ). FTICR mass spectra were acquired using a
236
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
solvent – DMSO-d6, and 1H NMR spectra were acquired after addition of 20 µl of deuterated trifluoroacetic acid. The results of integration for all regions of backbone protons of Y3 before and after labelling are represented in Table 1.
analyzed by ESI FTICR MS. The acquired data were used for formula assignments which yielded about 3000 formulas (CHO-only) for each product. They were further used for plotting Van Krevelen diagrams (Figure 1). It is clearly seen that humic-like substances obtained by phenolic polymerization represent the wide number of formulas. Thus, a different approaches to the synthesis of HS models, leading to monomolecular or low dispersed compounds, are required. On the over hand FTICR MS is a power tool for investigate not only molecular ensemble of natural HS. Using method of selective isotopic labeling in tandem of FTICR MS gives opportunity to make conclusions about structures of HS. These data are necessary to draw scheme of synthesis of low dispersed HS-substances.
Table 1. Integrals of backbone protons region of Y3 and Y3 labeled by H/D exchange reaction. Decrease in the proportion of carbohydrate and α-protons indicates the main direction of the H/D exchange in the chosen conditions
REFERENCES
(1) MacCarthy, P. Soil Sci. 2001, 166(11), 738-751. (2) Hertkorn, N., Ruecker, C., Meringer, M. et al. Analyt. Bioanalyt. Chem. 2007. 389, 1311-1327. (3) Kostyukevich, Y., Kononikhin, A, Popov, I. et al. Anal. Chem. 2013, 85, 11007-11013. (4) Kunenkov, E. Kononikhin, A. Perminova, I. V. et al. Anal. Chem. 2009, 81, 10106-10115. (5) Lindström, B., Sjöquist, B., Anggard E. J. Labelled Compd. Radiopharm. 1974, 10, 187-194. (6) Kovalevskii, D.V., Permin, A.B., Perminova, I.V., Kononov, D.V., Petrosyan, V.S. Vest. Mosk. Univ., Ser. 2: Khim. 2000, 41, 39-42.
Acknowledgments: This study was partially supported by the Russian Foundation for Basic Research (grant # 13-04-01853).
Figure 1. Van Krevelen diagrams for the synthetic humiclike products obtained from the assigned formulas (CHOonly) in each sample. Green dots represent MHQ-HA, purple dots - MHQ-FA. Green and purple circles represent location of lignins and tannins regions.
H/D-exchange reaction: the procedure used of H/D exchanging allows to obtain rich labeled compounds [5]. High resolution of FTICR MS allows to distinguish atom of D with mass 2.0141 amu and 2 atoms of H with mass 2.0156 amu. Thus a length of series of H/D exchanging for every odd peaks in Y3 mass-spectrum indicates the amount of the exchanged protons. For example, for m/z 341,12288 the lengh Series of H/D exchange reached 9 (Figure 2).
Figure 2. Series of H/D exchange for m/z=341.12288 For a complete quantitative description of the deuterium exchange, NMR spectra were acquired for sample Y3 before and after reaction. 1 H NMR Spectroscopy: to detect only backbone protons in the HS, the original sample preparation technique was used (6). In brief, prior to analysis, hygroscopic water was removed from the samples under reduced pressure using vacuum pipeline. The dried samples were dissolved in anhydrous aprotic
237
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Using van Krevelen Plots to Explore the Relationship Between Molecular Formula and Exact Mass E. M. Perdue(a) *, N. W. Green(b)
(a) Department of Chemistry, Ball State University, Muncie, IN 47306 USA (b) School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0340 USA * Corresponding author e-mail: [email protected] Keywords: van Krevelen plots, FTICR mass spectrometry, molecular formulae Abstract Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has enabled the acquisition of molecular-level information regarding the chemical compositions of humic acids, fulvic acids, and natural organic matter. The conversion of mass-to-charge ratios into molecular formulae is accomplished using specialized computer software, only some of which is widely accessible. While developing software for this purpose, the authors discovered important relationships in van Krevelen plots that led to an efficient algorithm for assigning a molecular formula to an exact mass. Three synthetic data sets and one actual FTICR-MS data set have been used to compare the performance of several existing computer programs and software that was developed in the authors’ laboratory. In the “brute force” approach, the number of moles of each isotope is varied from its lower limit to its upper limit in a series of nested loops:
Introduction Van Krevelen plots have been used to display bulk average chemical compositions of organic matter in coal, kerogen, rocks, soils, sediments, and natural waters. With the advent of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), molecular masses of individual components in these highly complex mixtures of organic compounds could be determined to such a high degree of accuracy that it was often possible to assign unique molecular formulae to individual peaks in a mass spectrum. Kim et al. (2003) first used a van Krevelen plot to display and interpret the compositional data that are generated by FTICR-MS for a humic substance. This highly structured plot exhibited numerous linear trends that were attributed to gain/loss of CH2, H2O, H2, and O. The striking linear features of this van Krevelen plot inspired Hertkorn et al. (2008) to make a similar plot for all possible combinations of C, H, and O within a molecular mass range of 200-700 Da. They found the same linear features throughout van Krevelen space. This presentation will demonstrate that additional details relating exact mass and molecular formula are revealed in van Krevelen plots and may be exploited to facilitate the assignment of molecular formulae to peaks in an FTICR mass spectrum.
for Carbon:=MinC to MaxC do for Hydrogen:=MinH to MaxH do for Oxygen:=MinO to MaxO do for Nitrogen:=MinN to MaxN do for Sulfur:=MinS to MaxS do for Phosphorus:=MinP to MaxP do CalculateErrorSquared;
If N nested loops are used to fit N isotopes to an exact mass, the computer program will be classified as a type N program. This presentation will also consider N-1, N-2, and N-3 programs, in which progressively fewer nested loops are used to fit N isotopes to an exact mass. . All of these programs attempt to find all possible molecular formulae for a given exact mass, subject to the user-imposed constraints on the number of moles of each element in a molecular formula. An N-1 program is obtained by eliminating one loop and solving directly for the moles of that element by forcing Eq. 1 to equal zero. One possible path to an N-2 program is elimination of both the C and H loops, so that the exact mass that remains after subtraction of the masses of all other atoms should be the mass of a hydrocarbon, in which all fractional mass is due to H. Once H is known, all remaining integer mass is due to C. The N-3 program is an application of principles gleaned from a re-analysis of the information content of van Krevelen plots to eliminate the C, H, and O loops. The approach will be explained fully in the presentation.
Experimental The mathematical challenge in obtaining molecular formulae from exact masses is to minimize the square of the difference between a measured exact mass (EM) and the sum of the product of moles (n i) and exact mass (EMi) of all the isotopes in the molecular formula:
Data Sets and Software Four data sets were used to compare the variety of computer programs to which the authors had access. The first data set contains all molecular formulae with nominal mass in the range of 150-1000 Da that consist of only C, H, and O and meet the conditions of 2 ≤ H
(1)
238
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
≤ 2C+2 and 0 ≤ O ≤ C+2. The second data set was generated by random additions of 13C, NH, S, and/or PH to the first data set. The third data set was generated by non-random additions of 13C, NH, S, and/or PH to a non-random subset of the first data set. The fourth data set was an actual FTICR-MS data set for Suwannee River fulvic acid (IHSS No. 2S101F).
much greater for the Full mode than for the CHO mode of calculation. In CHO mode, all versions of software generated a similar number of molecular formulae (113093– 126489); however, aggregate run times for processing the four data sets varied by four orders of magnitude (0.005–57.4 min). Conversion of CHOFIT and HR2 from N programs to N-3 programs decreased run times by factors of 1677 and 198, respectively. In Full mode, there was a much greater variability in the number of molecular formulae that were found (3871778–23143698), probably reflecting the extent to which mathematically valid but chemically questionable molecular formulae were detected and removed. Aggregate run times for processing the four data sets varied by more than three orders of magnitude (0.8–2599.5 min). Conversion of CHOFIT and HR2 from N programs to N-3 programs decreased run times by factors of 1130 and 3042, respectively. These results suggest that the algorithm in N-3 programs should realistically increase the performance of this type of software by three orders of magnitude.
Table 1. Characteristics of the four test datasets Nominal Mass, Da Data Set Count Range Median % 13CNSP* 1 53573 150-1001 790 0 2 101912 150-1273 804 47.4 3 14790 150-1435 802 27.6 4 28514 148-2000 447 Unknown * Percentage of formulae containing 13C, N, S, and/or P
Computer software included the N, N-1, N-2, and N-3 versions of the authors’ CHOFIT software. The Formula Calculator from the National High Magnetic Field Laboratory (NHMFL) at Florida State University (www.magnet.fsu.edu) and HR2.EXE, which is a component of the Seven Golden Rules Software from University of California at Davis (http://fiehnlab.ucdavis.edu/projects/Seven_Golden_R ules/Software/), were used. The source code of HR2.EXE was modified to convert it from an N program to an N-3 program by incorporating the authors’ N-3 algorithm. The proprietary software FormCalc used by P. Schmitt-Kopplin at Helmholtz Zentrum Muenchen was also used in this analysis. Results and Discussion Each data set was processed by each computer program, with the time of execution being measured as carefully as possible. When execution times were too small to be measured accurately, a data set was analysed repeatedly in a loop until an accurate measurement of elapsed time was possible, after which the execution time per analysis was calculated. The effect of N (the number of isotopes being used in a molecular formula) was investigated by processing each data set by each computer program in a “Full” mode and in a “CHO” mode. C, H, O, N, S, P, and 13C were used in Full mode, but only C, H, and O were used in CHO mode. Results for the CHO mode and the Full mode are in Table 2 and Table 3, respectively. The number of molecular formulae that are found and the run times of the calculations are
REFERENCES
(1) Kim, S., Kramer, R. W., and Hatcher, P. G. (2003) “Graphical method for analysis of ultrahigh-resolution broadband mass spectra of natural organic matter, the van Krevelen diagram”, Anal. Chem., 75, 5336-5344. (2) Hertkorn, N., Frommberger, M., Witt, M., Koch, B., Schmitt-Kopplin, Ph., and Perdue, E. M. (2008) “Natural organic matter and the event horizon of mass spectrometry”, Anal. Chem., 80, 8908-8919.
Acknowledgments: The authors especially express their gratitude to P. Schmitt-Kopplin for making his proprietary software available for this study. Table 2. Summary statistics for the CHO mode of analysis Allowed Range Total Run time Software of Composition Formulae (min) Original HR2 (N) C1-83H1-145O0-36 113093 6.133 Formula Calculator C0-xH0-xO0-36 126336 57.400 FormCalc (HZM) C0-83H0-∞O0-36 129351 0.041 CHOFIT (N) C1-mH2-mO0-m 126352 8.384 CHOFIT (N-1) C1-∞H2-mO0-m 126352 0.200 CHOFIT (N-2) C1-∞H2-∞O0-m 125785 0.016 CHOFIT (N-3) C1-∞H2-∞O0-∞ 126323 0.005 Modified HR2 (N-3) C1-∞H1-∞O0-∞ 119051 0.031 Subscripts x, m, and ∞ mean the limits are either unknown, calculated from the mass, or unlimited, respectively.
Table 3. Summary statistics for the Full mode of analysis Total Run time Software Allowed Range of Composition Formulae (min) 13 Original HR2 (N) C0-1C1-83H1-145O0-36N0-10S0-6P0-4 3930238 2433.4 13 Formula Calculator C0-1C0-xH0-xO0-36N0-10S0-6P0-4 7244622 386.9 13 FormCalc (HZM) C0-1C0-83H0-∞O0-36N0-10S0-6P0-4 23143698 8.2 13 CHOFIT (N) C0-1C1-mH2-mO0-mN0-10S0-6P0-4 5400522 2599.5 13 CHOFIT (N-1) C0-1C1-∞H2-145O0-36N0-10S0-6P0-4 5400522 114.5 13 CHOFIT (N-2) C0-1C1-∞H2-∞O0-36N0-10S0-6P0-4 5370792 3.8 13 CHOFIT (N-3) C0-1C1-∞H2-∞O0-∞N0-10S0-6P0-4 5387589 2.3 13 Modified HR2 (N-3) C0-1C1-∞H1-∞O0-∞N0-10S0-6P0-4 3871778 0.8 Subscripts x, m, and ∞ mean the limits are either unknown, calculated from the mass, or unlimited, respectively.
239
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Ecosystem-specific molecules in dissolved organic matter as fundament for new marker compounds V.-N. Roth (a) *, T. Dittmar(b), R. Gaupp(c), G. Gleixner(a)
(a) Max Planck Institute for Biogeochemistry, 07745 Jena, Germany (b) Research Group for Marine Geochemistry (ICBM-MPI Bridging Group), University of Oldenburg, Institute for Chemistry and Biology of the Marine Environment (ICBM), 26111 Oldenburg, Germany (c) Institute for Geosciences, Friedrich Schiller University, 07749 Jena, Germany * Corresponding author e-mail: [email protected] Keywords: DOM, FT-ICR-MS, tannin molecular formulae, non-metric multidimensional scaling, molecular biogeochemistry
Abstract Ecosystem activities are reflected in the molecular composition of dissolved organic matter. We tested if ecosystem-specific molecular fingerprints can be discerned in a comparative study of samples from a contrasting range of ecosystems: bog, forest, grassland and river. Ecosystem-specific molecules are the fundament to search for new marker compounds that allow a more detailed tracking of DOM origin and fate. We applied electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to resolve the molecular diversity of DOM. We applied nonmetric multidimensional scaling revealing an intrasystem similarity of the molecular composition that might be connected to a pH and vegetation influence. Studying ecosystem-unique molecular formulae, we demonstrated ecosystem-specific characteristics. For forest ecosystems tannin molecular formulae reoccurred to be of special importance and we suggest them as suitable marker compounds. Future studies have to include a higher diversity of ecosystems and sample number to evaluate new marker compounds and to separate the pH from vegetation influence.
from four major ecosystems of contrasting features: bog, forest, grassland and river. We applied multivariate statistical analyses to discern the molecular dissimilarities between ecosystems. Based on intra-ecosystem similarities, we identified ecosystem-unique molecular formulae for their subsequent characterization according to their chemical characteristics.
Introduction Molecular level understanding of DOM processes is needed to understand origin and fate of DOM. The highly dynamic mixture of small molecules that dissolved organic matter (DOM) is composed of is affected on its way from source to sink by ongoing alteration. On the one hand the physicochemical framework influences the DOM sources. On the other hand the individual ecosystems are defined by their biological properties. The corresponding prevailing conditions are reflected in the DOM composition (1), (2). As a consequence, ecosystem-specific molecular fingerprints should reside in the molecular composition of DOM which is the fundament to track the fate of DOM from the source on. Established marker substances are reduced to a limited number of molecules form substance classes such as ligninderived phenols, carbohydrates, amino acid or lipids. But more not yet identified markers should be present in the vast pool of DOM. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) was introduced in recent years to study the DOM composition on a broadband molecular level. Providing access to several thousand molecular formulae per DOM sample, FTICR-MS enables to elucidate molecular carbon cycle processes. We hypothesized that ecosystem-specific molecules exist and that it is possible to identify them by FTICR-MS. We used FT-ICR-MS data of DOM samples
Experimental River and bog water samples from a transect of 1000 km along the Yenisei River (3) were filtered with glass fibre filters (GF/F, 0.7 µm). Forest and grassland soil water samples were collected using glass ceramic suction plates (1-16 µm) at four different long-term monitoring sites that differ in vegetation and soil pH. All DOM samples were freeze-dried after sampling and redissolved in ultrapure water for solid phase extraction of DOM (4) prior to FT-ICR-MS analyses that were performed with a 15 Tesla FT-ICR-MS in negative ESI mode. The elements C, H, N, O and S were considered for molecular formulae assignment. To compare FT-ICRMS data of different measurement, we applied a conservative limit of detection for the whole set of samples (LODGroup, (3)). Applying LODGroup left 2721 different molecular formulae whereas the 240 ubiquitous formulae were excluded from statistical analyses.
240
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
To discern dissimilarities between samples, we applied nonmetric multidimensional scaling (NMDS) that does not assume specific relationships within the data. To test for the correlation between pH and the data set, we applied redundancy analysis (RDA).
might indicate similar ecosystem-specific chemical properties. There are two main groups of formulae that are unique for forest soil water DOM. The first expresses low H/C ratios near 0.5 and low O/C ratios between 0 and 0.7. The second expresses H/C and O/C that represent tannin molecular formulae. In a recent, but not yet published, study solely focused on the here included forest soil water DOM samples, we observed a correlation of certain tannin molecular formulae with the low pH forest soil sites. Reoccurring as unique substances at forest sites indicates that certain tannins might represent possible forest system-specific markers. In summary, we could demonstrate that ecosystem specific molecules exist in DOM. We confirmed recent results that pH is one of the environmental factors that drives DOM composition. In addition, we demonstrated the influence of biological systems represented by a vegetation influence. We advise to use future studies based on a higher number of ecosystems and samples to separate the pH and vegetation influence as well as to identify and evaluate new system-specific marker molecules.
Results and Discussion NMDS and RDA: [Figure 1] The NMDS ordination plot demonstrates that the four ecosystems are clearly separated according to their molecular composition. The forest soil DOM samples are subdivided according to the three individual sites along NMDS1 that indicates a possible vegetation gradient because the samples are ordered from 100 % conifer to grassland. Surface and soil water samples are separated by NMDS2. In addition to the indicated vegetation gradient, RDA demonstrated a pH gradient in the data set that explains 15.9 % of the variability. The direction of pH increase for surface and soil water samples coincides with NMDS1. Even if the correlation of pH with the molecular composition does not prove causal effects, we see strong indications for a real pH effect that was already shown in previous studies that focused on individual samples of this study. The mentioned studies a) examined the pH effect on bog and river DOM from the Yenisei transect (3) and b) revealed also a pH gradient in the DOM composition of forest soil DOM in an unpublished study.
Figure 2. Van Krevelen diagram of molecular formulae that uniquely occurred in one of the four major ecosystems and not in the others. Number of unique formulae: bog: 120, forest: 170, grassland: 196 and river: 76. It is indicated that tannin molecular formulae are important for forest systems. Modified version of (5).
Figure 1. Nonmetric multidimensional scaling ordination plot based on 66 measurements and 3 axes. The direction of pH increase is indicated by the pH arrow along NMDS1. NMDS2 separates surface water DOM from soil water DOM. Modified version of (5).
REFERENCES
(1) Kaiser, K.; Kalbitz, K. Soil Biol. Biochem. 2012, 52, 29-32. (2) Steinbeiss, S.; Temperton, V. M.; Gleixner, G. Soil Biol. Biochem. 2008, 40, 2634-2642. (3) Roth, V.-N.; Dittmar, T.; Gaupp, R.; Gleixner, G. Geochim. Cosmochim. Ac. 2013, 123, 93-105. (4) Dittmar T.; Koch, B.; Hertkorn, N.; Kattner, G. Limnol Oceanogr.-Meth. 2008, 6, 230-235. (5) Roth, V.-N.; Dittmar, T.; Gaupp, R.; Gleixner, G. Vadose Zone J. 2014, doi:10.2136/vzj2013.09.0162, in press.
Ecosystem-specific molecular composition: [Figure 2] Based on the clear separation of ecosystems due to their molecular DOM composition, we identified molecular formulae that were unique for each ecosystem and plotted them in a van Krevelen diagram. The unique formulae express differences in their elemental composition dependent on the ecosystem and mainly occur in distinct areas of the van Krevelen diagram. A clear trend of increasing H/C from forest to bog, grassland and river DOM is demonstrated. Having similar H/C and O/C ratios
Acknowledgments: Funding was provided by the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (GRK 1257 and SFB 1076).
241
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Potential and Constraints of Biochar for Soil Carbon Sequestration B. Glaser (a) *, C. Rumpel(b), C. Naisse(b), K. Wiedner(a), Y. Kuzyakov(c) (a) Martin-Luther-University Halle-Wittenberg, Institute of Agronomy and Biogeochemistry, von-Seckendorff-Platz 3, 06120 Halle/Saale, Germany.
Nutritional Sciences,
Soil
Bioemco, CNRS, UPMC (UMR 7618 CNRS-UPMC-ENS-UPEC-IRD-AgroParisTech), Thiverval-Grignon, France. (b)
(c)
University of Göttingen, Soil Science of Temperate Ecosystems, Büsgenweg, 37077 Göttingen, Germany
* Corresponding author e-mail: [email protected]
Keywords: Charcoal, soil organic matter, soil hydraulic properties, up to 6 keywords
Abstract Returning organic carbon to the soil in the form of (bio)char can sequester carbon in soil and increase its fertility. Recent research on biochar stability and degradation is summarized. Biochar stability strongly depends on chemical structure being more a function of technological process conditions rather than of feedstock. Pyrolysis or gasification chars (pyrochar) are by a factor of 10 – 15 more stable than hydrothermal carbonization products (hydrochars). Mean residence time of pyrochar is in the centennial to millennial range, while hydrochars are stable in natural environments only for decades. Pyrochars exhibited negative priming on natural soil organic matter while hydrochars showed a positive priming. Stability of pure chars can be predicted using the atomic H/Corg ratio, while for modified chars (e.g. composted, in soil or biochar fertilizers) molecular markers such as benzene polycarboxylic acids are required. use should be considered in addition to the direct benefits of carbon sequestration.
Introduction During the last two decades, interest in biochar research and application was strongly raised because of the “Terra Preta” phenomenon being a model for sustainable management of natural resources including biochar and nutrient-containing organic waste materials (1). Exponentially growing interest in pyrogenic C (biochar) is mainly connected with its importance for the global C cycle (2) and with its potential role as a C sink in soils and sediments for long periods of time, because its microbial decomposition and chemical transformation is very slow but also because of potential to improve soil fertility. The chemical and biological inertness of pyrogenic C and biochar is mainly based on polyaromatic chemical structure.
Experimental A range of experiments were carried out including characterization of material properties, laboratory and field incubations, a range of different chars including pyrochars, charcoal, and hydrochars and stable (13C) and radioactive (14C) labelling techniques.
Results and Discussion Chemical reactivity towards acid dichromate oxidation was lower for pyrochars than for hydrochars and related to the H/C ratio of the chars (6). Different feedstocks slightly influenced their reactivity (7). Our results showed that the chemical reactivity of Holocene charcoals increased with their time of exposure in soil, indicating that they may not be used as reference materials in terms of stability. Incubation experiments corroborated the use of atomic H/C ratio to predict the long-term stability of biochars and thus to calculate their C sequestration potential. Our results confirmed that the chemical and biological stability of pyrochars is much higher than that of
Biochar stability depends on its production conditions and thus, on its physico-chemical properties (3). In general, it is assumed that the stability of biochar is related to its aromatic structures (4). The proportion of aromatic C and, therefore, the intrinsic chemical recalcitrance of biochar increases with pyrolysis temperature and time (5). In addition, biochar application may possess additional carbon mitigation potential owing to indirect effects. For example, increases in soil organic carbon (SOC) stocks, and decreases in GHG emissions and fertilizer
242
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
been found down to 1 m depth in such soils, despite the climatic conditions strongly favoring decomposition and the lack of additional biochar additions during the last 500 years (1). However, MRT assessments from natural ecosystems or terra preta can only deliver orders of magnitude in accuracy, since there is no way to quantify the initial (repeated) biochar input to obtain a straightforward mass balance for terra preta. The longest laboratory biochar decomposition study also revealed MRT of about 2,000 years (8). However, MRT depends on material properties, especially the degree of aromatization. Hydrochar has a less aromatic structure and higher percentage of labile carbon species (3). Therefore, it decomposes similar to soil organic matter within decades.
hydrochars (6). During the early stage of soil incubation under laboratory conditions, hydrochar input induced a positive priming effect, indicating a stimulation of soil organic matter mineralization by addition of easily degradable carbon instead of sequestering carbon. In contrast, the pyrochar input induced a negative priming effect showing additional soil organic matter protection in addition to the higher stability of the char. However, for both chars, no priming effect after the first weeks of incubation was observed, indicating mainly apparent priming - a short-term phenomenon related with changes of microbial biomass turnover. Physical weathering of chars led to increasing O/C atomic ratios due to the surface oxidation and a loss of fine particles. A feedstock effect was observed for pyrochar with a massive C loss during weathering from corn stover compared to wood. Thus, the wide range of feedstock potentially used for the production of pyrochars and hydrochars requires a detailed investigation of main feedstocks types. Physical weathering effects on biological and chemical stability as well as priming suggests that it has consequences for the C sequestration potential at least at decadal timescale.
REFERENCES
(1) Glaser, B.; Haumaier, L.; Guggenberger, G.; Zech, W. Naturwissenschaften 2001, 88, 37–41.
(2) Forbes, M. S.; Raison, R. J.; Skjemstad, J. O. Sci. Total Environ. 2006, 370, 190–206.
(3) Schimmelpfennig, Sonja; Glaser, B. J. Environ. Qual. 2012, 41, 1001–1013. (4) Glaser, B.; Haumaier, L.; Guggenberger, G.; Zech, W. Org. Geochem. 1998, 29, 811–819.
Half–lives of fresh and weathered hydrochars were estimated between 8.4±0.9 to 10.4±0.3 years, and between 73.6±5.6 to 145.0±24.0 years for fresh and weathered pyrochars, respectively (6). Carbon sequestration potential including char mineralization, priming effect and physical weathering assessed for both chars, indicates that at the decadal scale, hydrochar and pyrochar would lead to similar increase of soil C storage. At the century scale, only pyrochar would have the potential to increase soil carbon sequestration.
(5) Brewer, C. E.; Schmidt-Rohr, K.; Satrio, J. A.; Brown, R. C. Env. Progress & Sustainable Energy 2009, 28, 386–396. (6) Naisse, C.; Alexis, M.; Plante, A.; Wiedner, K.; Glaser, B.; Pozzi, A.; Carcaillet, C.; Criscuoli, I.; Rumpel, C. Org. Geochem. 2013, 60, 40-44. (7) Wiedner, K.; Naisse, C.; Rumpel, C.; Pozzi, A.; Wieczorek, P.; Glaser, B. Org. Geochem. 2013, 54, 91–100.
(8) Kuzyakov, Y.; Bogomolova, I.; Glaser, B. Soil Biol. Biochem. 2014, 70, 229–236.
True C sequestration potential of biochar depends on several factors. Char from natural fires is usually the oldest carbon pool present in ecosystems. Mean residence time (MRT) of naturally generated char in Australian woodlands ranged between 718 – 9,259 years (8). The long-term stability of biochar has been shown in terra preta, being 500 – 7,000 years old (8). Considerable biochar stocks of 50 Mg C ha-1 have
Acknowledgments: We acknowledge financial contributions of EuroChar project (FP7-ENV-2010 Project ID 265179), ClimaCarbo project (BMBF 01LY1110B), COST Action TD1107 (Biochar as option for sustainable resources management) and International Humic Substances Society (IHSS).
243
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The colloidal instability is new characteristic of humic substances A. Popov(a)*, A. Luneva(b) (a)
Saint Petersburg State University, 199178, Russian Federation, St. Petersburg, 16th Line, 29, Department of soil science and soil ecology (b) Saint Petersburg State Agrarian University, 196600, Russian Federation, St. Petersburg, Pushkin, Peterburgskoye shosse, 2/1a. * Corresponding author e-mail: [email protected] Keywords: humic substances, colloidal instability, molecular weight Abstract. For the characteristic of colloidal properties of humic substances (HS) the threshold of aggregation instability (TAI) was proposed. Threshold of aggregation instability is certain concentration of HS at which begins a sedimentation of HS in strongly acidic solution. It was evaluated by mg of HS carbon per 1 litre of solution. For this purpose, the concentrated H2SO4 was added in extracting alkaline solution to pH ~ 1. Next a mixture was warmed till 70-80 ºС. Then to it the HS solution with known concentration was instilled up to formation of light sediment. The minimal quantity of HS was conformed to TAI. We were studied HS, which were extracted from the humus accumulative horizons of some soils by 0.1 M Na4P2O7 with 0.1 M NaOH. The extracted HS weren't divided into humic and fulvic acids. It was find an interrelation (r = –0,745) between TAI and average molecular weight of HS. Introduction Humic substances (HS) are amorphous darkcoloured organic nitrogen-containing organic substances of natural origin. In chemical respect they are randomized redox-heteropolymers mainly of agrilglycoproteid nature. Besides, HS are polyfunctional polyampholites―they have both negative charged groups and positive charged ones. As generally accepted, HS are colloids. The chemical composition together with functional groups causes the hydrophilic and lipophilic balance of HS molecules. Colloidal properties of HS are caused their three-dimensional structure (1, 2, 3). We take the view (1, 2, 3) that “macromolecules” of HS consist of the amphiphilic fragments with various molecular weights (MW). These fragments of HS, being sufficiently good surfactants, are capable to spontaneous association of molecules with education such three-dimensional supramolecular assemblies as micelles or "pseudomicelles" (1). The molecules in these micelles are come apart from each other by hydration shell. It was shown that the formation of humic pseudomicelles is influenced by the molecular sizes of the species involved (4, 5). A divide of humic material into humic acids (HA) and fulvic acids (FA) is connected with change of aggregative stability in strongly acidic solution (6). So, HA in strongly acidic solution are colloidal unstable humic material, and FA—colloidal stable one. Molecules of HA and FA can change the conformation (7). So HS molecules behave like spherical colloids at high concentration of a sample, low size рН or in the presence of a significant amount of electro neutral electrolytes. In turn at small concentration of a sample, with rather low ionic force and at higher values рН, humic acids take the branched form of linear colloids. In one's time M.M. Kononova (8) offered a method for characteristic of HS colloidal properties. This method was based on determination of coagulation threshold by solution of CaCl2. This method is rather difficult and one characterizes chemical affinity of HS with calcium ions. Thereupon the simple method
characterized HS colloidal properties was worked up. It allow to determine a new characteristic of colloidal properties of HS―threshold of aggregation instability (TAI) The aim of paper―to finds interrelation between TAI and size of the average MW of HS. Experimental The objects were a lot of soil soils of the European part of Russia and the Ukraine: Eutric Podzoluvisol (Umbric Albeluvisol Abruptic, soddy-podzolic soil), taiga zone (Leningrad Region); Rendzic Leptosol (Rendzic Leptosol Eutric, sod-calcareouse), taiga zone (Leningrad Region); Haplic Greyzem (Greyic Phaeozem Albic, grey forest soil), forest-steppe zone (Belgorod Region); Haplic Chernozem (Voronic Chernozem pachic, typical chernozem), forest-steppe zone (Belgorod Region); Calcic Chernozem (Voronic Chernozem Pachic, сhernozem ordinary), steppe zone (Voronezh Region); Haplic Kastanozem (Haplic Kastanozem Chromic, dark chestnut), the dry Taurida steppe (Kherson Region, the Ukraine). In the table 1 it is provided a depth of soil sampling. Table 1. The index of object and a depth of sampling. Object 1 2 3 4a 4b 5a 5b 6a 6b
The soil Eutric Podzoluvisol Rendzic Leptosol Haplic Greyzem Haplic Chernozem Haplic Chernozem Calcic Chernozem Calcic Chernozem Haplic Kastanozem Haplic Kastanozem
A depth of sampling, sm 5-15 10-15 2-10 5-20 20-40 5-18 18-32 5-10 10-20
Humic substances were extracted from the humus accumulative horizons by alkaline solution of sodium pyrophosphate (0.1 M Na4P2O7 with 0.1 M NaOH). The isolated HS weren't divided into HA and FA.
244
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
For the characteristic of HS colloidal properties the TAI was determined. Threshold of colloidal instability is certain concentration of HS at which begins a sedimentation of HS in strongly acidic solution. It was evaluated by mg of HS carbon per 1 litre of solution. For this purpose, the concentrated H2SO4 was added in extracting alkaline solution (в нашем случае 0.1 M Na4P2O7 with 0.1 M NaOH) to pH ~ 1. Next a mixture was warmed till 70-80 ºС. Then to it the HS solution with known concentration was instilled up to formation of light sediment. The minimal quantity of HS was conformed to TAI. Besides, a MW distribution of HS was determined by gel filtration. We were used dextran gels (Sephadex G-75, superfine) and the 0.05 M tris-buffer-acetic acid solution (pH ~ 8) with Triton X-100 as eluent for a HS MW determination.
This fact can speak that with increase the size of HS molecules the hydrophilic-lipophilic balance moves towards lipophilicity (or hydrophoby). The more MW of colloidal molecules, the less aggregation stability. As a result the molecules of HS with bigger molecular weight coagulate earlier. The main distinction of colloidal systems from true solutions is that degree of dispersion of colloidal particles is size changeable. Integration of particles gradually leads to that they become too large and lose those physical and chemical features, which are inherent in disperse particles. In disperse systems the specific surface of a disperse phase is very great. One of the most important consequences of a big surface of a disperse phase is that lyophobic disperse systems have an excess superficial energy, and, therefore, are thermodynamic unstable. Therefore in disperse systems the various spontaneous processes, which lead to reducing an excess energy. The processes of reduction of a specific surface due to integration of particles are most the general. Therefore when studying HS it is necessary to pay more attention to colloidal properties of these connections. The impotent implication of the obtained data is that division of HS into HA and FA has to be carried out at the certain (fixed) concentration of a humic material in solution. In this regard the ratio of CHA/CFA (carbon of HS to carbon of FA), which is used for characteristic of humus qualitative, is not correct. As in more diluted (light) solutions of HS the ratio CHA/CFA always will be less, than in more concentrated (dark) ones. The availability of the return close interrelation between the average molecular weight and values of threshold of aggregation instability confirm that HS are capable to form the structured colloidal micelles.
Results and Discussion The size of average MW and the value of TAI of HS are adduced in Table 2. Table 2. The average molecular weights and the threshold of aggregation instability of studied humic substances. Object
The soil
1 2 3 4a 4b 5a 5b 6a 6b
Eutric Podzoluvisol Rendzic Leptosol Haplic Greyzem Haplic Chernozem Haplic Chernozem Calcic Chernozem Calcic Chernozem Haplic Kastanozem Haplic Kastanozem LSD05
Average MW, kDa 17.2 14.9 15.6 11.0 13.8 14.4 14.7 17.8 10.1 2.44
TAI of HS, mg C•L–1 26.8 34.6 36.7 71.8 65.9 34.6 32.9 36.0 54.1 5.76
REFERENCES
(1) von Wandruszka, R. Soil Sci. 1998, 163, 921-930. (2) Tombacz, E. Soil Sci. 1999, 164, 814-824. (3) Piccolo, A.; Conte, P.; Cozzolino, A. Soil Sci., 2001, 166, 174-185. (4) Chilom, G.; Bruns, A.S.; Rice, J.A. Org. Geochem. 2009, 40, 455-460. (5) Engebretson, R.R. and von Wandruszka, R. Org. Geochem. 1997, 26, 759-765. (6) Popov, A.I. Humic substances: properties, a structure, formation. Saint Petersburg, Publishing house of the Saint Petersburg university, 2004 (In Russian). (7) Ghosh, K. and Schnitzer, M. Soil Sci., 1980, 129, 266-276. (8) Kononova, M.M. Soil Organic Matter: Its Role in Soil Formation and in Soil Fertility. New York, Pergamon Press, 1966.
It was revealed that that the size of average MW decreased with increase in value of a TAI of HS [Figure 1]. The coefficient of correlation was close and essential (r = –0,745).
– 1 TAI of HS, mg C•L
100 80 60 40
Acknowledgments: Professor V.P. Tsiplenkov for consultation.
20 0 5
10
15
20
25
Molecular weight, kDa
Figure 1. An interrelation between a threshold of colloidal instability and size of the average molecular mass of HS.
245
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characterization of humic substances fractionated by polarity and their biological effects on plant growth R.L. Sleighter (a) *, P. Caricasole (a), K.M. Richards (a), T. Hanson (a), P.G. Hatcher (a,b) (a) FBSciences, Inc., Norfolk, VA 23508 (b) Old Dominion University, Norfolk, VA 23529 * Corresponding author e-mail: [email protected]
Keywords: humic substances, solid phase extraction, FTICR-MS, NMR, plant growth, bioassays Abstract Humic substances were fractionated into three components (acid insoluble, acid soluble hydrophilic, and acid soluble hydrophobic), characterized by advanced analytical techniques (EEMs, NMR, FTICR-MS), and then evaluated for their biological effects on plant growth using bioassays. Approximately 15-20% of the carbon could be precipitated into the acid insoluble fraction. Upon fractionation of the acid soluble portion using C18 extraction, about 65% of the carbon was found to be hydrophilic, and the vast majority of iron was also found in the acid soluble filtrate. The acid insoluble portion was more aromatic and oxygen-poor than the acid soluble fraction. The hydrophilic filtrate was oxygen-rich and contained mostly tannin-like molecules, while the hydrophobic fraction was more lignin-like. During bioassay testing, the samples dominated by the filtrate portion yielded the best biological stimulation using seed treatment and soil and foliar applications (i.e., root/shoot elongation and higher plant masses). Elmer AAnalyst 200) and then characterized by UV/Vis absorbance and excitation emission matrix spectroscopy4 (EEMs, Horiba Scientific Aqualog), liquid-state proton nuclear magnetic resonance (1H NMR) spectroscopy (400 MHz Bruker Biospin Avance III), and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS, Apollo II ESI ion source on a Bruker Daltonics 12 Tesla Apex Qe). ESI is a soft ionization technique that yields molecular ions with little to no fragmentation5,6. FTICR-MS provides ultrahigh resolution and mass accuracy, which allows for the assignment of molecular formulas to the thousands of peaks detected in each spectrum7,8. The acid soluble portion that was fractionated using C18 was combined to give 5 different filtrate:retained ratios (100:0, 75:25, 50:50, 25:75, and 0:100). Whole and fractionated samples were tested for plant response using seed treatment and soil and foliar applications on wheat and cotton. Bioassays were evaluated in a randomized block design with 7 replicates per treatment and assessments (root/shoot lengths, fresh/dry masses) were statistically evaluated.
Introduction FBSciences, Inc. is a life science company with a global focus on improved crop yield and quality through improved plant health and efficiency, in part by treatment with humic substances (HS). HS are ubiquitous in the environment and have long been regarded as beneficial to soil fertility and plant growth1,2. They contain thousands of individual molecules, all varying in their individual structure, function, reactivity, and polarity. Here, the acid insoluble and acid soluble portions were isolated. Then, the acid soluble portion was fractionated using C18 solid phase extraction, which separates compounds based on polarity. Material that passes through the C18 is polar and is referred to as the filtrate, while the material that is retained by the C18 is non-polar3. Samples were chemically characterized using an array of advanced analytical techniques. After characterization, the original whole and fractionated samples were tested using bioassays in growth chambers at various carbon and iron rates and combinations. Bioassays are utilized as a tool for demonstrating the effects of the different sample types on plant activity, in an effort to correlate molecular level details with plant response (i.e., root and shoot elongation, fresh and dry weights, final emergence).
Results and Discussion About 15-20% of the carbon precipitated into the acid insoluble fraction. Upon fractionation of the acid soluble portion using C18 extraction, about 65-70% of the carbon was found to be hydrophilic. The vast majority of iron was also found in the acid soluble filtrate. EEMs results show that the fractionated samples all have humic-like peaks that vary in their intensities. Greater than 95% of the integrated fluorescence was from humic-like peaks A and C, with little contribution from peptide-like fluorescence (peak T). NMR characterization revealed that while there was significant overlap between the fractionated samples, the acid insoluble portion is more aromatic than the acid soluble and the C18 filtrate portion is
Experimental Terrestrial whole samples were acidified to pH<2, and precipitation was allowed to occur at room temperature for 48 hours. The acid insoluble fraction was separated from the acid soluble by centrifugation. The acid soluble fraction was C18 extracted to obtain the hydrophobic, non-polar fraction (i.e., retained) and the hydrophilic, polar fraction (i.e., filtrate). The 3 fractions (acid insoluble, acid soluble polar, and acid soluble non-polar) were analyzed for their dissolved organic carbon (DOC) concentrations (Shimadzu TOC-VCSN) and iron (Fe) contents (Perkin
246
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
more oxygenated than the retained, in agreement with the mechanism of polarity separation. A clear shift in the mass spectra was observed at each nominal mass region, where peaks with low mass defect had higher magnitudes in the polar filtrate and peaks with high mass defect were more prominent in the non-polar retain, indicating that the filtrate is more oxygenated and/or more aromatic. This shift was also observed in the van Krevelen diagrams (Figure 1). The acid insoluble portion was dominated by low O/C aromatics and condensed aromatics with low H/C ratios. The tannin component (O/C>0.6, H/C 0.5-1.5) dominates the acid soluble filtrate, while the lignin component (O/C 0.1-0.6, H/C 0.5-1.5) dominates the retained portion. Furthermore, the N- and S-containing components that are more polar are also detected in the acid soluble filtrate fraction. Bioassays using seed, soil, and foliar applications were compared to that of untreated checks. For the seed treatments and soil applications, the whole sample and samples dominated by the filtrate portion gave better root and shoot elongation. Samples that were dominated by the retained portion generally gave results worse than the untreated check. By altering the DOC and Fe rates, it was found that both were important parameters to consider to obtain optimal, synergistic plant stimulation. For foliar applications, all fractions at application rates lower than 1 mg/L C gave higher plant masses than the control. Further evaluation showed significant correlations between root/shoot and mass measurements with the ratio of the filtrate:retained fractions. However, it has yet to be determined if this is due to the types of carbon components in each of these fractions (i.e., hydrophilic vs. hydrophobic) or due to the higher metal concentration in the filtrate vs. the retained. While more research is necessary to discern the exact mechanism, this study is an example of enhanced plant growth during controlled application of humic substances that vary in their chemical components and concentrations.
2.5 a) Whole Sample 2822 formulas
H/C
2.0
1.5
1.0
0.5
2.5 0.0
0.2
0.4
0.6 O/C
0.0
0.2
0.4
0.6 O/C
CHO formulas CHOS formulas 0.8
CHON formulas CHONS formulas 0.0 b)1.0 Acid Insoluble 1.2 814 formulas
CHO formulas CHOS formulas 0.8
CHON formulas CHONS formulas 0.0 1.0 1.2
2.0
H/C
1.5
1.0
0.5
2.5 c) Acid Soluble Hydrophilic 2717 formulas 2.0
H/C
1.5
1.0
0.5
0.0
0.2
0.4
0.6 O/C
CHO formulas
CHON formulas
CHOS formulas 0.8
CHONS formulas 0.0 1.0 1.2
2.5 d) Acid Soluble Hydrophobic 2593 formulas 2.0
REFERENCES
1.5 H/C
(1) Aiken, G.R., MacCarthy, P., Malcolm, R.L., Swift, R.S. Humic Substances in Soil, Sediment, and Water. Wiley, New York, 1985. (2) MacCarthy, P., Clapp, C.E., Malcolm, R.L., Bloom, R.R. Humic Substances in Soil and Crop Sciences: Selected Readings. American Society of Agronomy, Madison, 1990. (3) Sleighter, R.L., Hatcher, P.G. Mar. Chem. 2008, 110, 140-152. (4) Fellman, J.B., Hood, E., Spencer, R.G.M. Limnol. Oceanogr. 2010, 55, 2452-2462. (5) Cech, N.B., Enke, C.G. Mass Spec. Rev. 2001, 20, 362-387. (6) Stenson, A.C., Landing, W.M., Marshall, A.G., Cooper, W.T. Anal. Chem. 2002, 74, 4397-4409. (7) Marshall, A.G., Hendrickson, C.L., Jackson, G.S. Mass Spec. Rev. 1998, 17, 1-35. (8) Sleighter, R.L., Hatcher, P.G. Fourier transform mass spectrometry for the molecular level characterization of natural organic matter: Instrument capabilities, applications, and limitations. In: Fourier Transforms- Approach to Scientific Principles. InTech, Vienna, 2011.
1.0
0.5
e)
0.0
0.2
Sample
0.4
0.6 O/C
CHO formulas
CHON formulas
CHOS formulas 0.8
CHONS formulas 0.0 1.0 1.2
O/C
H/C
DBE
W hole
0.59
0.91
13.0
DBE/C % CHO % CHON % CHOS 0.60
73%
13%
14%
Acid Ins oluble
0.40
0.79
14.6
0.65
89%
8%
3%
Acid Soluble Hydrophilic
0.67
0.89
12.4
0.61
61%
17%
22%
Acid Soluble Hydrophobic
0.46
0.97
13.4
0.56
86%
10%
4%
Figure 1. van Krevelen diagrams of the a) whole sample prior to fractionation and the b) acid insoluble, c) acid soluble hydrophilic, and d) acid soluble hydrophobic portions, along with e) the average O/C, H/C, DBE, and DBE/C for each.
Acknowledgments: We thank the COSMIC facility at ODU for instrument time on the NMR and FT-MS.
247
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Degradation of lignin by hydroxyl radical (a)
D.C. Waggoner *, H. Chen(a), A.S. Willoughby(a), P.G. Hatcher(a) (a) Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA USA 23529 * Corresponding author e-mail: [email protected] Keywords: Hydroxyl radical, lignin, FTICR-MS, humification Abstract
The generation of condensed aromatic molecules from lignin-derived organic matter after exposure to hydroxyl radicals has been demonstrated using ultrahigh resolution mass spectrometry. Although condensed aromatic compounds are common in soil and dissolved organic matter samples, their presence has largely been attributed to combustion processes. A non-pyrogenic route for the formation of condensed aromatic molecules from lignin is suggested here; specifically that hydroxyl radical oxidation of lignin is capable of producing black carbon-like compounds. Hydroxyl radicals are known to be produced by photochemistry in aqueous systems and enzymatic processes in soils and are therefore readily available for the alteration of lignin to more condensed aromatic material. particles larger than 0.7 μm. A Fenton reaction was then used to generate hydroxyl radicals in solution with the addition of Fe(II) salt and H2O2 after adjusting the pH to 3. The resulting solution was filtered, and any particles generated were redissolved in an ammonium hydroxide/water/methanol solution before being injected into a 12 T Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to obtain elemental compositions for the complex suite of molecules formed. Samples were analyzed using negative mode electrospray ionization (ESI), as described by Sleighter and Hatcher (8). The filtrate was additionally analysed using the same conditions after clean-up with cation exchange resin to remove inorganic salts. Results were compared to those obtained for extracted lignin not reacted with hydroxyl radicals. Peaks were assigned unique molecular formulas using a formula calculator (6).
Introduction Lignin, a phenolic biopolymer, accounts for roughly 20% of organic matter input into soils. It is estimated that lignin comprises 20-30 g kg-1 of woody and vascular plant material, for a global equivalent on the order of 3x1011 metric tons of lignin (1). Compared to most biopolymers, lignin is substantially more resistant to microbial degradation; however, several microorganisms are capable of breaking down lignin. Various white rot fungi are able to cleave lignin side chains as well as the aromatic ring during enzymatic degradation (2). It is believed that the microbial degradation of lignin occurs through a series of radical intermediates generated by fungal enzymes. In aquatic samples, lignin present as dissolved organic matter (DOM) has been shown to be removed during photo irradiation, either directly by UV degradation or through the generation of hydroxyl radicals in solution, which then degrade lignin (3). What happens to lignin following degradation in both soil and aqueous systems, however, is not well understood. A recent study has confirmed the removal of lignin type compounds upon photo irradiation of DOM; however, this study also noted the generation of condensed aromatic compounds, similar to black carbon (BC), not previously present in the sample (4). The source of black carbon in natural samples is usually attributed to the thermal oxidation of organic matter (fossil fuel combustion, biomass burning, etc.), making this observation very significant. Several molecular characterization studies of soil humic substances using ESI-FTICR-MS have found similar BC-like material, which was initially attributed to thermogenic processes (5-7). We propose that lignin, not pyrogenic sources, may be responsible for this observed BC through microbial and oxidative alteration, specifically hydroxyl radical oxidation.
Results and Discussion Figure 1 shows a van Krevelen plot of the formulas found in the initial base extracted wood obtained from the Great Dismal Swamp, Virginia, USA, and the same sample following exposure to hydroxyl radicals. We observe the creation of a significant number of new compounds which plot in several notable regions of the diagram. Many molecules plot in the more aliphatic region (H/C > 1.2), but these molecules are also present in the degraded wood, suggesting that they are already part of the sample prior to hydroxyl radical oxidation. Most notable, however, are molecules that plot in the region commonly associated with condensed aromatic structures (H/C < 0.6). Also notable is an abundance of new peaks that emerge at higher O/C but the same H/C ratio as aromatic components of lignin in the sample, and these are believed to derive from oxidized lignin. Previous studies (2) have shown that lignin oxidation by hydroxyl radicals involve ring hydroxylation and ring opening reactions, which produce unsaturated aliphatic carboxylic acids and
Experimental Lignin was isolated from ground wood samples using a base extraction method and filtered to remove
248
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
hydroxylated acids. These unsaturated (hydroxyl) acids can undergo cyclization reactions, decarboxylation, and dehydroxylation at low activation energies to produce aromatic acids, hydroxyaromatic acids, and aliphatic/alicyclic acids. All of these types of molecules would reasonably plot in the range of H/C and O/C space shown in Figure 1. While molecules that fall within the condensed aromatic region are commonly attributed to those of pyrogenic black carbon, these results would suggest an alternative formation mechanism for black carbonlike molecules is taking place involving hydroxyl radicals.
REFERENCES
(1) Thevenot, M.; Dignac, M.-F.; Rumpel, C. Soil Biology and Biochemistry 2010, 42, 1200. (2) Higuchi, T. Proceedings of the Japan Academy, Series B 2004, 80, 204. (3) Stubbins, A.; Spencer, R. G. M.; Chen, H.; Hatcher, P. G.; Mopper, K.; Hernes, P. J.; Mwamba, V. L.; Mangangu, A. M.; Wabakanghanzi, J. N.; Six, J. Limnology and Oceanography 2010, 55, 1467. (4) Chen, H. A., H.A.N.; Sanders, R.L.; Myneni, S.C.B., Mopper, K.; Hatcher, P.G. Science 2014 (in preparation). (5) Kramer, R. W.; Kujawinski, E. B.; Hatcher, P. G. Environmental science & technology 2004, 38, 3387. (6) Ohno, T.; He, Z.; Sleighter, R. L.; Honeycutt, C. W.; Hatcher, P. G. Environmental science & technology 2010, 44, 8594. (7) Ikeya, K.; Sleighter, R. L.; Hatcher, P. G.; Watanabe, A. Rapid communications in mass spectrometry : RCM 2013, 27, 2559. (8) Sleighter, R. L. H. P. G. In Fourier Transforms –Approach to Scientific Principles; Nikolic, G., Ed.; InTech: Rijeka, Croatia, 2011, p 295.
Figure 1. Van Krevelen plot of CHO-only formulas assigned to the FTICR-MS spectra of lignin extracted from Dismal Swamp wood. Points in red are of the initial extracted lignin, and hydroxyl radical reacted sample formulas are shown in black. In summary, the humic acid extract of wood collected in the Great Dismal Swamp was exposed to hydroxyl radicals generated via Fenton reaction using iron and H2O2. A portion of the extract was altered in such a way as to precipitate from solution. The precipitate generated was not initially present, nor in the sample after adjusting the pH to 3 or the addition of iron, but rather only formed when H2O2 initiated the formation of hydroxyl radicals. The presence of black carbon-like molecules formed only after hydroxyl radical oxidation suggests that lignin-derived material can be converted to condensed aromatic compounds through this pathway. The additional shift of the compounds to a higher O/C would suggest that significant oxidation is occurring via this process. The high O/C molecules are not observed in soil humic acids (6,7). It is possible that further reactions (e.g., dehydroxylation, decarboxylation, cyclization) would result in the generation of more condensed aromatic, BC-like compounds. The evidence presented herein suggests that lignin could be responsible for a large percentage of BC observed in soil and peat and is in fact occurring through hydroxyl radical oxidation.
249
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characterization by fluorescence of Oxisol organic matter under sugarcane bagasse ash application J. Cora (a) *, L. Campos(a), D. Milori(b), D. Oliveira(a), (a)
Soil Science Department – Sao Paulo State University, Campus Jaboticabal-SP, Brazil. Zip Code 14884-900 Embrapa Agricultural Instrumentation. P.O. Box 741. 13560-970, São Carlos-SP, Brazil * Corresponding author e-mail: [email protected] (b)
Keywords: residue, laser-induced fluorescence, total soil carbon, soil management Abstract Sugarcane bagasse ash from sugar and ethanol industry contain organic matter and plant nutrients that can play an important role in agricultural production and the maintenance of soil fertility. The present study has aimed to evaluate the total soil carbon content and the humification degree of SOM by laser-induced fluorescence, following sugarcane bagasse ash application. The highest rate (80 Mg ha-1 dry bases) of sugarcane bagasse ash application to the soil caused an increase in the total soil carbon of around 1% for all Oxisoils. The humification degree of of the SOM decrease with decreasing of soil clay content. However, the humification degree increase with increasing of rate of sugarcane bagasse ash. Sugarcane bagasse ash application to the soil accelerate SOM mineralization and caused decrease of more humified SOM resulting in high levels of labile carbon and low humification degree, a phenomenon known as "priming effect. Introduction The Brazilian industry of ethanol, sugar and citrus juice generate their own electrical energy by burning sugarcane bagasse. The process generates sugarcane bagasse ash. In 2013/2014, the sugarcane production was of 530 million tons. Considering that 1000 kg of sugarcane yields 250 kg of bagasse, which after burned the process generates 25 kg of bagasse ash (10%). The sugarcane bagasse ash production in 2013/2014 growing season was 12.5 million tons, approximately. It has been disposed on soil with out any criteria supported by scientific and/or environmental law. The relevance of recycling sugarcane bagasse ash for agricultural purposes is due to its high concentration of organic matter and some plant nutrients such as N, P, Ca, Mg, Zn, Cu, Mn, which can benefit both the soil and plants. This aspect is relevant in tropical regions where occur accelerated organic matter decomposition and predominance of low cation exchange capacity soils. Control of the qualitative and quantitative changes that occur in SOM under sugarcane bagasse ash application is important to ensure the environmentally safe and agronomically efficient use of sugarcane bagasse ash as a soil amendment. However, few studies have been done to evaluate qualitative e quantitative changes in SOM due to sugarcane bagasse ash application in the soil. The chemical recalcitrance is one of the mechanisms responsible for stabilization of soil organic matter. During humification process, the increase of aromatic and alkyl structures and the increase of conformational disorders are referred as the principal roles in the organic matter recalcitrance. Fluorescence spectroscopy has been used to assess the organic humification of tropical soil subjected to different management systems (Bayer et al., 2002; Milori et al., 2002). The present study has aimed to evaluate changes in the total soil carbon content and the degree of humification of SOM by laser-induced fluorescence, following sugarcane bagasse ash application. The information obtained from this study is important because there are few field experiments
with sugarcane bagasse ash applications in tropical soils, and there is a lack of knowledge to understand possible modifications in the characteristics of SOMin tropical soils under sugarcane bagasse ash application. Experimental Soil samples have been collected from three Oxisols with different texture (clayey, loamy, and sandy) located in Jaboticabal, São Paulo State, Brazil (21º15’22” S, 48º15’18” W, and 550-m altitude). The climate is Cwa (subtropical, mesothermic, with a hot and humid summer and a cold and dry winter; the average annual temperature is 22ºC and average annual rainfall is about 1400 mm), according to Köppen climatic classification. The soil samplings were taken at a depth of 0.10 m. The experimental design was a completely randomized with six treatments and four replications, totaling 72 experimental units. Treatments consisted of a control (without sugarcane bagasse ash) and five rates of sugarcane bagasse ash (5, 10, 20, 40, and 80 Mg ha-1 (dry basis). The sugarcane bagasse ash rates were mixed with soil. Each experimental unit consisted of a 1.000 mL pot, where 0.15 kg air-dried soil + sugarcane bagasse ash were incubated for 182 days in a dark environment with temperature of 25°C. Soil moisture was maintained to field capacity along the incubation period. The sugarcane bagasse ash presented pH 8.3, total carbon content 19% and silicon oxides of 82%. The sugarcane bagasse ash surface structural groups were determined by nuclear magnetic resonance (NMR) 13C, and presented alkyl (13.3%), C-susbs (23.3%), Aril (35.7%), Phenol (8.0%), Carboxil (13.2), and Carbonil (7.4). After incubation, the incubated soil were dried and sieved (100 mesh sieve) for determination of total carbon by thermal oxidation, using a Perkin Elmer PE 2400 Series II CHNS/O Analyzer. Humification degree of SOM (HFIL) was obtained by the ratio between the mean value of area under the spectrum of fluorescence emission and the soil total carbon content mean value for the different soils (Milori et al. 2002). Data were
250
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
subjected to analysis of variance, and the means were compared by Tukey test at 5% probability.
Table 1. Soil carbon contents of the Oxisoils with different textures under sugarcane bagasse ash application. Oxisoils Clayey Loamy Sandy Ash Rate Total soil carbon (%) 0 1,38 1,22 1,04 5 1,44 1,28 1,18 10 1,45 1,34 1,27 20 1,54 1,63 1,64 40 1,78 1,72 1,75 80 2,19 2,36 2,15
Results and Discussion The highest rate (80 Mg ha-1 dry bases) of sugarcane bagasse ash application to the soil caused an increase in the total soil carbon of around 1% for all Oxisoils (Table 1). The humification degree of the SOM decrease with decreasing of soil clay content. However, the humification degree increase with increasing of rate of sugarcane bagasse ash (Table 2). The laser-induced fluorescence emission spectra showed a single band (575 nm) for all Oxisoils. However, the fluorescence intensity was influenced by soil texture as sandy>medium>clay (Figure 1 A). Higher fluorescence is due to the higher amount of fluorophores present in the organic fraction of the soil at advanced stage of humification (Milori et al., 2006). The soil organic and mineral fraction interaction is higher in clayey soils due to the recalcitrant compounds be physically protected by occlusion in microaggregates (Milori et al, 2006; Ananyeva et al, 2013). The humification degree was similar for the 5 and 10 Mg ha-1 sugarcane bagasse ash applications rates for the clayey and the loamy Oxisoils (Table 2), except for the sandy Oxisoil (Figure 1 B). The substances present in the sugarcane bagasse ash may have promoted microbial activity to produce more humified organic compounds, decreasing the humification degree of the SOM with increasing of the ash rate. However, the sandy Oxisoil, presenting lower buffering capacity, the highest humification degree was observed for the lowest ash rate. According to Giller et al. (2009), substances present in some urban and industrial wastes applied to soil, while protecting SOMfrom microbial decomposition by direct chemical interactions, can also stimulate the decomposition by providing nutrients to the microorganisms. The addition of residue of organic or inorganic nature can accelerate mineralization and cause dilution of the more humified organic compounds resulting in high levels of labile carbon and low humification degree, a phenomenon known as "priming effect". As a conclusion, the highest rate (80 Mg ha-1 dry bases) of sugarcane bagasse ash application to the soil caused an increase in the total soil carbon of around 1% for all Oxisoils. The sugarcane bagasse ash applications to the soil caused a decrease in the degree of humification of the SOM for Oxisoils with different texture. The humification degree of of the SOM decrease with decreasing of soil clay content. However, the humification degree increase with increasing of rate of sugarcane bagasse ash. Sugarcane bagasse ash application to the soil accelerate SOMmineralization and caused decrease of more humified SOMresulting in high levels of labile carbon and low humification degree, a phenomenon known as "priming effect
Table 2. Analysis of variance (F test) of the humification degree of the SOMfor Oxisoils with different texture under sugarcane bagasse ash application. Oxisol (S) HFIL (u.a.) Clayey 23.109,46 c Loamy 29.954,20 b Sandy 52.002,66 a F 1.431,89** Sugarcane bagasse ash rates (R) 0 47.873,36 a 5 42.303,63 b 10 40.300,19 b 20 31.019,48 c 40 28.343,96 d 80 20.292,01 e F 328,90 ** CV 5,58 Interaction S x R 72,94 **
A
B
ash rate nm Figure 1. Laser-induced fluorescence emission spectra (excitation wavelength in 458 nm) (A) and humification degree (B) of the Oxsoils with different texture under sugarcane bagasse ash application REFERENCES
(1) Ananyeva, K.; Wang, W.; Smucker, A.J.M.; Rivers, M.L.; Kravchenko, A.N. Soil Biology and Biochemistry, 2013. 57, 868-875. (2) Bayer, C., Martin-Neto, L., Mielniczuk, J., Saab, S.C., Milori, D.M.B.P., and Bagnato, V. Geoderma 2002, 105, 81-92. (3) Milori, D.M.B.P., Martin-Neto, L., Bayer, C., Mielniczuk, J., and Bagnato, V. Soil Sci. 2002, 167, 739-749. (4) Milori, D.M.B.P., Galeri, H.V.A., Martin-Neto, L., Diekow, M., Gonzáles-Pérez, M., Bayer, C., and Salton, J. Soil Sci. Soc. Am. J. 2006, 70, 57-63.
Acknowledgments: The authors are grateful for São Paulo Research Foundation (Brazil), for the scholarship of the second author.
(5) Giller, K. E.; Witter, E.; Mcgrath, S. P. Soil Biology and Biochemistry. 2009, 41, 2031-2037. (6) Fontaine S.; Barot, S.; Barré, P.; Bdioui, N.; Mary, B.; Cornélia, R. Nature. 2007, 450, 277–280.
251
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Cobalt and aquatic humic substances: complexation assays and ecotoxicology tests with Ceriodaphnia dubia C.H. Watanabe (a) *, A.H. Rosa(a), R. Fracácio(a), V.S. Lira(a), T.R. Sá(a), E.S.J. Gontijo(a), A. S. C. Monteiro (b).
(a) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Av. Três de Março, 511, Alto da Boa Vista, 18087-180, Sorocaba, SP – Brazil (b) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Rua prof. Francisco Degni, 55, Quitandinha, 14800-060, Araraquara, SP – Brazil * Corresponding author e-mail: [email protected] Keywords: toxicity, aquatic humic substances, cobalt, Ceriodaphnia dubia. Abstract Studies of processes capable of reducing the concentration of free metal ion, for example, complexation reactions, show the possibility of a significant decrease in toxicity. However, few studies in the literature showing the influence of the SHA in particular fractions of different molecular sizes, the toxicity of substances found in aquatic environments. Thus, the aim of this study was to evaluate the behavior of cobalt metal with the natural concentrations of SHA by chemical and biological parameters. To check the chemical behavior tests were carried out complexes concentrations and different matrices and biological assays were conducted using the toxicity test organism Ceriodaphnia dubia to determine acute and chronic toxicity data. The results presented can assist on development of water quality criteria and can contribute to the improvement of the quality assessment of freshwater. Introduction The release of pollutants into surface waters and modify the characteristics of the environment can change aquatic life present. Metals represent a class of chemical compounds that in light of anthropogenic activities, has been consistently found in bodies of water in concentrations exceeding those recommended in present legislation. Cobalt is an essential metal that plays an important role in biochemical reactions essential for life (1), but became toxic if given in high doses as well as cumulative , with their long-term exposure, which causes numerous adverse effects (2). Studies of processes capable of reducing the concentration of free metal ion, for example, complexation reactions, show the possibility of a significant decrease in toxicity. In this scene, Humic substances (HS) are the most important class of natural complexing agents, acting on sorption and biodegradation of several compounds (4). It is estimated that half of the dissolved organic carbon (DOC) disposed in shallow ocean waters and it is refractory organic material of the type SH. The study of humic substances based on molecular size, usually performed by ultrafiltration systems, can be understand the behavior of complex molecules such as humic substances being developed techniques of molecular fractionation (5). The ecotoxicological assessment has been a tool that allows the analysis of environmental quality, the associate ecotoxicological risk and concentration of contaminants in the using of acute and chronic toxicity tests with aquatic organisms. Emphasizing this issue, the present study evaluates the toxicity of cobalt in presence and absence of aquatic humic substances taking into account chemical and biological parameters by using test organism and complexation assays, respectively.
Sample of aquatic humic substances (AHS) was characterized and extracted of waters by Sorocabinha River, localized in the city of Iguape-SP in Brazil, region characterized by high concentrations of natural organic matter. Procedures of humic substances extraction were based on adsorption/desorption principle. The chromatographic method on packed with XAD resin column was used for such extraction where the sorbed fraction is eluted with an aqueous solution of sodium hydroxide 0.1 mol L-1. (6) The water extracted humic substances were subjected to molecular fractionation by a system ultrafiltration (UF) separating the AHS extract in five fractions: F<5kDa, 5
Experimental
252
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
freshwater C. dubia were performed assays lasting 7 to 8 days, at a temperature controlled 23 and 27 degrees and photoluminescence 16h light and 8h dark environment, occurring exchange of the test solution every two days as well as food given daily as established by norm. The assays allowed identification of acute and chronic toxicity through the parameters mortality and reproduction, respectively.
60 50 40 30 20 10 0
F>100
30
5
Treatment
Figure 3. Reproduction data of cobalt concentration exposures using AHS.
Response of concentration identifies high interferences of cobalt as in presence as absence AHS. Data of reproduction obtained through the assays indicate AHS generate a improvement compared to cobalt concentrations isolated. Furthermore, the tests matrix and AHS didn’t interfere on mortality or reproduction in some cases stimulating on reproduction. Toxicity assays with ultrafiltration fractions: The response of toxicity assays considering molecular size are disposes on Figure 4 and show toxicity in all fractions when exposed lonely in Co 100µg.L-1 concentration.
F<5
Fraction (kDa)
Figure 1. AHS fractions according molecular size.
Comparing data on literature show similar behaviour of fractions distribution on ASH extracted. Complexation assays: The results of complexation assays are disposes on Table1. Total Co (µ L-1)
Free C (µ L-1)
Complexation (%)
50
54.20
38.43
29,09
50*
52.13
2.97
100
110.03
55.23
94,30 49,80
100*
103.90
4.44
95,73
Reproduction
Solution (µg.L-1)
120
* Matrix contained reconstitute water.
100100 80
80
70
60 40 20
Control
13.5 0
0 0.3 0
0
Reproduction
F<5
5
0 Mortality
10
30
F>100
Figure 4. Reproduction and mortality results
The results of complexation assays shows different behavior enter matrix composed by reconstituted water and ultrapure water solution observing efficiency complexation on the first. This can be justified due to the introduction of nutrients that may have made easier this complexation.
Therefore, the present work showed AHS acted in the reduction concentration of cobalt followed toxicity reduction considering distribution fractions, but the same didn’t ensue exposing the organisms on fractions individually.
Toxicity assays with Ceriodaphnia dubia: Figure 2 shows the result of mortality obtained after assays of toxicity in the concentrations 50µg.L-1 and 100µg.L-1 of Co. Great party of assays reported acute effect, showing elevated toxicity acute to C. dubia in this concentrations.
REFERENCES
(1) Siegel, F.R Environmental Geochemistry of Potentially Toxic Metals. Springer, 2002 (2) Simonsen, L. O.; harbak, H.; bennekou, P.. Cobalt metabolism and toxicology: A brief update. 2012 (3) Grassi, M. T.; Cad. Tem. of Chem. New Sch. Esp. Ed. 01– 40, 2001. (4) Pereira, G. F.; Implantação de testes de ecotoxicidade para a avaliação de tecnologias de tratamento visando a redução da toxicidade de efluentes da indústria minero-metalúrgica. In: XVI In. Cie.. Rio de Janeiro , 2008. (5) Rocha, J.C.; rosa, A.H.; In: Substâncias húmicas aquáticas: interações com espécies metálicas, UNESP Ed., 2003.
80 Mortality (%)
100
100
0
Table 1.Complexation obtained from Co concentrations before (Total) and after (Free) ultrafiltration procedures by 1kDa membrane..
60 40 20 0
(neonates/organism)
Percentage (%)
Results and Discussion Characterization of AHS: The fractions percentage obtained from AHS extracted after ultrafiltration procedures are disposes in Figure 1.
of
18 16 14 12 10 8 6 4 2 0
(neonate/organism)
Reproduction
The results of reproduction (number neonates/organism) are disposes on Figure 3.
Acknowledgments: Thanks to FAPESP and CNPq to financially support this work.
Treatment
Figure 1. Compartive data of mortality exposures of Co and Ceriodaphnia dubia.
253
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Comparison of Structural Properties of Humic-Like Substances in Atmospheric Fine Aerosol Isolated by Different Methods D. Asakawa (a) *, N. Fujitake(b)
(a) Osaka City Institute of Public Health and Environmental Sciences, 8-34, Tojo, Tennoji, Osaka, 543-0026, Japan (b) Faculty of Agriculture, Kobe University, 1-1, Rokkodai, Nada, Kobe, 657-8501, Japan * Corresponding author e-mail: [email protected] ([email protected]) Keywords: Humic-Like Substances, Atmospheric Fine Aerosol, NMR, TMAH py-GC/MS, HPSEC
Introduction
Experimental Molecular size distribution: The HPSEC chromatograms of the HULISs and humic substances are shown in Fig. 1.
254
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
°; acquisition time, 0.839 s; repetition time, 2.5 s; line broadening, 50 Hz; scan number, 10,000–20,000. Assignments of carbon species were divided by dotted lines.
°
Proportions of ca
Chemical composition: TMAH py-GC/MS showed the differences in the composition between HULISs and humic substances. T
–
255
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Effect of Humic Substances on Fungal Melanin Formation
VA Terekhovа (ab) *a, AA Belik (b), NG Neizvestnaya (c), MM Gladkova (b), OS Yakimenko (b), GA Kalabin (c) (a)
Institute of Ecology and Evolution RAS, Moscow, Russia Lomonosov Moscow State University, Soil Science Faculty, Moscow, Russia (c) Peoples’ Friendship University of Russia * Corresponding author e-mail: [email protected] (b)
Keywords: humic products, dark pigments, micromycete, Cladosporium cladosporioides, NMR spectroscopy Abstract Fungal pigment melanin play a special role in hypotheses of soil humification processes, being debated as precursors of soil humic substances (HS) due to their similarities in many properties. This fact generated discussions about their mutual transformations. The objective of this study was to analyze the influence of HS on formation and composition of dark-colored fungal pigments. The pure culture of melanin-containing micromycete Cladosporium cladosporioides was cultivated with and without addition of potassium humate. Fungal biomass was determined, melanin was extracted from mycelium and analyzed using 13C NMR spectroscopy. The obtained data allowed assuming that HS can modify the melanin structure of Cladosporium cladosporioides and probably change the way of fungal melanin synthesis. Introduction Many fungal species produce dark-colored pigment melanin, which plays an important role in fungi physiology and in ecosystem functioning as a whole. Melanin production by fungi contributes to the virulence of pathogens of humans as well as those of food crops. Fungal melanins are amorphous polymers, which can be synthesized by two pathways. One is synthesis from endogenic substrates using 1,8dihydroxynaphthalene and this pheomelanins contain only C and O atoms. Another pathway involves L-3,4dihydroxyphenylalanine and resulting eumelanins can contain N atoms. But the detailed structure of melanins is still poorly understood. Fungal melanins play a special role in hypotheses of soil humification process. Certain similarities of humic substances (HS) and melanins in terms of chemical and physical properties generated discussions about their mutual transformations. Attempt to understand this question was done using comparative analysis of biodegradation ability of melanins and HS (1). Other data suggest that pheomelanins can be precursors of HS because of their similar elemental content, spectral properties, and low content of polysaccharides (2).
Experimental Cultivation of C. cladosporioides.The pure culture of melanin-containing micromycete Cladosporium cladosporioides was cultivated in Chapek nutrient medium (content, g/l: Sucrose - 30, NaNO3 3, KH2PO4-1, MgSO4*H2О0.5, KCl 0.5,FeSO4*7H2O - 0.01, agar-agar - 15). Fungal biomass for melanin extraction was cultivated in microbiological vessels (mattresses) with liquid Chapek medium under stationary conditions for 24 h in the dark during 14 days as described earlier (3). Inoculate as suspension containing 1,5-2,0*106 of spores per ml was brought into a vessel with 200 ml of medium. Inoculate was obtained by washout by the distilled sterile water from the surface of culture of the fungal culture which has been grown up in test tubes, then suspension was filtered through the sterile dense gauze filter and content of fungal propagules, represented mostly by conidia, was counted up in Gorjaev chamber. Accumulation of a biomass and melanin structure were studied at cultivation of micromycetes in following treatments: (МЕ1) – pure Chapek medium (control); (МЕ2) - Chapek medium with addition of potassium humate (PowHumus), 5 mg/l; (МЕ3) Chapek medium with addition of potassium humate (PowHumus), 10 mg/l; (МЕ4) Chapek medium with addition of potassium humate (PowHumus), 10 mg/l and nanodiamonds (ND), 5mg/l. Potassium humate (PH) «Powhumus» (produced from leonardire by Humintech Ltd.) was added from sterilized solution into liquid Capek medium. Composition of PH: potassium humate 80-85%, K2O – 12%, Fe – 1%, N – 1,3%, other elements – 1,1%, water – about 10%, pH - 9-10, Nanoparticles of detonation diamonds (ND) brand SDND (Single Digit NanoDiamond), (PLSDND, PlasmaChem GmbH, Germany) were added into liquid Capek medium as sterilized water suspension in concentration of 5 mg/l. The tested NDsample is characterized by uniformity of the basic physical and chemical properties, namely: the average particles diamond size is 3,5 nanometers, the specific surface area is 360 m2/g, negative surface charge (the zeta-potential in water is 40 мВ). The surface of ND-
However till now many details of synthesis, localization of melanin in fungi cells, their ability to incorporate into soil HS substances and also possible transformation of high-molecular humic components under influence of micromycetes remain debatable. The reasons for that is the high variety of melanin pigments at different fungi species as well as the complexity and variability of HS in soils and other environments. Previously, it was found that addition of HS to nutrient growth medium causes a different respond of dark-colored and hyaline cultures of micromycetes. Thus, potassium humate «Powhumus» restricted mycelial growth of melanized micromycetes (Alternaria alternata, Cladosporium cladosporioides, Phoma glomerata), but did not affect the growth of hyaline fungi (Fusarium oxysporum) (3). The objective of this study was to analyze the influence of HS on formation and composition of dark-colored fungal pigments.
256
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
particles is saturated with O-containing functional groups. (5) After 2 weeks of incubation Cladosporium cladosporioides fungal biomass was separated by filtering from a nutrient medium, wrung out on a filter paper, weighed and frozen at -6 C. Part of mycelia was dried for calculation of absolute-dry weight in different treatments. From the rest of mycelia biomass melanin was extracted. . Extraction of fungal melanin (FM) from Cladosporium cladosporioides was carried out according to the standard technique described earlier (2)] 13 С NMR spectroscopy. About 200-400 mg of melanins were dissolved in 5 ml of 0.3М NaOD/D2O. Mixture was placed in ultrasonic bath for 30 min at 30ºС, further centrifuged (10 min at 4500 r/min), solution was separated from precipitate and transferred to an ampoule for NMR-spectroscopy (diameter 10 mm). 13С NMR spectra were registered at temperature 20ºС using spectrometer «JNM-ECA 600» "Jeol" (Japan) with working frequency 150 MHz for nucleus 13С, delay time 8 sec, number of scannings is103-104. Processing and transformation of spectra were carried out using the software “DeltaTM” (JEOL USA, Inc.). Distribution of C-atoms was defined by integration of corresponding spectral areas as following (ppm.): 220-190 – С atoms of ketones and quinones (C = O); 190-164 - C of the carboxyl, ester and amide groups (COO); 164-149 - carbon of O, N-substituted aromatic moieties (ArO, ArN); 149-111 - carbon of unsubstituted and C-substituted aromatic moieties (Ar); 111-94 - carbon bonded by single bonds to two heteroatoms (OCHO); 94-45 - carbon bonded by a single bond to the heteroatom and mainly included in carbohydrate moeties (AlkO); 45-0 - carbon of aliphatic (alkyl) fragments, non-hetero (Alk). (4,5). Results and Discussion Fungal biomass and C. cladosporioides malanins were different in treatments with no additions and with PH and ND. The results show that the potassium humate inhibited fungal biomass formation: 2-fold decrease was observed from 6,8 g/L in control and 3,4 g/L under HS. It corresponds with earlier received data about inhibiting ability of HS in relation to pigmented fungi and other strains of C. cladosporioides and even other species (Alternaria alternata). ND in the tested concentration did not demonstrate significant inhibition of С cladosporioides growth. Under favorable environmental conditions, usually no hyperactivity of melanin-containing fungi is observed. Increase of their share is typical for stressful conditions. We consider this phenomenon as one of mechanisms of maintenance of sustainable functioning of environment due to HS-influence (8). Development of melanin-containing fungi promotes accumulation of HS, providing with that their known protection functions. The effect of HS on melanin synthesis was analyzed using 13С NMR spectroscopy to determine structure of molecular fragments of pigments. As a whole, integrated intensity of signals of separate ranges of the chemical shifts corresponding to various molecular fragments differ slightly among all the samples
(Table). However, for melanin ME 2 , which differs from others by a double decrease of fungal mycelium biomass, an increase of C- aromatic fragments at the lowest content of C-alkyl groups is observed. Also the minimum total intensity of signals of Alk-O and Alk groups (about 42 % against 45, 47 and 50 % for samples 1, 3 and 4, accordingly) is showed for ME2. Probably, this fact characterizes changes of structure and a way of melanin synthesis of under a HS-action. Table Content of structural fragments of melanin molecules in 13С NMR spectrum ranges, % Fragments of melanin molecules
220-190, С=О 190-164, СОО 164-149, ArO 149-111, Ar 111-94, OCHO 94-45, AlkO 45-0, Alk
Treatment
МЕ1
МЕ2
МЕ3
0,5±0,4 20,7±0, 2 3,1±0,9 21,1±0, 4 9,6±0,5
1,3±0,3 17,6±0,5
1,2±0,8 18,4±1,4
1,9±0,5 24,2±1,4
2,1±0,8 20,5±0,4
13,4±0,2
10,6±0,2
17,2±0, 9 27,8±0, 4
22,2±1,0
22,7±2,0
19,4±0,4
24,3±0,1
МЕ4 2,0±1,0 14,9±1, 5 0,7±0,4 21,2±0, 2 11,0±0, 8 29,2±1, 2 20,9±0, 1
According to 13C NMR data, an increase of aromatic carbon moieties combined with lower signals of alkyl carbon groups were showed for melanin synthesized in the presence of HS. Also the smaller integrated signal intensity for Alk-O and Alk groups were observed for these melanins (about 42 % versus 45 in the control). Conclusions Comparison of structural features of Cladosporium cladosporioides’ melanin revealed the effect of HS which was shown as a transformation of structural fragments of molecules (compared to control) at background of fungi growth inhibition. Action of ND was not toxic for С cladosporioides. 13 C-NMR revealed high degree of similarity between melanins and HS. An increase of aromatic carbon moieties combined with lower signals of alkyl carbon groups were showed for melanin synthesized in the presence of HS. Also the smaller integrated signal intensity groups Alk-O and Alk were observed for these melanins (about 42 % versus 45 in the control). The obtained data allowed assuming that humic substances can modify the melanin structure of Cladosporium cladosporioides and probably change the way of fungal melanin synthesis REFERENCES (1) Zavgorodnyaya Yu A., Demin V.V., Kurakov A.V. Organic Geochemistry, 2002, 33, 347-355. (2) Meuzelaar H.L.C., Haider K., Nagar B.R., Marthin J.P. Geoderma, 1977, 17, 3, .239-252. (3) Fedoseeva EV, Patsaeva SV, Terekhova VA Mikologiya i Fitopatologiya, 2009 , 43, 3, 243-250. (4) Kalabin GA, Kanitskaya LV, Kushnarev DF Chemistry, 2000 – 408. (5) Kholodov VA, Konstantinov AI, AV Kudryavtsev, Perminova IV Eurasian Soil Science. 2011, 9, 1064-1073.
Acknowledgments: This work is supported by Russian Foundation for Basic Research (grant No 1204-01230- a).
257
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Soil organic carbon after applying roasted mate tea residue F.B. Pereira(a), K.C. Lombardi(a)*, O.T. Mendes(a)
(a) Universidade Estadual do Centro-Oeste – UNICENTRO, Campus Irati, PR 153, km 7, Riozinho, Irati, Paraná, Brazil, Zip Code 84500-000. * Corresponding author e-mail: [email protected] Keywords: oxidizible fractions, residue, agroindustry, Ilex paraguariensis Abstract Most conventional methods for organic C determination aim the maximum oxidation and recovery of C. However, measures of total C content may not be a sensitive indicator of soil quality changes. Methods that extract preferentially the more labile C allows to evaluate in more detail the changes caused by soil organic management practices. This study had the objective to evaluate the total organic carbon (TOC) and oxidizible fractions of soil organic C (F1, F2, F3, F4), after applying different doses of roasted mate tea residue (0, 40, 80 and 120 t.ha-1) by incorporation, at three depths (0-5, 5-10 and 10-15 cm). The results show the roasted mate tea residue incorporation increases the COT and oxidizible fractions of soil organic C (F1, F2, F3, F4), suggesting the residue decomposition. All the chemical variables evaluated were statically higher with the addition 80 and 120 t.ha -1 of residue. Introduction Soil organic matter (SOM) management can be an effective way to preserve the soil quality, aiming to improve soil nutrient pool and structure stability, enhance cation exchange and water holding capacities, retain toxic elements, and increase C storage providing energy source to the heterotrophies microorganisms (1). The organic management can be accomplished by industrial wastes application such as the roasted mate (Ilex paraguariensis A. St.-Hill.) leaves, which is a residue from the ready-to-drink line of mate tea industry. Besides improving soil C content, that also provides a proper destiny for this industrial residue promotes the recycling and reduces material waste in the product processing (2). Total content C measures may not be a sensitive indicator of soil quality changes. Adopting, therefore, methods that extract preferentially the more labile C, might allow better evaluate the changes caused by soil organic management practices (3). Chan et al. (2001) proposed a modification in the Walkley & Black (1934) classical method of organic C determination, separating four fractions with decreasing degrees of oxidation, by the increasing concentration of sulfuric acid. F1 and F2 fractions are the most labile C form, called light fraction, related to availability of soil nutrients and macroaggregates stability (4). F3 and F4 fractions are related to the chemical stability and large molecular weight compounds, from the decomposition and humification of OM (5). This study aimed to evaluate the changes in the total organic carbon (TOC) and fractions of soil organic C after applying different doses of roasted mate tea residue by incorporation.
according to the climatic classification of Köppen, the climate is Cfb (temperate). Soil was incubated in PVC columns (height=30 cm, ᴓ=10 cm), with five compartments (height=5 cm). To fill the columns was used a NITSSOLO VERMELHO Distrófico típico (6): i) 0-10 cm: A horizon soil; ii) 1025 cm: B horizon soil. The mate tea residue is derived from the extraction of roasted mate tea concentrated, used in the manufacture of liquid line of the company (products "ready to drink"). The residue C:N ratio is 73:1. The experiment was set in randomized block design (RBD), with two factors (doses of residue and depths), three repetitions and four treatments: - T1: control treatment, without residue application. - T2: incorporation of 10 t/ha of residue. - T3: incorporation of 20 t/ha of residue. - T4: incorporation of 40 t/ha of residue. The residue was incorporated at 15 cm depth. After 200 days of residue application (July 2013), soil sampling was performed at the depths 0-5 cm, 5-10 cm and 10-15 cm. Samples were oven-dried at 50ºC, ground and sieved at 0,212 mm (65 Mesh). TOC was determinate using an adapted method (3). 0.25 g soil were placed into a test tube with 2,5 mL of K2Cr2O7 0,167 Mol L-1 and 3,75 mL of sulfuric acid concentrated. The test tubes were placed into the digester. The Cr2O7- excess were titrated with (Fe(NH4)2(SO4)2.6H2O) 0,2 Mol L-1 and Diphenylamine 1% indicator. In this work, soil oxidizible C fractions was extracted using an adapted method (3). 0,5 g were placed into 125 mL flasks with 10 mL of K 2Cr2O7 0,167 Mol L-1 and the corresponding amounts of sulfuric acid concentrations of 3, 6, 9 and 12 Mol L-1. We obtained the following fractions: (F1) - C oxidized by the acid concentration of 3 Mol L-1; (F2) - difference between the C oxidized by the acid concentrations 6 Mol L-1 e 3 Mol L-1; (F3) - difference between the C oxidized by the acid concentrations 9 Mol L-1 e 6 Mol L-1; (F4) -
Experimental The experiment was established in December 2012 in the city of Irati, Paraná, Brazil, (25 º 27' 56" S and 50º 37' 51" W). The altitude is 950 meters and,
258
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
difference between the C oxidized by the acid concentrations 12 Mol L-1 e 9 Mol L-1. ANOVA was performed to evaluate possible statistical differences between doses, application modes and factors interaction. The Bartlett test was applied to verify the variances homogeneity and the Tukey test at 1 or 5 % probability for evaluation of average. Results and Discussion The interaction between the factors doses of residue and depths was significant only for COT, F1 and F2. Both factors, in separate, were significantly different to F3 and only the factor doses of residue were significantly different to F4. Total Organic Carbon: [Table 1] COT was statistically higher with addition of 80 and 120 t.ha-1 residue dose at three depths evaluated, keeping the values elevated in the soil profile, indicating the residue decomposition during the experiment. However, the decomposition of a residue with a high C:N ratio (73:1) may result in nitrogen deficiency to the plants; a way to avoid this problem is the residue composting before applying in the soil, to reduce the C:N ratio. Oxidizible Organic Carbon Fractions: F1 fraction content [Table 1] were statically higher with addition of 80 and 120 t.ha-1 residue dose at three depths evaluated, keeping the values elevated in the soil profile. The F1 decrease from the superficial depth to the second one resulting from the production of water-soluble organic acids with low molecular weight during the residue decomposition process, which are leached at soil profile (7). Since F1 fraction is considered a good indicator of quality soil changes (8), the F1 increase with residue incorporation at three depths suggests this application way was adequate to promote the SOM mineralization and nutrients availability. There were significant changes for F2 fraction only at third depth [Table 1]: the higher organic C contents were found with incorporation of 80 and 120 t.ha-1 residue dose, and the treatment with 80 t.ha-1 dose did not differ from the treatment with 40 t.ha-1 residue dose. As all the incorporated doses were statically different from the control treatment, the results indicates a formation of F2 fraction during the residue decomposition at this depth. The highest contents of F3 fraction were found with addition of 80 and 120 t.ha-1 residue dose [Table 2]. In depth, the F3 highest content was found at depth 5-10 cm. This result suggests the dose increase elevated the soil F3 content and the formation of this fraction was favored in depth. For F4 fraction [Table 2] only the dose factor were significantly different: the highest doses incorporated, 80 and 120 t.ha-1, promoted higher contents than the control treatment. The F3 and F4 fractions results indicate during the residue decomposition more stable carbon compounds are formed, contributing with the soil C stock in the medium and long term.
Table 1. Interaction averages of soil total C contents and averages of soil oxidizible organic C fractions F1 and F2 contents, after application of roasted mate tea residue doses (factor 1), by incorporation, in depth (factor 2). *significant at 5% probability by Tukey test (0,01=
COT (g.dm-3)
F1 (g.dm-3)
F2 (g.dm-3)
Variation sources Doses (t.ha-1) 0 40 80 120 Fcalc p-value
0-5 25,54 bA 26,67 bA 35,89 aA 35,39 aB
Doses (t.ha-1) 0 40 80 120 Fcalc p-value
0-5 7,71 bA 7,73 bB 9,48 aB 10,59 aB
Doses (t.ha-1) 0 40 80 120 Fcalc p-value
0-5 6,14 aA 6,13 aA 8,03 aA 6,97 aB
Depth (cm) 5-10 10-15 26,38 bA 21,67 cB 28,58 bA 28,09 bA 36,86 aA 35,95 aA 39,26 aA 36,94 aAB 2,7307* 0,0389 Depth (cm) 5-10 10-15 8,67 bA 8,04 cA 9,30 bA 10,56 bA 12,55 aA 13,02 aA 12,64 aA 12,56 aA 3,0735* 0,0244 Depth (cm) 5-10 10-15 5,15 aAB 4,02 cB 5,05 aA 6,65 bA 5,54 aB 7,71 abA 6,81 aB 9,22 aA 3,8027** 0,0095
Table 2. Averages of soil oxidizible organic C fractions F3 and F4, to roasted mate tea residue doses factor and F3 averages to depth factor. * significant at 5% probability by Tukey test (0,01=
F3
F4
Depth (cm)
F3
0 40 80 120 Fcalc p-value
6,19 b 7,07 b 9,88 a 10,12 a 60,3762 ** <0,001
5,10 c 5,57 bc 7,58 a 7,51 ab 6,5972 ** 0,0024
0-5 5-10 10-15
8,57 b 9,66 a 6,72 c
Fcalc p-value
45,4351** <0,001
REFERENCES
(1) Bayer, C.; Mielniczuk, J. Fundamentos da matéria orgânica do solo: ecossistemas tropicais e subtropicais. 2008, 2, 1-5. (2) Pereira, F.B.; et al. Functions of natural organic matter in changing environment. 2012, 165-167. (3) Mendonça, M.; Matos, E.S. Matéria orgânica do solo: métodos de análise. 2005, 14-23. (4) Chan, K.Y.; Bowman, A.; Oates, A. Soil. Sci.. 2001, 166, 61-67. (5) Loss, A. et al. R. Bras. Ci. Solo. 2009, 1, 57-64. (6) EMBRAPA. Sistema brasileiro de classificação de solos. 2009. (7) Vilar, P.F. Atributos bioquímicos de um argissolo amarelo cultivado com adubos verde e de um latossolo húmico após aplicação de calcário. 2013. (8) Majumder, B. et al. Soil. Sci. 2008, 72, 775-785.
Acknowledgments: CNPq and Fundação Araucária for financial support.
259
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Fluorescence Spectroscopic Characterization of a Hydrophobic Base by DAX-8 Fractionation in a Biologically Treated Municipal Sewer Effluent T. Kusakabe(a) *, N. Wada(a), Y. Shimizu(a)
(a) Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga 5200811, Japan. * Corresponding author e-mail: [email protected] Keywords: EEM fluorescence spectroscopy, DAX-8 fractionation, hydrophobic base (HoB), effluent organic matter (EfOM), biologically treated municipal sewer effluent (BTSE), water reclamation Abstract The water reuse is critically important in the sustainable water use system both in (semi)arid regions and rapidly urbanized areas. A biologically treated municipal sewer effluent (BTSE) or secondary effluent is spotlighted as a vital and plentiful water source in urban areas. Effluent organic matter (EfOM) in the secondary effluent has to be well understood to optimize water reclamation technologies depending upon local demands relevant to water quantity and quality, safety/risk, and energy consumption. The objective of this research is to investigate the fluorescence spectroscopic characteristics of a hydrophobic base (HoB) operationally defined by the DAX-8 fractionation in a secondary effluent from a municipal wastewater treatment plant (WWTP) in Japan. The results implied that using only EEM fluorescence spectroscopy for characterizing EfOM potentially achieves an inappropriate interpretation. Fulvic-like and humic-like peaks in the EEM spectrum of EfOM are caused not only by hydrophobic acids (HoA), but also by hydrophobic bases (HoB). combination of these, etc. depending upon local demands relevant to water quantity and quality, safety/risk, and energy consumption. With these considerations, the objective of this research was set to investigate fluorescence spectroscopic characteristics of a hydrophobic base (HoB) operationally defined by the DAX-8 fractionation in a secondary effluent from a municipal wastewater treatment plant (WWTP) in Japan.
Introduction Fluorescence spectroscopy has been widely applied to characterize and trace dissolved organic matter (DOM) and its fractions in the aquatic environments for decades (1). Its advantages over UV-Vis absorption spectroscopy are high sensitivity and the power to differentiate chromophoric DOM (CDOM) and/or fluorophores in samples. Excitation-emission matrix (EEM) fluorescence spectroscopy has been extensively used in combination with a multivariate analysis such as parallel factor analysis (PARAFAC) (2-3). More recently, fluorescence spectroscopic techniques are recognized as a powerful monitoring tool for water/wastewater treatment fields despite practical problems including the inner filter effects and fluorescence quenching by matrix components in water samples (4). Isolation and fractionation of DOM and aquatic humic substances has been generally applied to investigate these physico-chemical and spectral properties, reactivity, interaction, bioavailability, and environmental behaviour. DOM is comprehensively fractionated based upon hydrophobic-hydrophilic and acid-neutral-base properties by using a series of resin adsorption columns for further analyses and characterizations (5). The water reuse is critically important in the sustainable water use system both in (semi)arid regions and rapidly urbanized areas. A biologically treated municipal sewer effluent (BTSE) or secondary effluent is spotlighted as a vital and plentiful water source in urban areas (6). Effluent organic matter (EfOM) in the secondary effluent has to be well understood to optimize water reclamation technologies such as membrane filtration, chemical oxidation, and a
Experimental Methodologies Water sample was collected from a municipal WWTP with the conventional activated sludge process in Japan in June, 2013, and was filtered with precombusted Whatman GF/B. The HoB fraction was operationally defined as a fraction back-eluted from a DAX-8 resin column (bed volume = 3 mL) with 0.1 N HCl (ca. 9 mL) after passing the prefiltered water sample (200 mL) through the column at neutral pH (5, 7). DAX-8 fractionation was conducted in duplicate. Fluorescence measurements were carried out by a Hitachi F-4500 spectrofluorometer with a 150 W Xe lamp. EEM spectra were acquired by scanning both excitation (Ex) and emission (Em) wavelengths from 200 to 600 nm. Instrumental parameters were slit width of 5 nm both for excitation and emission sides; scan speed of 2,400 nm/min; PMT voltage of 700 V; and response time of 0.1 sec. The blank spectrum was collected with MQ water on each measurement date, and then was subtracted from all EEM spectra. In order to correct for any fluctuations in instrumental conditions, measured fluorescence intensity was divided by intensity of a water Raman scattering peak at Ex/Em = 350/395 nm.
260
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Excitation wavelength (nm)
Results and Discussion Dissolved organic carbon (DOC) concentration of the secondary effluent was 5.6 mgC L-1. DAX-8 fractionation demonstrated that the HoB fraction accounts for only 1.3% of DOC in the sample. EEM spectrum of HoB was shown in Figure 1. Fulvic-like and humic-like peaks, which were assigned as hydrophobic acids, were unexpectedly appeared even in the EEM spectrum of HoB in the secondary effluent. The hydrophobic acid (HoA) fraction accounted for 28.9% of DOC in the sample. Fulvic-like and humiclike peaks were detected in the EEM spectrum of HoA by definition.
Emission wavelength (nm)
Figure 1. EEM spectrum of hydrophobic base (HoB) fraction in the biologically treated municipal sewer effluent (secondary effluent). Overall, these results implied that using only EEM fluorescence spectroscopic characterization of EfOM potentially achieves an inappropriate interpretation. Fulvic-like and humic-like peaks in the EEM spectrum of EfOM are caused not only by hydrophobic acids (HoA), but also by hydrophobic bases (HoB). Further data accumulation and investigation are necessary for scientific understanding of the role and function of EfOM in wastewater reclamation. References
(1) Coble, P.G. Marine Chemistry 1996, 51, 325-346. (2) Bro, R. Chemom. Intell. Lab. Syst. 1997, 38, 149-171. (3) Stedmon, C.A.; Bro, R. Limnol. Oceanogr.: Methods 2008, 6, 572-579. (4) Henderson, R.K.; Baker, A.; Murphy, K.R.; Hambly, A.; Stuetz, R.M.; Khan, S.J. Wat. Res. 2009, 43, 863-881. (5) Leenheer, J.A. Environ. Sci. Technol. 1981, 15, 578-587. (6) Shon, H.K.; Vigneswaran, S.; Snyder, S.A. Critical Reviews in Environ. Sci. Technol. 2006, 36, 327-374. (7) Imai, A.; Fukushima, T.; Matsushige, K.; Inoue, T.; Ishibashi, T. Jpn. J. Limnol. 1998, 59, 53-68.
261
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characterizing the Chemical Composition of Bacterially-Secreted Organic Matter as a Function of Carbon Sources L. Aristilde*, S. S. Sasnow, H. Wei
Department of Biological and Environmental Engineering, College of Agricultural and Life Sciences, Cornell University, Ithaca, NY 14853 USA * Corresponding author e-mail: [email protected] Keywords: soil bacteria; carbon metabolism; carbon cycling. Abstract Using liquid chromatography-mass spectrometry, we characterized the distribution of low-molecularweight organic molecules secreted by the ubiquitous soil bacterium Pseudomonas putida grown on three organic substrates commonly found in soils: glucose, citrate, and succinate. We identified 92 distinct extracellular metabolites based on both accurate mass-to-charge ratios and retention times. These metabolites included amino-containing compounds important in nitrogen cycling, and polycarboxylates and polyphosphates known to be involved in metal complexation and mineral dissolution. Hierarchical clustering revealed that the relative secreted amounts were carbon-source dependent whereby the metabolism of the main organic carbon source seemed to dictate the chemical diversity of the organic secretions. These findings shed light on the link between nutrient availability, carbon metabolism, and organic matter cycling mediated by soil bacteria. Introduction Soil bacteria are intimately involved in the cycling of organic matter (12). The purpose of the present study was to shed light on how carbon utilization determines organic matter chemistry secreted by soil Pseudomonas, which are aerobic gram-negative bacteria that are ubiquitous in natural soils and waters. These bacteria are well known for exhibiting metabolic diversity (2). Of special interest is their colonization in the rhizosphere because many soil Pseudomonas are proven plant growth-promoting bacteria (3). The rhizosphere is rich in organic nutrients including glucose and organic acids secreted by plant roots to recruit microorganisms (4), which in turn secrete organic molecules including N-containing and metal-scavenging molecules beneficial to the plant and to the surrounding microbial community (5). Here we investigated the influence of three carbon sources (glucose, citrate, succinate) common in soils on the chemical composition of secreted organic molecules by Pseudomonas putida. We combined nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry to measure the substrate consumption and characterize the secreted organic molecules.
based on their accurate mass-to-charge ratios (74≤ m/z ≥ 808) and retention retention times. The accuracy of metabolite detection was ascertained via standard measurements of commercially available compounds. Results and Discussion Growth Phenotype and Carbon Consumption: Similar doubling time was measured during exponential growth on glucose, citrate, and succinate (respectively, 2.95±0.09, 2.91±0.09, 2.89±0.09 h). However, total carbon consumption differed. While there was near complete consumption of citrate and succinate, only one third of the glucose total carbon was consumed. The lack of positive correlation between growth kinetics and carbon consumption for all three substrates suggested different metabolic fates of the organic substrate, consistent with the fact that glucose enters the carbon metabolic network via the upper glycolysis whereas citrate and succinate enters via the tricarboxylic acid (TCA) pathway (Figure 1A). Organic Secretions and Carbon Metabolism. The profile of the 92 extracellular metabolites indicated that carbon metabolism may play a role (Figure 1B). In the presence of glucose, increased levels of glycolytic (glucose-6-phosphate, gluconate, 3phosphoglycerate, dihydroxy-acetone-phosphate) and pentose-phosphate pathway (ribose-5-phosphate, xylulose-5-phosphate, sedoheptulose-7-phosphateP, histidine) metabolites were consistent with the entry of glucose via upper glycolysis (Figures 1). In the presence of citrate, TCA cycle-associated amino acids (asparagine, threonine, methionine, ornithine) were relatively high. However, the levels of these amino acids were lower in the presence of succinate. Succinate initial catabolism directly feeds into lower glycolysis, which may explain the elevated amounts of some glycolytic compounds in the presence of succinate. Noticeably, growth on succinate led to a relatively high amount of nucleosides such as cytidine, inosine-, uridine- and guanosine-phosphates. Due to the similar bacterial growth on the substrates, the difference in the extracellular nucleoside content was
Experimental P. putida (strain KT2440) were grown (20 °C) in minimal growth medium (pH 7.0) containing glucose, citrate, or succinate as the sole organic carbon source (308 mM of carbon) and adequate mineral salt concentrations. Growth was monitored by measuring the optical density of the cells at 600 nm. The depletion of organic substrates in the growth medium was monitored using high resolution 1H nuclear magnetic resonance. To characterize extracellular metabolites, we analyzed filtered samples by reversedphase ion-pairing liquid chromatography coupled with electrospray ionization high-resolution accurate-mass mass spectrometer operated in negative mode. Parameters were according to a previous study (6) with some modifications. We targeted 92 compounds
262
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
A Glycolysis
Gluconate
G6P
R5P
6-PG
F6P
F6P
S7P
Ru5P CO2
FBP
B
His, IMP, UDP, UMP, IMP, GDP
Glucose
Xu5P
KDPG
GAP
E4P
Citrate GAP
DHAP
F6P
GAP
NADH
Pentose-phosphate pathway
1,3-bisPG ATP 3-PG
Ser, Gly, Cys
PEP
Trp, Tyr, Phe
ATP
lactate Pyr
CO2, (ATP)
hydroxybenzoate
Ala, Leu, Val
CO2, NADH Ac-CoA
CO2
citrate
CO2
oxaloacetate Glyoxylate
GAP
Citrate
shunt
NADH
Ac-CoA malate
Asp, Asn, Thr, Lys, Met, Ile
Xu5P aconitate
glyoxylate
fumarate succinate
Succinate
isocitrate CO2, NADH 2-ketoglutarate CO2, NADH, GTP
Glu, Gln, Pro, Orn, Arg
Tricarboxylic acid cycle
Figure 1. (A) Schematic of the central carbon metabolism of P. putida showing the point of entry of the different substrates (gray boxes), the catabolic routes via glycolysis, the pentose-phosphate pathway, and the tricarboxylic acid cycle. (B) Unsupervised hierarchical clustering of the extracellular metabolome (92 metabolites) for each growth condition; the two columns for each condition represent data obtained from two independent biological replicates. Abbreviations in A are as explained in part B or as follows: 2-keto-3-deoxy-6-phosphogluconate (KDPG); fructose-6-phosphate (F6P), glyceraldehyde-3-phosphate (GAP); erythrose-4-phosphate (E4P); 1,3-bisphosphoglycerate (1,3-BisPG); acetyl-CoA (Ac-CoA); nicotinamide adenine dinucleotite (NADH); adenosine triphosphate (ATP); guanosine triphosphate (GTP); amino acids are shown as their three-letter codes. In B, red-colored metabolites were included in the metabolic network illustrated in part A. I.; Lehmann, J.; Manning, D.,A.C.; Nannipieri, P.; Rasse, D.P.; Weiner, S; Trumbone, S.E. Nat. 2011, 478, 49-56. (2) Poblete-Castro, I.; Becker, J.; Dohnt, K.; dos Santos, V.; Wittmann, C.Appl. Microbiol.Biotechnol. 2012, 93, 22792290. (3) Molina, L.; Ramos, C.; Duque, E.; Ronchel, M. C.; Garcıa, ́ J. M.; Wyke, L.; Ramos, J. L. Soil Biol. Biochem. 2000, 32, 315-321. (4) Jones, D. Plant Soil 1998, 205, 25-44. (5) Archana, G.; Buch, A.; Kumar, G. N. In Satyanarayana, T.; Johri, B. N., Eds. Springer Netherlands: 2012; pp 35-53. (6) Lu, W.; Clasquin, M. F.; Melamud, E.; AmadorNoguez, D.; Caudy, A. A.; Rabinowitz, J. D. Anal. Chem. 2010, 82, 3212-3221. (7) Lehmann, J.; Solomon, D.; Kinyangi, J.; Dathe, L.; Wirick, S.; Jacobsen, C. Nat. Geosci. 2008, 1, 238-242.
likely due to differences in the intracellular carbon metabolism. Thus, the chemical diversity of the organic secretions was partly controlled by the overlap between the biosynthetic steps of the secreted compounds and the initial metabolic steps of the main carbon source. Our findings set the stage for further investigation towards deciphering the link between the intracellular metabolic dynamics and the chemical composition of bacterially-secreted organic matter, which may contribute to the nanoscale spatial heterogeneity in soil organic matter composition (7). REFERENCES
(1) Schmidt, M.W.; Torn, M.S.; Abiven, S.; Dittmar T.; Guggenberg, G.; Janssens, I.A.; Kleber, M.; Kögel-Knabner,
263
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
UV-VIS spectroscopic properties of humic acids from the rusty soils under Pinus silvestris natural forest and managed by clear-cutting E. Jamroz(a)*, M. Jerzykiewicz(b), J. Weber(a), A. Medynska – Juraszek(a), I. Cwielag – Piasecka(a), J. Bekier (a) (a)
Wroclaw University of Environmental and Life Sciences, Institute of Soil Science and Environment Protection, Grunwaldzka 53; 50-357 Wroclaw, Poland (b) Wroclaw University, Faculty of Chemistry, F. Joliot-Curie 14, 50-383 Wroclaw, Poland * Corresponding author e-mail: [email protected] Keywords: humic acids, UV-VIS, forest soils, clear-cutting
Abstract.
Aim of the study was to characterize humic acids from the soils covered by Pinus silvestris in natural forest ecosystems and after clear-cutting in the lowlands, middle region of Poland. Soils in the study area were covered by coniferous forests, coniferous mixed forests and broadleaf mixed forests. Results shows that forest harvesting enhances mineralization and humification processes which lead to form humic acids of higher molecular weight in Oa horizons of poor coniferous and coniferous mixed stands. Higher values of E4/E6 in humic acids from A horizons in the natural forest stands, confirm that in poor pine ecosystems soil organic matter is characterized by humic substances of lower humification degree in comparison to the harvested. Introduction Polish forest resources are an important part of the natural environment of the country. They are also a significant part of the geographic space, occupying 30.4% of the country area, and are mainly publicly owned (81.3%). Along with shelterbelts, forests are second only to agriculture as a form of land use in Poland. The main species in the lowlands is Pinus silvestris, prevailing in 79% of forest stands. Forest ecosystems are an important source of carbon and play a major role in the global C cycle. The average C content of forest soils is about 120 t/ha when the mean of all ecosystem soils is about 80 t/ha (Hedde et al., 2008). Hugh looses of C in the forest ecosystems can be caused by harvesting. Thus in the last decades scientists have been trying to evaluate what is the effect of various disturbances, on carbon stocks. Clear-cutting alters carbon cycle through inducing changes in soil organic matter (SOM) decomposition (Falsone et al., 2012). Properties of SOM in forest ecosystems can differ depending on the parent rock, soil type, climate, altitude, type of management and especially on the tree species, particularly in the forest floor horizons (Lal, 2005; Ussiri and Johnson, 2007). The composition of litter differs widely among plant species and consists mainly of a mixture of polysaccharides and lignin, aliphatic biopolymers and tannins (Kögel-Knabner, 2002). Aim of the study was to characterize humic acids from the soils covered by Pinus silvestris in natural forest ecosystems and after clear-cutting in comparison to the coniferous and broadleaf mixed forest stands.
Material and methods Soils in the study area were covered by coniferous forests (Pinus s.), coniferous mixed forests (Pinus s., Picea a.,.) and broadleaf mixed forests (Quercus p , Fagus s., Pinus s., Picea a.). Two sampling areas in each forest stand were chosen: the harvested and undisturbed – natural forests. Explanation of the objects: CNF – coniferous natural forest, CMNF – coniferous mixed natural forest, BMNF – broadleaf mixed natural forest, C-C – clear cutting. All soil profiles were described as Brunic Arenosols developed from loose quartz sand. Humic acids were extracted from the litter (Oa) and mineral (A) soil horizons according to the method recommended by International Humic Substances Society. Soil organic carbon was analysed using CS-mat 5500 analyser, total nitrogen was determined by Kjeldahl method using Buchi N analyzer. Elemental analysis of HA was performed for C, H, N with a Perkin-Elmer 2000 instrument. O was calculated with the mass balance. UV-VIS spectra were recorded using Hach-Lange spectrophotometer DR – 5000. Results and Discussion In humic acids from Oa horizons of the forest stands after clear-cutting lower content of carbon and higher content of oxygen was found (Tab.1). The concentrations of hydrogen differed between litter and mineral horizons and were lower in humic acids extracted from A horizons in comparison to the Oa. Harvesting affected content of hydrogen in HA as well. In the analyzed soil horizons lower content of H in the forest stands after clear-cutting was found. Higher values of H/C, indicating higher aliphacity was found in HA from Oa horizons under natural
264
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
forest stands, what points to more intense processes of soil organic matter transformations after clearcutting forest management than in natural – undisturbed ecosystems. Table 1
Elemental analysis of humic acids from rusty soils under natural pine forest and managed by clear-cutting. Explanation in the text.
Object 1C-C 1CNF 2C-C
Soil horizon
N
C
H
O
H/C
O/C
Oa
1.61 33.20 47.13
18.07
1.42
0.54
A
1.96 35.96 41.87
20.21
1.16
0.56
Oa
1.47 32.60 50.79
15.15
1.56
0.46
A
1.78 35.79 42.29
20.13
1.18
0.56
Oa
1.30 33.61 49.01
16.08
1.46
0.48
A
2.04 35.01 41.26
21.69
1.18
1.27 32.87 49.92
15.94
1.94 35.40 41.82
20.84
2CMNF Oa A
0.62
4.33
1.52
0.48
2C-C
2.98
4.35
1.18
0.59
2CMNF
6.73
3.81
3CC
3.02
3.96
3BMNF
1.20
3.82
Our results show that humic substances are good indicators of environment disturbance caused by human activity, such as type of forest management.
Absorbance
2C-C 2CMNF
REFERENCES Falsone G., Celi L., Caimi A., Simonov G., Bonifacio E. 2012. The effect of clear cutting on podzolisation and soil carbon dynamics in boreal forests (Middle Taiga zone, Russia). Geoderma, 177-178, 27-38. Hedde M., Aubert M., Decaens T., Bureau F. 2008. Dynamics of soil carbon in a beechwood chronosequence forest. Forest Ecology and Management, 255, 193-202. Kögel-Knabner I. 2002. The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter. Soil Biology & Biochemistry, 34, 139-162. Lal R. 2005. Forest soils and carbon sequestration. Forest Ecology and Management, 220, 242-258 Ussiri D.A.N. & Johnson C.E. 2007. Organic matter composition and dynamics in a northern hardwood forest ecosystem 15 years after clear-cutting. Forest Ecology and Management, 240, 131-142.
3C-C 3BMNF
nm
0
nm
250 280 365 465 496 533 574 619 665
Figure 1. Effect of clear-cutting on the optical density of humic acids extracted from Oa horizons of the rusty soil. Explanations: CNF – coniferous natural forest, CMNF – coniferous mixed natural forest, BMNF – broadleaf mixed natural forest, C-C – clear cutting
Absorbance
7
3
A 4.18
1
4
Oa
6.16
2
5
Object
3.79
1CNF
3
6
E4/E6 values of humic acids from rusty soils under natural forest and managed by clear-cutting. Explanation in the text.
1C-C
1C-C
4
Table 2
1CNF
6 5
UV-VIS spectroscopy (Fig.1 and 2) shows that in poor forest stands, harvesting increases mineralization and humification processes which lead to form humic acids of higher molecular weight in Oa horizons. Higher values of E4/E6 (Tab. 2) in humic acids from A horizons in the natural forest stands confirm that in poor pine ecosystems soil organic matter is characterized by humic substances of lower humification degree, and humic acids with lower weight in comparison to those, where clearcutting was the form of management.
1C-C 1CNF 2C-C 2CMNF
Acknowledgments: The project was financed by the National Science Center (grant no. N N305 155937)
3C-C 3BMNF
2 1 0
250 280 365 465 496 533 574 619 665
Figure 2. Effect of clear-cutting on the optical density of humic acids extracted from A horizons of the rusty soil. Explanations: CNF – coniferous natural forest, CMNF – coniferous mixed natural forest, BMNF – broadleaf mixed natural forest, C-C – clear cutting
265
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Growth of gold nanoparticles in presence of humic substances as revealed by optical spectroscopy methods E. Shirshin (a)*, A. Polyakov(b), V. Lebedev(b), A. Goldt(b), E. Goodilin(b, c), V. Fadeev(a) and I. Perminova(c)
(a) M.V. Lomonosov Moscow State University, Faculty of Physics, 119991, Leninskie gory 1/2, Moscow, Russia (b) M.V. Lomonosov Moscow State University, Faculty of Materials Science, 119991, Leninskie gory 1/73, Moscow, Russia (c) M.V. Lomonosov Moscow State University, Faculty of Chemistry, 119991, Leninskie gory 1/3, Moscow, Russia * Corresponding author e-mail: [email protected] Keywords: humic substances, gold nanoparticles, plasmon resonance, fluorescence spectroscopy, energy transfer Abstract We report the study of gold nanoparticles (AuNPs) growth in presence of humic substances (HS) using optical spectroscopy methods – dynamic light scattering (DLS), UV-visible absorption and fluorescence spectroscopy. During the AuNPs synthesis, the evolution of surface plasmon resonance peak was observed, followed by the HS fluorescence band deformation, which can be attributed to excitation energy transfer in the HS-AuNPs system. Au nanoclusters transformation was also monitored by the relative changes of particle size distribution calculated from DLS data, indicating Au seed aggregates of random complex morphology in the beginning of the synthesis process. This fact was also confirmed using transmission electron microscopy. growth. Finally, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to monitor colloid particles size in the HS-AuNPs system.
Introduction Gold nanoparticles (AuNPs) are extensively used in different sensing applications due to the presence of surface plasmon resonance (SPR), which is responsible in certain cases for fluorescence emission and Raman scattering enhancement. Recently, AuNPs synthesis based on reduction of Au(III) species by HS was described (1, 2). While being a polydisperse heterogeneous system, HS possess a unique ability to bind various contaminants, e.g., polyaromatic hydrocarbons (PAH). Hence, HS-coated nanoparticles can serve as nanosensors for pollutants detection, e.g., using SERS detection, where HS represent itself a universal linker between analyte molecules and AuNPs. Moreover, HS are supposed to prevent nanoparticles aggregation. Wet chemistry is widely used for AuNPs preparation. At the same time, the mechanisms of metal nanoclusters transformation following this process are still debatable; it is known that complex structures are formed as the intermediates even in the case of common citrate synthesis (3). When using HS, the situation becomes even more complicated; HS are known to form supramolecular structures in presence of metal ions (4), hence, its conformation should be expected to change significantly during the synthesis process. Here, we report the study of AuNPs growth in presence of HS using optical spectroscopy methods. UV-visible (UV-vis) absorption spectroscopy was used mainly to investigate the characteristics of the SPR peak, indicating the changes of nanoclusters shape. The use of fluorescence spectroscopy, both steady-state and time-resolved, allowed monitoring conformational changes of HS during Au seeding and
Experimental Potassium humate was purchased from Sakhalinsky Humates (Russia). According to manufacturer’s information, potassium humate was produced by alkaline extraction from leonardite. HAuCl4 was purchased from Sigma. All solutions were prepared using purified water (Milli-Q RG, Millipore). In a typical synthesis aqueous HAuCl4 solution was added to hot (70oC) solution of potassium humate under vigorous stirring and pH was set to 7.0 using 0.1 M KOH. The HS and Au concentration in the reaction mixture were of 100 mg/L and 5*10 -4 M, respectively. The heating and stirring were continued for 6 h; the beaker containing reaction mixture was coated with foil in order to avoid possible photodecomposition of Au(III) species. UV-vis absorption spectra of the HS-AuNPs system were obtained in the flow-cell using the custom-built spectrophotometer, allowing performing the measurements with 10 s time resolution. For fluorescence studies, the samples were collected each 15 minutes and measured using Fluoromax-4 (Horiba Jobin Yvon) spectrofluorometer (excitation wavelength 266 nm). Time-resolved measurements in the picosecond domain were performed using the time-correlation single photon counting technique (TCSPC, Becker&Hickl) at two excitation wavelengths (280 and 405 nm). DLS measurements of the aliquots of reaction mixture were carried out at 70oC using Zetasizer Nano ZS (Malvern) apparatus equipped with a He–Ne laser (laser power 4 mW, wavelength 632.8 nm). Particle morphologies were
266
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
also studied using Carl Zeiss Libra 200 MC transmission electron microscope operating at 200 kV.
excitation wavelength are presented in Figure 3. The final HS-AuNPs system demonstrated a decrease of fluorescence intensity in the longer wavelength region, where the SPR peak in the absorption spectrum is located. Moreover, the ratio of integral fluorescence intensities for the left (450-500 nm) and right (500550 nm) areas of the fluorescence spectra showed an increase during the synthesis process, following the increase of SPR peak intensity in the absorption spectra.
Results and Discussion DLS and TEM: The results of DLS measurement for the initial potassium humate solution demonstrated the presence of only one particle size distribution (PSD) peak centered at ca. 220 nm which was ascribed to HS dynamic supramolecular structures (Figure 1).
Figure 3. Fluorescence emission spectra for the aqueous HS
solution and HS-AuNPs system and absorption spectrum for HS-AuNPs.
Figure 1. Particle size distributions as measured by DLS at different times after HAuCl4 addition to HS solution.
This fact can be explained as a consequence of excitation energy transfer (EET) from HS to AuNPs leading to fluorescence quenching. In case of a homogeneous donor system (e.g., dye) EET should result in an overall fluorescence decrease without a spectral deformation. Hence, HS represent itself an inhomogeneous system, where only a part of chromophores strongly interact with AuNPs. To further investigate the nature of photophysical processes leading to the deformation of fluorescence spectrum, we performed time-resolved measurements in the picoseconds domain. Surprisingly, the fluorescence lifetimes of the HS and HS-AuNPs systems occurred to be close to each other, indicating the fast EET in the second case. At the same time, the average fluorescence lifetime τ varied depending on the registration wavelength λ. Upon 280 nm excitation, we observed the monotonous increase of τ at higher λ, while for the 405 nm excitation the decrease of τ at higher λ values was observed. This fact indicates the complex nature of fluorophores interaction in HS particles.
In two minutes after HAuCl4 addition the second PSD peak corresponding to AuNPs appeared. During the first 20 minutes this peak has low intensity and its position shifts randomly in 10-50 nm range indicating formation of Au seed aggregates of random complex morphology as was also demonstrated by transmission electron microscopy (Figure 2A). Then the position of this peak became quite constant (15-20 nm) and its relative intensity growths significantly leading to decrease of HS-associated peak amplitude and indicating formation of huge number of monodispersed quasispherical AuNPs (Figure 2B).
Figure 2. A) Au seed aggregates with random complex
REFERENCES
morphology formed in 3 min after HAuCl4 addition to HS solution and B) resulting quasispherical AuNPs formed after 6h of synthesis.
(1) Alvarez-Puebla, R. A.; dos Santos Jr, D. S.; Aroca, R. F. Analyst 2007, 132(12), 1210-1214. (2) Baigorri, R.; García-Mina, J. M.; Aroca, R. F.; Alvarez-Puebla, R. A. Chem. Mater. 2008, 20(4), 1516-1521. (3) Mikhlin, Y.; Karacharov, A.; Likhatski, M.; Podlipskaya, T.; Zubavichus, Y.; Veligzhanin, A.; Zaikovski, V. J. Colloid Interface Sci. 2011, 362(2), 330-336. (4) Engebretson, R. R. and von Wandruszka, R. Kinetic aspects of cation-enhanced aggregation in aqueous humic acids. Environ. Sci. Technol. 1998, 32(4), 488-493. (5) Del Vecchio, R.; Blough, N. V. Environ. Sci. Technol. 2004, 38(14), 3885-3891.
UV-vis Absorption and Fluorescence Spectroscopy: Absorption spectra of the HS-AuNPs system exhibited gold nanoparticles SPR peak, which demonstrated a trend to spectral blue shifting, narrowing and intensity increase during the synthesis process. The SPR peak position at 524 nm (Figure 3) was ascribed to the monodisperse spherical nanoparticles. We also note that the slope of the absorption spectra in the semilogarithmic scale increased during the synthesis process, indicating, according to (5), the increase of the average HS particle size (or the length of the conjugated chromophores chain). Fluorescence spectra of HS aqueous solution and the final HS-AuNPs system obtained for the 266 nm
Acknowledgments: This work was supported by M.V.Lomonosov Moscow State University Program of Development. The authors are grateful to Russian Foundation for Basic Research (grant №13-04-01853).
267
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Transport of POM in Japanese River Systems after Heavy Rain Events by Typhoon in 2011-2013 S. Nagao (a)*, T. Suzuki(b), S. Ochiai(a)**, S. Tomihara(c), A. Kirishima(d), M. Kanamori(b), T. Uemura(b), Y. Miyata(a), A. Goto(e), T. Hasegawa(e), M. Yamamoto(a) (a)
Wake, Nomi, Ishikawa 923-1224, Japan. Low Level Radioactivity Laboratory, Institute of Nature and Environmental Tecnology, Kanazawa University (b) Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Graduate School of Natural Science and Technology, Kanazawa University. (c) 50 Tatsumi-cho, Onahama, Iwaki, Fukushima 971-8101, Japan. Environmental Aquarium Aquamarine Fukushima. (d) 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan. Institute of Multidisciplinary Research for Advanced Materials, Tohoku University. (e) Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Faculty of Natural System, Institute of Science and Technology, Kanazawa University. * Corresponding author e-mail: [email protected] **Present address: Rokkasho-mura, Aomori 039-3212, Japan. Institute for Environmental Sciences. Keywords: Organic matter, Carbon isotope, Nitrogen isotope, C/N ratio, Migration behaviour. Abstract Variations in organic carbon and total nitrogen concentrations, and relationship between C/N ratio and isotope composition (13C and 15N) were studied in riverine suspended solids from the Abukuma River system in Japan. The samplings were carried out at the upper, middle and lower reaches before and after rain event by typhoons in 2011-2013. There is a negative correlation between C/N ratio and 13C, and C/N ratio and 15N after the Typhoon Roke in 2012. On the other hand, in normal flow condition, there is no correlation between C/N ratio and 13C of suspended solids. These results indicate that the transport of particulate organic matter (POM) in these river basins is controlled by their watershed conditions and supply of POM in rain event. a watershed area of 5400 km2. The other rivers have short river length of 43-78 and small watershed from 106 to 749 km2. Annual mean water discharge of Abukuma River is 192 m3/sec (observation year of 2008-2009) at Iwanuma Observatory of lower river area. All of rivers flow into the coastal region in Pacific Ocean. The sampling location is presented in Figure 1. The river researches were carried out after rain events by Typhoon Roke in September 2011, Typhoon Guchol in June 2012 and the rain front in October 2013. The typhoon precipitated more than 400 mm of rain daily in parts of eastern and western Japan by Typhoon Roke. Fukushima Prefecture had rainfall of 100–200 mm during September 15–22. The daily rainfall on September 21 was 137 mm by the impact
Introduction Riverine organic matter plays an important role on geochemical processes within drainage basins and a source of organic matter to river systems and continental margin sediments (1,2). The age and source of organic matter exported to the ocean by rivers are important factors for understanding the global cycling of modern and ancient carbon (3). It is well documented that high export of POM from land to ocean occurs in temperate river systems due to heavy rainfall and steep slopes (4-6). However, it is still remained for the understanding behavior of POM in river systems after rain events. The 13C tracer has proven to be simple and very useful for dissolved organic carbon and particulate organic carbon in riverine, estuarine and marine environments. The multiple use of 13C, 15N, C/N ratios and biomarkers adds a second dimension to studies of carbon cycling in surface aquatic environments (7-8). The aim of this study is to understand the fate and geochemical behavior of particulate organic carbon (POC) in river systems after heavy rain events. We selected five river systems in Fukushima Prefecture in Japan during 2011-2013. Experimental Five river systems, Abukuma River, Uta River, Niida River, Natsui River and Same River were set up as monitoring sites in Fukushima Prefecture, Japan. The Abukuma River has a river length of 239 km and
Figure 1. Sampling location of the river research in this study.
268
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
high flow conditions. There is downward trend in C/N -13C values (correlation factor, r of 0.90), and 15N13C values (r= 0.99) from upper to lower Abukuma River after the heavy rain event. Variation range for the rain event samples is smaller and lower in 13C and 15N than those of low flow condition. These results indicate that after rain events the contribution of POM from watershed varies downward from upper to lower river. The results of other river systems will be reported in our presentation.
of Typhoon Roke at Iwaki city, located in the southern coastal region of Fukushima and in a watershed of the Natsui River. This value is about one-tenth of the annual mean rainfall (1409 mm for 1981–2010). The Typhoon Guchol has rainfall of 80-90 mm on 19-20 June 2012 in Fukushim Prefecture. After rain events, about 40 l of water were taken into polyethylene containers and then transferred to the laboratory. Suspended particles in river water samples were filtered with No.5A and 0.45µm filters (a diameter of 152 mm). The suspended solids were collected from the filters. In normal flow condition, river water samples were filtered with Whatmann GF/F filters (a diameter of 47 mm) Organic carbon contents were determined using a elemental analyzer. Prior to analysis for the riverine suspended solids, calcium carbonates were removed by adding 0.1 M HCl solution and rinsed with Milli-Q water. The 13C and C/N ratios of organic matter were determined for the samples removed carbonates using an isotope ratio mass spectrometer. 13C is given as the per mil deviation from the isotopic composition of the VPDB standard. 15N is given as the per mil deviation from the nitrogen isotope composition of atmospheric N2. Results and Discussion Downward variation trend: Figure 2 shows variation trend in characteristics of organic matter in suspended solids just after the heavy rain event on 20 June 2012. The sampling was carried out at almost maximum of water discharge at each monitoring station. The POC ranges from 23 to 41 mg/l and 3.4 to 3.9%. On the other hand, 13C and 15N values of the upper site has lower values than those of the middle and lower sites.
Figure 3. Variations in C/N versus 13C and 15N versus 13C values of POM at low flow condition In April 2012 () and high flow condition in June 2012 after heavy rain by Typhoon Guchol (). REFERENCES
(1) Hedges, J.I.; Ertel, J.R.; Quay, P.D.; Grootes, P.M.; Richey, J.F.; Devol, A.H.; Farewell, G.W.; Schmidt, F.W.; Salati, E. Science 1986, 231, 1129-1131. (2) Raymond, P.A.; Bauer, J.E. Org. Geochem. 2001, 32, 469-485. (3) Raymond, P.A.; Bauer, J.E. Nature 2001, 409, 497500. (4) Kao, S.; Liu, K. Limnol. Oceanogr. 1996, 41, 17491757. (5) Masiello, C.A.; Druffel, E.R.M. Global Biogeochem. Cycle 2001, 15, 407-416. (6) Higueras, M., Kerherve, P.; Sanchez-Vidal, A.; Calafat, A.; Ludwig, W.; Verdoit-Jarraya, M.; Heussner, S.; Canals, M. Biogeosciences. 2014, 11, 157-172. (7) Guo, L.; Macdonald, R.W. Global Biogeochm. Cycles 2006, 20, GB2011, doi:10.1029/2005GB002593. (8) Nagao, S.; Aramaki, T.; Seki, O.; Uchida, M.; Shibata, Y. Nucl. Instr. Methods Phys. Res. B 2010, 268, 1098-1101.
Figure 2. Variations in POC, C/N ratio, 13C and 15N of organic matter in riverine suspended solids of the Abukuma River after heavy rain by Typhoon Guchol on 20 June, 2012.
Acknowledgments: The research was partly funded by a Grant-in-Aid for Science Research, No. 24310009 and 2411008 of the Japanese Ministry of Education, Culture, Sports, Science and Technology. Radiocarbon analyses were careried out under the Shared Use Program of JAEA Facilities.
Comparison between high and low flow conditions: Figure 3 shows distributions of C/N and 13C, and 15N and 13C of riverine suspended solids at low and
269
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Literature Review of Humic Substances in Animal Feeds R. Taylor
(a)
*, K. Smedley(b), M. Soni(c)
(a) 400 South 200 East Emery UT 84522, USA (b) 5200 Wolf Run Shoals Rd.Woodbridge, VA 22192 (c) 749 46th Square Vero Beach, FL 32968, USA * Corresponding author e-mail: [email protected] Keywords: Humic substances, peat, soft coal, humic acid, fulvic acid, animal feed. Abstract Humic substances have been studied as an ingredient in animal feeds for over a century. Observations in most studies showed some benefit to animals when humic substances included in their diets, especially those in controlled environments. Due to the various compositions of these heterogeneous substances there is limited understanding of the mode of action or why these substances can be beneficial in an animal’s diet. The lack of understanding of humic substances by feed regulators has created significant regulatory obstacles that prevent inclusion of these compounds into animal feeds. This paper presents some of the accumulated knowledge of different humic substances in animal feeds and discuses the need for future work. government agencies freely interchanged the term Introduction Humic substances are part of an animal’s every-day “peat” with “humus”. Dreyer (3) quoted an article environment and could arguably be a necessary published by the United States Department of the substrate that is omitted in controlled animal Interior (Bureau of Mines Bulletin No. 16 “The Uses environments. Containing both complex carbons and of Peat”) as one of the first known published articles minerals, humic substances are added to animal feeds on the extensive use of peat in animal feeds He also around the world as a trace mineral source, anti-caking included a table on food analysis of various grains, agent, non-nutritive carrier, mycotoxin binder, forages, and humus. The table detailed a comparison nutritive diluent, or pH adjuster. of feed components like protein, fiber, ash and The forms of humic substances included in feed carbohydrates (5). vary in the compiled research. Studies using peat, The feeding of peat prior to 1958 is further humic extracts, coal and soft coal all indicate some confirmed by McCandlish (6) who reviewed the component of either humic and/or fulvic acid. feeding of molasses soaked in peat for better handling of the sugar. The humic components of peat were later detailed Review and Discussion One present obstacle in the United States is the lack by Klavins (7). He discussed the organic matter of GRAS status (Generally Recognized as Safe) of changes from recognizable biochemical compounds to humic substances for use in animal feeds. The U.S. humic substances, with the concentration of humic Food and Drug Administration (FDA) enacted the substances increasing in direct correlation to the depth Food Additives Amendment of 1958. Ingredients fed of the deposits, reaching concentrations as high as to animal prior to that date were added to a GRAS list 80% of the total content of peat, with humic acids making them exempt from new regulatory generally ranging from about 27 to 50% requirements. Humic substances were not included in A report from the Texas Agricultural Experiment the GRAS list. After the GRAS list was established in Station stated that peat concentrations in swine feeds 1958 new regulatory paths were developed to as low as 2 to 5% are beneficial, while about 45,000 determine the safety of new animal feed ingredients. tons of commercial livestock feed were sold annually This new federal path is primarily used for created in England containing about 25 peat sources. A study ingredients with predictable Lewis structures and does on piglets investigated the effect of feeding peat not favor natural compounds found in humic preparation to piglets on growth and physiological and substances like humic and fulvic acid. biochemical indices (8). 6-day-old piglets received Animals consume humic substances in various feed containing either 1 of 3 types of peat at 0.03% forms in their natural environment without dietary concentration, antibiotics, or no supplementation from soil and aquatic sources. Humic supplementation. Treatment continued until the piglets reached the age of 100 days. At Day 28 weight substances can comprise up to 70% or more of topsoil differences were not significant, but by day 60 the organic matter (1). Studies on grazing animals have piglets receiving peat or antibiotics showed indicated that soil intake ranges from 3-8%; and the significantly greater growth as compared to the control USDA study indicates that it is not a passive activity; piglets. At termination on day 100, the antibiotic but animals actively search for soil consumption (2-4). group and 2 of the 3 peat groups exhibited As a supplement component of livestock feed there significantly higher weights than did the other groups. are numerous reports of peat being used as an ingredient since the late 1800's, where the authors and Additionally, supplementation of the diet with peat
270
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
resulted in a significant decrease in mortality, and no adverse effects were noted in physiological or biochemical measures. Other humic-containing substances like soft coal and coals have also been used prior to the 1958 Act. In some early studies performed during 1930-31, effects of coals on swine growth were investigated. In a feeding study, mixtures of charcoal, slack coal, wood ashes, ground limestone and bone meal at a 2:1 ratio with salt were fed ad libitum to swine (9). The swine consumption of slack coal was double that of wood ash/salt and charcoal/salt mixture, three times the amount of limestone/salt mixture, and five and one half times more than the bone meal/salt mixture consumed. Another report made reference to the feeding of slack coal to swine (10). These reports indicate the safe consumption of humic substances. Feeding coal to swine was a common practice for a long time. An early study found no significant benefit to piglet daily weight gain after 136 to 145 days when soft or hard coal was mixed into their basic rations (11). In a review article, Trckova et al. (12) reported that feeding of peat preparation as a supplement to piglets exerted a beneficial effect on their growth and general health status. Piglets fed with mixtures containing peat preparations also showed a lower mortality rate. Based on the review of experiments in rats, mice, calves, and piglets, these authors reported that the toxicity of humic substances is extremely low. Furthermore, gross and histochemical examinations from various studies confirmed the safety of humic acids in relation to blood, cardiovascular and endocrine systems and also to other vital organs. These preparations did not produce allergic responses or interfere with other medications and were not embryotoxic. Pisarikova et al. (13) investigated the effect of 3% sodium humate in the diet of growing pigs to determine apparent nutrient digestibility. The results showed significantly lower (P < 0.01) digestibility of crude protein (82.2 ± 1.92 vs. 87.9 ± 2.05%) and crude fat (32.0 ± 2.81 vs. 49.3 ± 2.25%) in the control group compared to the experimental group with sodium humate. The results confirmed the increased sorption (absorption and adsorption) ability gained by adding sodium humate fed to pigs. The study suggests that sodium humate aided sorption of nitrogen containing substances, which could lead to decreased amounts of ammonia released into the environment of the stables. No adverse effects of sodium humate were noted. To demonstrate the potential use of humic substances as iron source, humic shale was compared as a supplemental source of iron in a swine ration (14). This study indicated that the iron from the mined humic shale was bioavailable, calculated at approximately 71% of the iron sulphate as a standard. Another study conducted at lower levels of humic acid fed in the ration also demonstrated iron bioavailability when compared to iron sulfate (15). One point of concern from the study was that the swine receiving humic acid in their ration were required to be euthanized and incinerated to prevent the potential exposure of the humic substance from entering the food chain. In another study using a humic supplement intended for animal use, finishing steer calves were fed several diets that included increasing levels of humic
271
substances (173 ppm iron). Adding more humic substance in the ration also increased the levels of iron. The cattle that received the humic acid supplement in their diet responded with statistically significant increase in hemoglobin over the control or nonsupplemented steers; suggesting further bioavailability of iron from the humic substances (16). These studies indicate that humic substances when fed at levels up to 1% of feed intake provide bioavailable source of iron. Conclusions Animals not in a controlled environment are eating humic substances both actively and as a part of their normal diet. As the animal feed industry grows, the biggest challenge will be for scientists to share with regulators a better understanding of humic substances and how they interact with animals. More importantly the scientific community could further demonstrate that humic substances can benefit animals in controlled feed environments. One need from industry is for the scientific community to aid in establishing GRAS status for humic substances. This can be done for humic substances if a consensus from the scientific community is made regarding the safety of these substances in animal feeds. This effort might help industry avoid the added cost of o euthanizing and incinerating the study animals. REFERENCES
(1) Schnitzer, M. In: M. Schnitzer and S.U. Khan (eds.), Soil Organic Matter, Elsevier, New York, 1978 pp. 1-64. (2) Healy, W.B., New Zealand J. Agric. Res. 1968 11, 487-499; (3) Mayland, H. F.; Florence, A.R.; Rosenau, R.C.; Lazar, Y.A.; (4) Turner, H.A. J. Range Manag. 1975. 28(6), 448-452; Fries, G.F.; Marrow, G.S. Dairy Sci. 1982, 65, 611-618. (5) Dreyer, E.C. J. Amer. Peat Soc. 1918, 101. (6) McCandlish, A.C. The Feeding of Dairy Cattle. 1922, 165. (7) Klavins, M.; Sire, J.; Purmalis O.; Melecis, V. Mires and Peat, 2008, 3, 1-15. (8) Fuchs, B.; Orda, J.; Pres, J.; Muchowicz, Archivum Veterinarium Polonicum, 1995, 35, 97-107. (9) Thompson, C.P. In: Biennial Report, Director of Oklahoma Agricultural Experiment Station 1930-32. (10) Bohstedt, G., J. Anim. Sci., 1931, 222223. (11) Grummer, R.H.; Meyer, J.H.; Bohstedt, G., J. Anim. Sci., 1951, 10, 543-544. (12) Trckova M,; Matlova, L.; Hudcova, H.; Faldyna, M.; Zraly, Z.; Dvorska, L.; Beran, V.; Pavlik, I. Vet. Med. Czech. 2005, 50, 361-377. (13) Pisarikova1, B.; Zraly, Z.; Herzig, I. Acta Vet. Brno. 2010, 79, 349-353. (14) Kim, S. W.; Hulbert, L.E.; Rachuonyo, H.A,; McGlone, J.J. J. Anim. Sci. 2004, 17, 12661270. (15) Kim, S.W. Report provided by Live Earth Products Inc. 2009, pp 1-12. (16) Chirase, N.K.; Greene, L.W.; McCollum, F.T.; Auvermann, B.W.; Cole, N.A. Proceedings, Western Section, Am. Soc. Anim. Sci. 2000, 51 1-4. Acknowledgments: The Humic Products Trade Association (HPTA) funded the compilation of the research.
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Soil humus composition in mountain grasslands with different land-use intensity E. Filcheva (a), M. Zhiyanski(b) *, L. Naydenova(b)
(a) Institute of Soil Science, Agrotechnology and Plant Protection “N. Poushkarov” (b) Forest Research Institute – Bulgarian Academy Sciences * Corresponding author e-mail: [email protected] Keywords: grasslands, soil humus quality, land-use intensity Abstract Soil humus is a dynamic characteristic greatly vulnerable to land use and climate and with important feedbacks to the atmospheric greenhouse gas balance and the rate of climate change. The increased demand for accurate soil carbon stocks assessments and predictions of its changes as a result of land use/cover and climate change has triggered large-scale and long-term measurements of soil organic matter specifics. We studied the soil humus composition is four mountain grasslands, differentiated according to the land-use sub-type and land-use intensity. Two pastures – with intensive (Pi) and extensive grazing (Pe) and two meadows- managed (Mm) and unmanaged (Mu) were objects of present study. Humus composition was analyzed following the methodology of Kononova-Belchikova. Our results showed that the highest total carbon content was localized in the organic-mineral soil horizon and decreased toward deeper soil. The aggressive and mobile fulvic acids predominated in Pi, Mm and Mu, while humic acids were higher in Pe. Humic acids are “free” and bonded with R 3O3 and no Ca-bonded humic acids were established. The values of total org.C and C-extracted by 0.1 N NaOH was similar in most of studied horizons. The highest total carbon content estimated at 14.04 % was determined in A-horizon of soil in pasture with extensive grazing, compared with other studied sites, including the intensive pasture, where this value was 4.8 %. The higher grazing disturbance in Pi leads to increase root biomass in patch areas and in inter-patch upper soil related with decrease of soil humus content. We supposed that the reduced amount of litter input with increased recalcitrance to decomposition provoked the reduction of organic carbon content and in changes in its composition under intensive grazing. The extensive pasture in mountain areas is better land-use approach in the perspective of soil humus quality improvement. The managed meadow in mountain areas accumulated more carbon in superficial soil, but the composition of soil humus is similar to this in unmanaged grassland. Introduction The organic compounds of the soil to which the term soil humus is applied, is a groups of special scientific interest. Humic substances are defined as a series of relatively high-molecular-weight, yellow to black colored substances, formed by secondary synthesis reactions (humification) of plants, animals, and microbial decay products (1). Humic substances play a very important role in both soil fertility and the ecological environment because of their large contribution to various soil properties (2). Many abiotic and biotic factors can influence the quantity and quality of soil humus substances by regulating and controlling the humification of organic matter. The humic (HA) and fulvic acids (FA) are components of the soil organic matter (SOM) and their quantity and chemical characteristics depend on the specific climatic conditions (3), the altitude, the type of vegetation (4), as well as on the different silviculture and land-use management (5). Humic acid (HA) is the main extractable component of humic substances (6). The carbon ratio of HA:FA has been used as a turnover indicator to describe the intensity of humification process of soil organic matter (7). However, not enough data are available regarding the quantitative and qualitative features of soil humic fractions as affected by different intensities of land-use in mountain grassland areas.
The aim of this investigation was to study soil humus composition in mountain grasslands formed on Cambisols from the region of northern Central Balkan (Bulgaria) and to present the role of different land-use intensity on humus characteristics. Experimental The northern slopes of high mountain zone of Central Balkan Mountains were chosen for investigation within an altitude diapason between 1000 m and 1600 m a.s.l. The region characterized with mean annual temperature between 0-8.5 ºC and precipitation >800 mm. Four experimental grasslands with different land-use intensity – two mountain meadows and two mountain pastures – were included in the present research (Table 1). The sites are characterized by the same soil type – Humic Cambisols (8). The applied approach in site selection gave the possibility for comparative analyses. Table 1. Characteristics of studied grasslands Site Pi Pe
272
Land-use Intensive pasture Extensive pasture
Altitude (m) 1400 1550
Vegetation Grass – Nardus stricta
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Mm Mu
Managed mountain meadow Unmanaged mountain meadow
1200 1070
Na4P2O7 + 0.1M NaOH (HA+FA) was same as 0.1N NaOH extracted one, and the extracted with 0.1N H2SO4 showed similar values in the both studied pastures. Un-extracted carbon content was higher in Pe compared with Pi. The higher grazing disturbance in Pi leads to increase root biomass in patch areas and in inter-patch upper soil related with decrease of soil humus content. We are explaining this with the fact that the reduced amount of litter input with increased recalcitrance to decomposition provoked the reduction of organic carbon content and in changes in its composition under intensive grazing. The optical characteristics showed more condensed humic acids in the Pi and Pe which refer to better physical properties of this soil profiles.
Grass – Nardus stricta, Phleum alpinum, Poa ursina, Festuca supina, Carex laevis etc.
In each experimental site totally 4 representative soil profiles have been prepared and analyzed. The soil pH was determined by the standard ISO 10390:2005 (9). The soil carbon in samples was analyzed according to the modified method of Turin (oxidation with K2Cr2O7/H2SO4 solution in thermostat at 120ºС, 45 min. with presence of catalyst Ag2SO4 and titration with (NH4)2SO4.FeSO4.6H2O, and phenylanthranilic acid as an indicator) and accelerated method of Kononova-Belchikova (10, 11, 12). The criteria of soil humus composition were determined according to Orlov, Grishina (13).
Conclusions The results allowed comparison of soil humus composition in mountain meadows and pastures with different land-use intensity. Considering analyzed data it could be concluded that the extensive pasture in mountain areas is better land-use approach in the perspective of soil humus quality improvement. The managed meadow in mountain areas accumulated more carbon in superficial soil, but the composition of soil humus is similar in both managed and unmanaged grasslands. We summarized that different intensity of land-use influenced the quality and composition of soil humus in mountain grasslands.
Results and Discussion The total carbon content was highest in the upper soil horizon in each site (Pi, Pe, Mm, Mu) and decreased toward deeper soil (Table 2). The pH of studied Humic Cambisols was acid varying from 4.57 – 4.76 in Ahorizons to 4.89-5.85 in deeper horizons. The depth differentiation of soil horizons in both managed and unmanaged meadows was similar, while for unmanaged the B2C was reached. In managed meadow the effect of different practices applied were visible up to B-horizon, while in unmanaged one the typical distribution of soil humus was observed. According to the ratio between HA and FA in meadows’ profile soil humus could referred to humicfulvic type up to 40 (45) cm depth and humic acids are “free” and R2O3 bonded. In Mu the type humus in B2 was determined as fulvic-humic type, with predomination of humic acids totally bonded with Ca. The total organic carbon extracted by 0.1M Na4P2O7 + 0.1M NaOH (HA+FA) showed similar values to those obtained with extraction with 0.1N NaOH and only in very deep layers small differences were observed. The trend of decrease of un-extracted organic carbon was observed in both Mu and Mm grasslands. In the soils under meadows the total organic carbon was twice higher in the managed one than in unmanaged, which could be explained by higher content of organic residues which are deposited on superficial soil due to harvesting activities performed at least four times during the vegetation period. Meanwhile the humus composition did not differ, which is related with the same type of grass coverage. The studied pastures with different mode of utilization – intensive and extensive also characterized by wellexpressed differences in soil humus accumulation and composition. The total organic carbon contents in A and B-horizons were higher in Pe (14.04 %, 7.99 %) than in Pi (Pi – 4.08%, 1.22%, respectively. The soil humus type under intensive-used pasture could be referred as humic-fulvic, while under extensive use more favourable soil conditions were observed with predomination of HA and fulvic-humic type of humus. In the both pastures the humic acids are “free” and bonded with R2O3 and there were no Ca-bonded forms. The total organic carbon extracted by 0.1M
REFERENCES (1) Stevenson, F.J. Humus Chemistry. Genesis, Composition, Reactions Wiley, New York. 1994, 496 (2) Filcheva, E. Characteristics of soils in Bulgaria by content, composition and organic matter stocks. Grouping of soils in Bulgaria. ISBN 978-954-8702-11-9. “Minerva”Sofia. 2007, p. 191 (Bul). (3) Garcia, I.; Sirnon, M., Polo, A. Anales Edafol. Agrobiol. 1985, 44b, 81-82. (4) Howard, P. J. A.; Howard, D. M., Lowe, L. E. Soil Biol. Biochem. 1998, 30, 285–297. (5) Miglierina, A.; M., Rossel, R. A. Commun. Soil Sci. Plant Anal. 1995, 26, 19-20. (6) Buurman, P.; Nierop, K.G.J., Kaal, J., Senesi, N. Geoderma. 2009, 150, 10-22. (7) Rivero, C.; Chirenje, T., Ma, L. Q., Martinez, G., Geoderma. 2004, 123, 355-361. (8) WRB. World soil resources reports No.103, FAO, Rome. 2006. (9) ISO 10390:2011. BDS. Soil quality -- Determination of pH. (10) Kononova, M. Soil Organic Matter. 2nd Ed. – Pergammon press Inc. 1966, 544. (11) Kononova, М.; М., Belchikova, N. P. Practicum on soil management. “Коllоs”. 1973, 125-127 (Rus). (12) Filcheva, E.; Tsadilas, C. Commun. of Soil Sci. and Plant Analysis. 2002, 33, 3-4, 595-607. (13) Orlov, D. S.; Grishina L. A. Practicum on humus chemistry“. Moscow University. 1981, 258-259 (Rus).
Acknowledgments:
The work is a part of project “Land-use and management impacts on carbon sequestration in mountain ecosystems” financed under the Bulgarian-Swiss Research Programme (“BSRP”), 2011-2016, between the Government of Bulgaria and the Swiss Federal Council.
273
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Table 2. Content and composition of organic matter. Designations: a - % of the soil sample mass, b - % of the total carbon mass, c - % of the total humic acids, Optical index: E4/E6, HA - humic acids, FA- fulvic acids.
Profile
Mm А Mm В Mm С Mu А1 Mu В1 Mu В2 Mu В 2С Pi А Pi В Pe А Pe В Pe С
Depth, сm
pH (H2O)
Total carbon, %
2 – 9 (12)
4.76
5.30
9 (12) – 40 (45)
4.89
2.64
40 (45) ↓
5.19
1.25
2-17(23)
4.57
3.93
5.07
1.25
5.85
0.46
5.39
0.30
4.68
4.80
5.3
1.22
2 – 9 (12)
4.7
14.04
9(12) – 26 (30)
5.17
7.99
26(30) ↓
5.06
1.82
17(23)37(40) 37(40)79(85) 85 ↓ 2 – 26 (29) 26 (29)45(50)
Organic carbon (%) extracted with 0.1M Na4P2O7+0.1M NaOH total
Humic acids
Fulvic acids
1.76 33.21 0.77 29.17 0.43 34.40 1.30 33.08 0.50 40.00 0.18 39.13
0.78 14.72 0.39 10.61 0.18 14.40 0.53 13.49 0.21 16.18 0.10 21.74
0.98 18.49 0.49 18.56 0.25 20.00 0.77 19.59 0.29 23.20 0.08 17.39
0.12 40.00
0.00
0.12 40.00
1.34а 27.92b 0.43 35.25 4.59 32.69 1.92 24.03 0.72 39.56
0.66 13.75 0.16 13.12 2.93 20.87 1.00 12.52 0.30 16.48
0.68 14.17 0.27 22.13 1.66 11.82 0.92 11.51 0.42 23.08
Organic carbon (%) humic acid fractions CHA/CFA
free and R2O3 bonded
Cabonded
0.80
100.00
0.00
0.57
100.00
0.00
0.72
100.00
0.00
0.69
100.00
0.00
0.72
0.18 85.71c
0.03 14.29
1.25
0.00
100.00
-
0.00
0.00
0.97
100.00
0.00
0.59
100.00
0.00
1.76
100.00
0.00
1.08
100.00
0.00
0.71
100.00
0.00
274
Unextracted organic carbon (%)
Extracted with 0.1N H2SO4 (%)
3.54 66.79 1.87 70.83 0.82 65.60 2.63 66.92 0.75 60.00 0.28 60.87
0.15 2.83 0.10 3.79 0.09 7.20 0.23 5.85 0.09 7.20 0.04 8.70
0.18 60.00
0.03 10.00
3.46 72.08 0.79 64.75 9.45 67.31 6.07 75.97 1.10 60.44
0.16 3.33 0.13 10.65 0.23 1.64 0.19 2.37 0.16 8.79
Optical index (E4/E6 ) total HA
0.1 N NaOH
5.15
4.83
4.57
3.97
3.78
3.77
4.84
4.55
4.04 3.66
4.11 -
-
-
4.16
4.12
3.52
4.19
4.09
4.04
3.42
3.75
3.82
3.91
Organic carbon (%), extracted with 0.1 N NaOH 1.76 33.21 0.77 29.17 0.43 34.40 1.30 33.08 0.48 38.40 0.16 34.78 0.07 23.33 1.34 27.92 0.43 35.25 4.59 32.69 1.92 24.03 0.72 39.56
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Influence of Different Sources of Humic Acids, Applied Pre-treatment and Modifications on Their Behaviour in Aqueous Solutions M. Kalina*, M. Klučáková, V. Enev
Brno University of Technology, Faculty of Chemistry, Materials Research Centre; Purkyňova 464/118, 612 00 Brno, Czech Republic * Corresponding author e-mail: [email protected] Keywords: aggregation, dynamic light scattering, humic acids, methylation, particle size Abstract Humic acids are natural compounds often called as black gold of nature. They participate in natural processes. Significant is their role on transport of beneficial and harmful species in nature. Humic acids structural modelling is object of research for many decades and still a lot of key information are still missing or deficient. For structural modelling and also for possible future application of humic acids parameters as particle size and conformation seem to be essential. This contribution is dealing with study of both these parameters measured by means of dynamic light scattering method. The first part of the work is investigating behaviour of lignite and soil humic acids in aqueous solution. As a reference sample IHSS leonardite standard was utilized. Following part of the work describes effects of purification and selective modification of studied humic acids. Both these parameters significantly influence behaviour of humic acids in aqueous solutions. Introduction Humic acids are remarkable natural compounds, which play significant roles in natural processes (1, 2). This fact hand by hand with humic acids colloidal size, rich natural availability and relatively low-cost extraction techniques create from them extremely important material for practical applications. Particle size and shape as well as molecular weight can be listed as main parameters that must be taken into account in the case of investigation of the structure, reactivity and also in the possible future application of HA. Application of light scattering techniques in the area of humic particle characterization upraises recent years as novel technique. This area of research is still encountered by few experimental difficulties mainly caused because of the heterogenic and polydisperse character of humic materials.
All studied humic acids were characterized by means of elemental composition (CHNSO Microanalyser Flash 1120 Carlo Erba) and the content of ash. More details can be found in (1, 2, 3). For purposes of particle characterization all studied solid humic acids were dissolved in alkali solution of −1 0.1 NaOH to get final concentration of HA 1 g·cm . Particle size distributions for all studied humic acids dispersion were obtained using Zetasizer Nano ZS. The measurements were carried out at temperature (25.0±0.1) °C. Results and Discussion Characterization of studied humic acid samples Comparison of all studied HA samples by means of their elemental composition are shown in [Table 1]. Table 1: Elemental composition of utilized atomic % (normalized to ash-free sample) sample C H O N name 40.0 40.7 18.2 0.8 LHA1 43.9 48.1 0.9 0.4 LHA2 40.6 40.3 18.0 0.9 LHA3 44.5 37.6 16.7 1.0 LHA4 37.3 46.8 14.5 0.8 mLHA 47.0 32.5 19.8 0.3 IHA 45.5 37.9 15.9 0.6 mHA 38.5 41.4 17.2 2.7 SHA1 38.8 40.9 17.2 2.9 SHA2 37.6 42.5 16.4 3.2 CHA
Experimental Humic acids, studied in this work, were obtained in the process of alkaline extraction from different sources materials. Samples marked as LHA1 and LHA2 were isolated from South-Moravian lignite. IHA represent IHSS leonardite standard. LHA1 were repeatedly washed with water and finally dried in oven at 50 C (marked as LHA3). Furthermore washed sample of LHA3 was purified by means of the method of freeze-drying (LHA4). Separately, washed samples of LHA1 respectively IHA were modified by selective methylation of oxygen-containing functional groups (mainly carboxylic and phenolic) using TMS-N2 (trimethylsilyl diazomethane) (2). Samples marked as SHA1 – SHA2 were isolated from soil. These humic acids were sampled from upper humic horizon. cHA humic acids were isolated from compost (1).
HA samples in S 0.3 6.6 0.2 0.2 0.1 0.4 0.1 0.2 0.2 0.2
Ash (%) 30.5 41.2 26.6 2.0 27.1 2.6 2.6 1.1 0.1 0.4
Particle size distribution analysis The main part of this work was dealing with the utilization of dynamic light scattering for basic particle characterization and aggregation study of selected humic samples. Intensity size distribution
275
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
450 400 350 300 250 200 150 100 50 0
average particle size (nm)
LHA1 LHA2 IHA SHA1 SHA2 CHA
Connection between ash content and particle distribution of humic acids can be seen in [Figure 2]. Washing and the freeze-drying decreased the ash content of LHA1 from 30.5 % to 2 % (LHA4). It is obvious that the purification steps decreased significantly mainly the peak of huge aggregates round 5 m. These aggregates can be formed in solutions by contribution of inorganic impurities that can interact with humic acids. scattering volume (%)
0
Reactivity, solubility and aggregation processes in humic acids are also deeply connected to the content of functional groups of humic acids. Both most reactive functional groups of HA (carboxylic and phenolic) were modified in the process of selective methylation using TMS-N2 (5). The FT-IR spectra of HA and MHA confirmed the success of the methylation process. Results can be found in (5). Obtained volume size distributions for both studied HA are shown in [Figure 4]. The process of methylation increased only the peak intensities of particles round 1000 nm, which lead us to idea that methylation does not increase the aggregation of humic acids but it only somehow modify the conformation of HA in the solutions.
Figure 1: Average particle size determined for lignitic humic acids (LHA1 and LHA2), IHSS leonarite standard (IHA), soil humic acids (SHA1 and SHA2) and compost HA (CHA)
20 15 10 5 0
100 1000 10000 particle size (nm) Figure 4: Volume particle size distribution for lignitic humic acids (LHA1 - full line) and their methylated form (mLHA – dashed line).
20
10
Conclusion The results of this study shed new light on effects of purification and modification of humic acids on their behaviour in aqueous solutions. Particle size distribution must be taken into mind as one of the basic parameters in the case of structural study and also in the case of possible future application of humic acids. Presented method proved to be suitable also for basic comparison of humic acids isolated from different matrices.
15 10 5 0
400 350 300 250 200 150 100 50 0
10 20 30 40 50 ash content in material(%) Figure 3: Average particle size vs. ash content of the humic acids samples
scattering volume (%)
average particle size (nm)
as the basic outcome of DLS measurement can be converted, using Mie theory, to a volume distribution, which describes more clearly the distribution of mass in the sample. Refractive index of lignitic humic at 632.8 nm (wavelength of laser in Zetasizer Nano ZS) particles is necessary to know for the recalculation (1.439±0.013). First results in [Figure 1] show the comparison of humic acids isolated from different source matrices. The highest average particle size was determined for lignite humic acid LHA1. This sample contained high ratio of inorganic ash, which probably contributed to higher aggregation of the sample. Almost the same was observed for lignite HA sample LHA2. The average particle sizes were lower for leonardite IHSS standard (IHA) and significantly lower for soil (SHA1 and SHA2) and compost (CHA) humic acids.
100 1000 10000 particle size (nm) Figure 2: Volume particle distribution of lignite humic acids (LHA1 – full line), washed HA (LHA3 – dashed line) and freeze-dried HA (LHA4 – dotted line). 10
References
(1) Enev, V.; Pospíšilová, L.; Klučáková, M. Soil and Water Research, 2014, 9, 9-17. (2) Klučáková, M.; Kalina, M.; Sedláček, P.: Journal of Soils and Sediments, 2014, 14, 368-376. (3) Peuravuori, J.; Žbánková, P.; Pihlaja, K. Fuel Process. Technol., 2006, 87, 829-839.
Results of [Figure 2] lead us to the idea to plot average particle size of humic acids vs. ash content of HA samples. The results indicate significant correlation between both studied parameters.
Acknowledgments: This work was supported by Ministry of Education, Youth and Sports, Project LO1211.
276
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Chemical composition of humic acids extracted from soils influenced by ancient charcoal production in Rio de Janeiro - Brazil A.F. Rodrigues(a) *, E.H. Novotny(b), R.R. Oliveira(a) (a)
Pontifical Catholic University, R. Marques de São Vicente, 225, CEP 22451-900, Rio de Janeiro, Brazil. Embrapa Soils, R. Jardim Botânico, 1024, CEP 22460-000, Rio de Janeiro, Brazil. * [email protected] (b)
Keywords: charcoal kilns; Terra Preta de Índios; pyrogenic carbon; 13C NMR. Abstract During the late nineteenth century, some areas in Rio de Janeiro – Brazil – has undergone remarkable transformation of the forest arising from ancient charcoal activity carried out by ex-slaves. Nowadays, in the area of abandoned ancient kilns, it is possible identify charcoal in the soil. The charcoal weathering, in another anthropic soil, the Terra Preta de Índios (TPI), generated a peculiar soil organic matter, i.e very recalcitrant and with high cation exchange capacity (CEC), resulting in a resilient soil against degradation by intensive use. Thus, the structural comparison, by 13C Nuclear Magnetic Resonance, of the humic acids (HA) from paleo-charcoal kiln and from TPI facilitate to confirm the hypothesis that the natural weathering of charcoal in the soil generate this peculiar organic matter. The result showed that the HA from paleo-charcoal kiln in Rio de Janeiro show similar features from TPI, i.e: policondensed aromatic rings heavily functionalised with carboxylic groups. Introduction From a historical perspective, the present environmental heritage is a product of past human populations’ relationships with their environments (1). Our starting point in examining these interrelationships is the study of the paleo-territories of slaves, ex-slaves, and charcoal producers in the 19th and 20th centuries in Rio de Janeiro’s mountain forests. Until the early 19th century, the mountains near the city, the Pedra Branca Massif, were largely used for sugarcane production and lumber and firewood extraction. In addition, our fieldwork indicated that the Pedra Branca Massif had been heavily populated from the 19th century until the beginning of the 20th century; our research located 157 abandoned ancient (paleo) charcoal kilns (2) and 61 home charcoal maker ruins among the forest slopes. Civil construction was one of the largest charcoal consumers at the turn of the 19th century (especially the stonemasons), and charcoal forges multiplied throughout the city with the exponential growth of industries and civil construction. The only traces that they left were vestiges in the landscape as the black soil in and around the paleo - charcoal kilns (3). So, the objective of this study was to investigate the consequences of the weathering of coal in the soil of the Pedra Branca Massif. Investigations on soils with charcoal presence have attracted the interest of scientist from different fields, such the literature has shown news perspectives of using this resource in improving soil quality and the potential to mitigate the anthropogenic green house effect, by the storage of the carbon in the soil (4). The Amazon TPI also are soils with charcoal residues, of anthropogenic origin from pre-Columbian period, which have higher fertility and resilience than the adjacent ordinary soils (5). These characteristics are provide by the peculiar organic matter of TPI, rich in pyrogenic C (6), important also for C sequestration. Therefore, we face the challenge of investigate humic
acids from anthropic soils, as this influenced by ancient charcoal kiln, contemplated in the present work, drawing an analogy with TPI. Experimental It was selected a representative site from more than 157 charcoal kiln areas reported on the Pedra Branca Massif. However, update of this number indicates around 920 (data not published). It was sampled the center of the area under influence of the ancient charcoal kiln (center), and beyond two points: the dump (local deposition of charcoal residues not used for marketing); and surrounding soil, as control. In these points samples were collected at different depth: dump (0-10 cm, 10-20 cm and 40-60 cm), center of the paleokiln (0-10 cm, 10-20 cm and 20-40 cm); and control soil (0-10cm, 10-20 cm and 20-60 cm). The samples were dried and sieved (2 mm). After that, HA were extracted and purified by a selective extraction method (6). Briefly, an exhaustive extraction was done with NaOH solution adjusted at pH 7. HA samples were analysed by solid state 13C Nuclear Magnetic Resonance (NMR) spectroscopy. Results and Discussion The 13C NMR spectrum of HA extracted from control soil [Figure 1] is similar to the ones from ordinary HA from Tropical soils, exhibiting intense peaks from labile groups, such as: holocellulose (O and di-O- alkyl groups), probably partially oxidized to glucuronic acids (aliphatic carboxyl); lignin (aryl; Oaryl and methoxyl signals); and fatty acids (alkyl and aliphatic carboxyl). Already the HA samples rich in ancient charcoal, for example from dump site [Figure 1], display a significantly higher content of polycondensated aromatic structures than control samples, represented by the signal at 128 ppm, these structures give greater stability for the soil organic matter of these soils, and thus a greater potential for C
277
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
sequestration. Moreover, these samples exhibited carboxylic acids region (160-180 ppm) broader than control samples, due to an unresolved shoulder of upfield shifted carboxylic signal (around 165 ppm), this shoulder can be attributed to aromatic carboxylic acids. These structures guarantee a high fertility (high CEC) but in a recalcitrant form (aryl-carboxyl) resulting in a higher resilient soil, against cultivation, than the control one (6).
However, the TPI HA does not have significant peaks of labile materials, seen in higher quantity in the dump, because this TPI is a cultivated area, with important lost of labile organic matter (5).
Figure 3. 13C spectra of HA from dump and TPI, in the 0-10 cm layer. The interaction human-nature not always is destructive (Homo devastans) and, in this studied case, produced HA structurally more recalcitrant (polycondensated aromatic structures) and with high capacity of adsorb nutrients cations (carboxylic groups), besides, this reactive groups probably are recalcitrant, since they are linked directly to the polycondensated aromatic structures, similar to HA found in TPI, another emblematic example of synergic interaction human-nature, since TPI are soils with higher: resilience; fertility and; sustainability than the adjacent control soils. It is important to highlight that besides the ex-slaves, have contributed to the construction of Brazil with its workforce, also left legacy (even if unintentional) soil with charcoal, which have peculiar characteristics and serve as model for thinking about more sustainable agricultural models, similar to the pre-Colombian Amazon indigenous people, and their TPI legacy.
Figure 1. 13C spectra of HA from ancient charcoal kiln and surrounding soil, in the 0-10 cm layer. The spectra of the HA samples from the dump and center of ancient charcoal kiln (10-20 cm layer) [Figure 2] are similar, as both have carboxylic carbons from labile materials (aliphatic carboxyl – 172 ppm), as well as the typical aryl-carboxyl shoulder. In these samples the condensed aromatic structures are prevalent. However, these samples also shows peaks at 105 ppm (di-O-alkyl) and 72 ppm (O-alkyl), typical of carbohydrates (holocellulose), and the peak of the center of the kiln is narrower, indicating that the material is better preserved, while the broader peak of dump indicates that the material is more humified. This may be because the center is a flat area and thus a place of greater deposition of litter, less altered organic material.
REFERENCES
(1) Worster, D. Estudos Históricos. 1991, 4, 198-215. (2) Oliveira R.R. Revista Internacional Interdisciplinar. 2011, 8, 286- 315. (3) Oliveira, R.R. Rio de Janeiro: Editora PUC-Rio, 2005, 1, 230. (4) Lehmann, J.; Joseph, S. Biochar for Enviromental. Management. 2009, 1-5. (5) Novotny, E.H.; deAzevedo, E.R.; Bonagamba, T.J.; Cunha, T.J.F.; Madari, B.E.; Benites, V.M.; Hayes, M.H.B. Environ. Sci. Technol. 2007, 41, 400 - 405. (6) Novotny, E.H.; Hayes, M.H.B.; Madari, B.E.; Bonagamba, T.J.; deAzevedo, E.R.; deSouza, A.A.; Song, G.; Nogueira, C.M.; Mangrich, A.S. J. Braz. Chem. Soc. 2009, 20, 1003-1010. (7) Araujo, J.R.; Archanjo, B.S.; deSouza, K.R.; Kwapinski, W.; Novotny, E.H.; Achete, C.A.. Biol. Fert. soils. 2014, In press.
Figure 2. 13C spectra of HA from ancient charcoal kiln and dump, from the 10-20 cm layer. Comparing the 13C spectra of the HA samples from the superficial layer of the area of dump with HA from TPI (7) [Figure 3], it is verified a high content of polycondensated aromatic structures (intense and broad band at 128 ppm) and carboxylic groups (band at 172 ppm) with typical broad and asymmetric signal of aromatic carboxyl in both samples.
Acknowledgments: The authors are grateful to the CNPq for the financial support.
278
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The Influence of Organic Matter and Clay Minerals on Size of Surface Area of Different Peptization Level in the Meadow Soil of the Middle Priamurje, Russia Matiushkina L.A. Institute of Water and Ecology Problems, Far Eastern Branch, Russian Academy of Sciences, 65 Kim Yu Chen St., 680000 Khabarovsk, Russia Tel. +7 4212 227573; Fax +7 4212 325755; E-mail: [email protected] Keywords: meadow podbel; water-peptizable clay; aggregated clay; surface area; phyllosilicates; humic matter; nonsilicate iron Abstract. The studied soil was the meadow podbel (planosol in the FAO system), periodically over moistured with atmosphere precipitation. The soil profile revealed well-marked genetic horizons. Soil clay (particles < 2 µm) was sequentially divided into water-peptizable (WPC) and aggregated (AC) sub-fractions according to their ability to peptizate in water. Specifics of the quantitative content of WPC and AC in the major horizons of this soil were determined Surface area was estimated in WPC and AC samples by adsorption of water vapor. OM content and composition of clay minerals were estimated by X-ray diffractometry. Higher (1.5-2 times) surface area of aggregated clay particles is consistent with a higher content of phyllosilicates with a swelling smectite component, humic matter and non-silicate iron. These aggregating factors are less important for particles of water-peptizable clay. soil horizons were investigated: humus-accumulative AUg, eluvial ELnn,g and illuvial BTg with 20, 24 and 52% of clay respectively (according to the pipette method). WPC and AC fractions (particle size < 2 µm) were identified with the method of fractional peptization of clay in water (3). WPC particles peptizate, when soil is simply mixed with water (1:40) without any mechanical or chemical actions. Water was removed with a siphon. To extract AC fractions the remained wet paste-like sample was then kneaded in a weakly alkaline media (to destroy aggregates). Vapor absorption in WPC and AC samples was studies with the weight method (2). The obtained complete adsorption-desorption isotherms were in the range of relative pressures P/Ps from 10-3 to 1 at 25o C. The surface area was calculated from the adsorption part of isotherms with the BET method (4). Data on chemical and mineralogical composition of WPC and AC were already obtained (5, 6). В определено содержание Total carbon of OM in WPC and AC was estimated using the method of potassium bichromate oxidation and non-silicate iron was calculated with the Mehra and Jackson method of dithionite-citrate-bicarbonate extraction. Both methods had spectrophotometer measuring.
Introduction. From the point of view of the soil matrix organization the composition and surface of finedispersed soil minerals compose a mineral matrix, which governs soil processes including the distribution of various substances by separate “local soil centers” (1). In many soil studies it is important to know soil dispersion and surface area, as they determine, e.g., the ability to absorb nutrients, gases, water vapor (2). It is well known that fine-dispersed soil particles (< 1-2 µm) may be differentiated according to their properties, such as the ability of attracting various substances, water. Most often finedispersed particles are grouped by their size into fractions of fine dust (5 -2 µm) and clay (< 2 µm), which in its turn is subdivided into pre-colloids (2 0.2 µm) and colloids proper (< 0.2 µm). The present research was focused on the alternative method of clay particle differentiation, which is their ability to peptizate in water. The surface area, content of humic matter, non-silicate iron and mineralogical composition were further analyzed in the obtained soil fractions. Materials and methods The investigated soil profile is located on the flat part of the second above-floodplain terrace of the Amur River in the south-western part of the MiddleAmur Lowland, 150 km to the south of the City of Birobidzhan. The studied soil was formed on the lake-alluvial clay underlying meadow sedge and reed grass vegetation. The soil profile is characterized with a periodical surface over-moistening due to atmospheric precipitation in spring and during summer monsoon rains. According to the Russian national soil classification this soil type is described as the meadow podbel, and in the FAO system it corresponds to the planosol. In the Russian Far East south meadow podbels are used to grow crops only after they have been ameliorated. The following three
Results and discussion Specifics of WPC and AC content and distribution in the meadow podbel profile are presented in the table. WPC content increases down the profile and has a maximum at a depth of 90-110 cm, indicating a high water-peptizing capacity of a parent rock, which is mid-Quaternary lacustrine-alluvial clay. AC prevails over WPC along the entire profile. AC distribution along the profile reveals two evident maximums. The first maximum is in humusaccumulating horizon AUg, where total carbon content is 4.9%.
279
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The second maximum is in illuvial horizon BTg, where total carbon content is 1.5%. The following data show significant differences in the composition and properties of WPC and AC. The Figure illustrates the difference in adsorption ability of WPC and AC both at low relative pressures of water vapor (am), and at the marginal absorption (as). AC show higher adsorption of water vapor than WPC. The surface area of AC particles, calculated with am, exceeds the surface area of WPC in horizons Aug, ELnn.g, BTg by 64, 95 and 28 m2/g, respectively. Our earlier data (6) show the differences in mineralogical composition of water-peptizable and aggregated clays. AC are characterized by a high content of phyllosilicates with smectite layers. The content of smectite minerals (mica-smectite, kaolinite-smectite) is particularly high in the AC of horizon BTg. WPC have clastic components: micas-hydromicas, kaolinite, finely dispersed quartz, feldspars, plagioclases, amphiboles, as well as micashydromicas with low smectite layers and strongly deformed. Similar differences between waterpeptizable and aggregated clays were also found in the content of non-silicate iron and organic matter. The table clearly shows that the studied clay fractions of the upper horizon AUg contain almost equal amounts of non-silicate iron. Figure Isotherms of water vapor adsorption of WPC (point lines) and AC (unbroken lines) in meadow podbel, Priamurje, Russia. 1 and 3 – desorbtion, 2 and 4 –. adsorption. A – horizon AUg; B – horizon ELnn.g; C – horizon BTg.
Table The Value of Surface Area (S), Content of Organic Carbon (C), Carbon of Humic Acids (CHA) and Nonsilicate Iron (Fe2O3 d) in Water-peptizable (WPC ) and Aggregated (AC) Clays of Meadow Podbel, Priamurje, Russia. Clay particles < 2 µm WPC AC
Horizon; depth, cm AUg ELnn,g BTg Aug ELnn,g BTg
0 – 14 22 – 30 90 – 100 0 – 14 22 – 30 90 – 100
Content of clay particles /% 1.6 9.9 19.2 38.0 19.0 39.7
S of clay particles / m2/g 113.7 125.8 225.0 177.5 220.5 253.0
However, non-silicate iron content in AC of the underlying soil layers is approximately 2.4 times greater. An important factor, which may affect the surface of clay fractions of meadow podbel, is the content of humic substances. A relatively high content of humic substances corresponds to higher surface values in AC fractions. In humusaccumulative horizon AUg about 60% of the total soil humus is part of AC and only 2% is associated with WPC. Noteworthy is a high content of humic acids in AC humus and the sum of their fractions (CHA) is 30 times larger than in the WPC humus.
Organic C /% 9.9 3.2 1.4 13.3 3.6 1.5
CHA / % organic C clay particles 0.88 8.00 3.47 25.93 17.87 24.79
Fe2O3 d /% 2.10 1.79 2.19 1.99 6.56 3.88
(2) Shein, E.B. and Karpachevsky, L.O. (Ed.). Theory and methods of soil physics. 2007. Grif & K. Moscow. p.p. 130-165. . (3) Matyushkina, L.А.; Studies of ColloidChemical Soil Properties and Mineral Composition of Fine-Dispersed Fractions of Soils and Soil-Forming Rocks. In: Biogeochemical and Geoecological Studies of Terrestrial and Fresh Water Ecosystems. 2002. Vol.12. Dalnauka, Vladivostok. 67-79. (4) Brunauer, S.; Adsorption of gases and vapours. V. 1. Physical adsorption. 1948. Изд-во иностр. лит. Moscow. 784 p. (5) Chizhikova, N.P.; Kharitonova, G.V.., Matyushkina, L.A., Sirotskii, S.E. Eurasian Soil Science. 2004, 37, 8, 876-888. (6) Chizhikova, N.P.; Kharitonova, G.V., Matyushkina, L.A., Konovalova, N.C., Stenina, A.S. Eurasian Soil Science. 2013, 46, 8, 885-896
References (1) Zubkova, ТА. and Karpachevsky, L.О. Matrix Organization of Soil. 2001. Rusaki, Moscow.
280
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characterization of organic matter in areas under different covers in the Savannah biome (SP) - Brazil M. C. Piccolo (a) *, W. Bieluczyk(a), L. C. Reis(a), M.G. Pereira(b), F. C. Balieiro(c)
(a) Laboratório de Ciclagem de Nutrientes, CENA-USP, Av. Centenário 303, CEP 13400970, Piracicaba-SP, Brasil. (b) Laboratório de Gênese e Classificação do Solo, UFRRJ, BR 465, Km 7, CEP 23897000, Seropédica–RJ, Brasil. (c) Embrapa Solos, Rua Jardim Botânico, 1024, CEP 22460000 Rio de Janeiro-RJ, Brasil. * Corresponding author e-mail: [email protected] Keywords: carbon fractionation, soil quality, oxisols, tropical agriculture, environment preservation. Abstract. The identification and quantification of labile and recalcitrant soil organic matter (SOM) forms can be used to evaluate the impacts of management on soil quality (SQ). This study characterized the SOM through chemical and granulometric fractionation in areas located in the Savannah biome. Preserved, cultivated and environmental recovering areas were evaluated. For all fractions of SOM, except for the particulate organic carbon (POC), the preserved areas showed higher levels. The SOM had a positive correlation with the cation exchange capacity and negative with bulk density of soil (p ≤ 0.01). The results showed that SOM improved the physical and chemical SQ, and crops reduced the levels of carbon (C) in SOM fractions, negatively affecting the SQ. Introduction The need to increase the food production, coupled with technological innovations in agriculture, has resulted in breakthrough on the Brazilian agricultural frontier. The main areas currently exploited are located in the Savannah biome, which originally covered an area of 2.100.000 km2, and from this total, about 49% of the area is already deforested. São Paulo is Brazilian state with the highest levels of deforestation of the Savannah vegetation, with reduction of about 90% of the original area (1). Oxisols embrace approximately 50% of the Brazilian Savannah. These soils are characterized by having high acidity and low contents of essential nutrients to plants (2). Increasing the levels of soil organic matter (SOM) in Oxisols means increasing the cycling of nutrients, cation exchange capacity (CEC), water availability, and improve soil aggregation and soil biological activity (3). The SOM is a heterogeneous mixture of organic substances with different composition, lability and functions in soil. Considering these aspects, the SOM can be fractionated, chemically an/or physically. To evaluate these compartments can set, or even predict, the function of soil in carbon (C) storage or loss in systems (4). One of these methods is the chemical fractionation of SOM. It consists in obtaining the humic fractions by differential solubility. Fulvic acid fraction (FAF) and the humic acid fraction (FAF) fraction represent the portion of SOM soluble in alkaline solution. The humin fraction (HUM) is recalcitrant and connected to the minerals of soil (5). Granulometric fractionation separates the MOS by size. It is widely used in Brazil because of its methodological facility, low cost and successful results. Particulate organic carbon (POC) has the size of 53-2000 µm, composed of SOM recently added to the soil. The C associated with minerals (CAM), (< 53 µm) is a recalcitrant SOM, bound to soil minerals (6). Given the above, this study aims to characterize the SOM in areas with different degrees of anthropic
impacts, evaluating a dystrophic Oxisol in the Savannah biome. Within this context preserved, cultivated and environmental recovery areas were selected, aiming to generate information on quality characteristics of SOM and soil in these settings. Experimental The study was conducted in areas located in the city of São Carlos, São Paulo (21º57’56’’ S 47º51’10’’ W) in subtropical highland climate (Cwa). The soil was classified as dystrophic Oxisol (Typic Haplustox) and presented Sandy clay loam textural class (USDA) with 288 g kg-1 clay, 31 g kg-1 silt and 681 g kg-1 sand. Six areas were selected for the study: (i) semideciduous forest (SF), preserved at least 50 years; (ii) preserved Savannah (PS), with transition between “Cerrado sensu stricto” and “Savannah forest”, preserved since 1970; (iii) recovery of the Savannah (RS), classified as “Cerrado sensu stricto”, with management aiming at regeneration of native species since 1997; (iv) Eucalyptus (EUC), planted in 1994 for commercial purposes, with a single liming being held in 2005; (v) pasture (PAST), implanted after cutting down the forest in 1992. Urochloa decumbens was seeded and remained in the area for 15 years, after this period a singular soil amendment with lime and NPK was done, seeding the Urochloa brizantha in the area; (vi) Sugarcane (SUC), set in 1974. The SUC harvesting was performed with the practice of burning up to 1980, and after, mechanically and without burning. Liming and fertilization practices were based on soil chemical analyzes. Soil sampling was conducted in 2012, with five replicates (n=5) in the 0.0 to 0.2 m layer. Chemical fractionation of humic substances by differential solubility technique was performed. The HAF and FAF were extracted and the residue was reserved for determination of C in the form of HUM (5). The granulometric fractionation of SOM, separated the POC and CAM (6). To support the discussion related to the fractions of SOM density (Bd) and cation
281
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
exchange capacity (CEC) were determined (7). Finally, the mean attributes were compared by Tukey's t test at 5% probability.
Comparing to the other areas the area of EUC had the lowest concentrations of C in HAF and HUM. HUM had the largest contribution (between 62 and 69%) for the total humic substances. This means that even the FAF+HAF combination has not exceeded the levels observed for the HUM. For the granulometric fractionation between 87 and 89 % of the TOC was composed by CAM. The HUM and CAM are recalcitrant fractions and strongly stabilized with mineral soil matrix, having a half-life ranging from decades to hundreds of years (4). Therefore, the HUM and CAM, due to its stability, stay longer in Savannah soils, where the edaphoclimatic conditions favor the decomposition and mineralization of SOM. Correlations of SOM fractions with Bd and CEC: All variables were correlated at less than 1% probability (p ≤ 0,01) [Table 1]. The fractions of the SOM inter-related positively with each other and with the TOC, showing that its dynamics walk in parallel on the evaluated systems. Humic and granulometric fractions of SOM showed positive correlations with soil CEC. On the other hand, the Bd correlations with the attributes were always negative. These results in the assessed areas that increases in the levels of SOM and/or its humic and granulometric fractions improves quality. Such affirmative is supported by the decreased levels of Bd and increased CEC.
Results and Discussion Granulometric fractions of SOM: [Figure 1] The contents of total organic carbon (TOC) and CAM were higher in the area of SF, followed by the area of PS. The lower values of these attributes were found in the area of EUC. Areas of SR, PAST and SUC had intermediate levels. The increases in the levels of TOC and CAM usually occur only in long-term (4), and the management modified these attributes, mainly in EUC area, generating the found gradient. On the other hand, fertilization used in areas of PAST and SUC, could have promoted improvements in soil fertility and increase of TOC levels through gramineae roots. No differences were found POC contents, indicating similar C input for all the areas in the layer evaluated. POC
Organic Carbon (g kg-1)
25
CAM
20
b
15
a
10
0
bc
bc
c
bc
5
a
a
a
a
a
a
SF
PS
RS
EUC
PAST
SUC
Table 1. Pearson Correlation (r) between the attributes of the Savannah soil with different vegetation covers in São Carlos (SP-Brazil) TOC FAF HAF HUM POC CAM CEC FAF 0.82 HAF 0.85 0.71 HUM 0.84 0.70 0.86 POC 0.63 0.47 0.74 0.71 CAM 0.98 0.81 0.78 0.78 0.46 CEC 0.68 0.62 0.61 0.65 0.50 0.65 Bd -0.58 -0.52 -0.54 -0.55 -0.48 -0.54 -0.78
Land Uses
Figure 1. Granulometric fractions of soil organic carbon of the Savannah soil with different vegetation covers in São Carlos (SP-Brazil). POC: particulate organic carbon. CAM: carbon associated with soil minerals. SF: semideciduous forest. PS: preserved Savannah. RS: recovery of the Savannah. EUC: eucalyptus. PAST: pasture. SUC: sugarcane. Note 1: The letters compare the averages (n=5) of the attributes between areas by the Tukey t test at 5% probability. Note 2: error bars refer to the TOC contents.
TOC: total organic carbon. FAF: fulvic acid fraction. HAF: Humic acid fraction. HUM: humin. POC: particulate organic carbon. CAM: organic carbon associated with soil minerals. CEC: Cation exchange capacity; Bd: Bulk density. Note: All variables were correlated at less than 1% probability.
Land Uses
Humic fractions of SOM: [Figure 2] The carbon content in humic fractions were higher in the area of SSF, followed by the area of PS. When evaluated the FAF, the SUC area had the lowest concentrations of C. SUC PAS T EU C RS
FAF
HAF
c abc
bc
bc abc
b a
SF 0
39 %
c
bc bc
47 % c
49 %
ab
ab a
2,5
TOC- (FAF+HAF+HUM)
46 %
bc
bc c
PS
HUM
REFERENCES
(1) IBGE, Informação Geográfica 9, 2012 (online): http://www.ibge.gov.br/home/geociencias/recursosnaturais/id s/default_2012.shtm (2) Vendrame, P.R.S.; Brito, O.R.; Guimarães, M.F.; Martins, E.S.; Becquer, T. Anais da Academia Brasileira de Ciências. 2010, 82, 1085-1094 (3) Zech, W.; Senesi, M.; Kaiser, G.G.K.; Lehmann, J.; Miano, T.M.; Miltner, A.; Schroth, G. Geoderma 1997, 79, 117-161. (4) Wander, M.M.; (ed) Magdof, F.; Weil, L. Adv. in Agroec. CRC press, Boca Raton, FL, 2004, 67-102. (5) Benites, V.M; Madari, M.; Machado, P.L.O.A. Embrapa Solos, 2003, comunicado técnico 16, 1-7. (6) Cambardella, C. A. and Elliott, E. T. Soil Sci. Soc. Am. J. 1992, 56, 777-783. (7) EMBRAPA, Manual de Métodos de Análise de Solo, 2a ed., Rio de Janeiro, 1997, 1-212.
41 % a
5
7,5
51 % 10
12,5
15
17,5
20
22,5
Organic Carbon (g kg-1 )
Figure 2. Humic fractions of organic carbon of the Savannah soil with different vegetation covers in São Carlos (SP-Brazil). FAF: fulvic acid fraction. HAF: humic acid fraction. HUM: humin. TOC-(FAF+HAF+HUM): soil carbon not recovered by the humic fractions, also indicated in bars in percentage. SF: semideciduous forest. PS: preserved Savannah. RS: recovery of the Savannah. EUC: eucalyptus. PAST: pasture. SUC: sugarcane. Note: The letters compare the averages (n=5) of the attributes between areas by the Tukey t test at 5% probability.
Acknowledgments: We thank the São Paulo Research Foundation (FAPESP) for supporting the project 2012/13484-3 and UFSCAR for allowing us to conduct this research.
282
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Modern Concepts of Humin M. H. B. Hayes(a)* and R. S. Swift(b) (a)
Carbolea, CES, University of Limerick, Ireland Queensland Alliance for Agriculture and Food Science, University of Queensland, Qld 5072, Australia * Corresponding author e-mail: [email protected] (b)
Keywords: Humin, Humic Substances, NMR spectroscopy Abstract: Humin is a substantial component of soil organic matter, often comprising 50–70 per cent of the total OM. Within the classical operational definitions of humic substances, it has been implicitly assumed that the composition of humin would be similar to humic acid but with less oxygen-containing functional groups and higher molecular weight accounting for its non-extractability. Evidence has emerged indicating that humin has higher concentrations of aliphatic-C than humic and fulvic acids but this has proved difficult to confirm due to problems with separation and solubility. These obstacles have been overcome by a) the removal of humic and fulvic acids by exhaustive extraction and b) the novel use of DMSO/H2SO4 as a solvent for humin. Examination of humin by NMR spectroscopy has confirmed that it is dominantly aliphatic in nature with little aromaticity. The most likely source of humin is from cutin and suberin and related compounds in plants. thorough removal of the alkali soluble HSs, then the so-called humin extract will include components of HSs. Exhaustive extraction with 0.1 M NaOH alone will not isolate all of the HSs
Introduction In the classical definitions Humus was considered to represent the components of Soil Organic Matter (SOM) that are so transformed as to bear no morphological resemblances to the materials of origin. Humic Substances (HSs) are defined as the amorphous, brown, macromolecular substances differentiated on the basis of solubility properties into Humic Acids (HAs), precipitated at pH 1 from solution in aqueous base; Fulvic Acids (FAs), soluble in aqueous media at all pH values, and Humin the humified materials that are insoluble in aqueous media. Compounds belonging to recognisable classes, such as polysaccharides, peptides that can be synthesized by microorganisms or can arise from modifications of similar compounds in the plant debris, and are not covalently linked to the ‘humic core’ are not considered to be HSs. The concept of HSs as macromolecular materials has been challenged and there is an alternative proposal of pseudo-macromolecular assemblies arising from intermolecular associations(1). Any humified material recovered from soil, after exhaustive extractions in aqueous base could, on the basis of the classical definitions, be considered to be humin. On that basis, Kononova (2) estimated that humin composes more than 50% of SOM, and Hedges and Keil (3) considered as humin 70% of the OM in sediments. It has generally been held that soil humin components have intimate associations with the clay sized fractions (4). The various studies of humin were based on residues after extractions in aqueous sodium hydroxide (NaOH) and the IHSS isolation procedure involves extractions in 0.1 M NaOH. Although Rice and MacCarthy (5) by using methylisobutyl ketone (MIBK) in a two-phase system, introduced the concept of an organic solvent as ‘extractant’ for humin, the more usual approach was to regard humin as the organic matter recovered after dissolving the associated silicates in a HCl/HF or following an HF pre-treatment. If the treatment does not involve a
Results and Discussion The 13C NMR spectra using cross polarisation with total suppression of sidebands (CP/TOSS) for the humin fraction of a calcareous coarse sandy loam brown earth soil, from which humic and fulvic acids had been removed by sequential and exhaustive extraction at pH 7.0 and 10.6, then at pH 12.6, and with 0.1 M NaOH + 6 M urea, are shown in Fig. 1.
Figure 1. CP/TOSS 13C NMR spectra of HAs isolated in 0.1 M NaOH at pH 12.6, and in 0.1 M NaOH + 6 M urea (Urea HA) from a cultivated, maize-amended soil. The spectra show close similarities between the HAs isolated in the base and base + urea systems. The soil had been amended with 8% maize (Zea mays L) stover prior to incubation for two years (with periodic mixing and irrigation) and the spectra show significant evidence for partially transformed/humified lignin (Oaromatic, 140-150 ppm, and methoxyl, 56 ppm). The NMR provides evidence also for aliphatic hydrocarbon (alpha, 10-35 ppm) carbohydrate (OC 60-90 ppm and
283
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
anomeric C, 105 ppm), aromaticity (120-140 ppm), and carboxyl/ester/amide (160-185 ppm) functionalities. Song et al (6) used DMSO (94%) + 98% H2SO4 (6%) following the exhaustive extraction in the base/urea solvent. A significant amount of material was recovered as a humin precipitate when the solution was diluted with water to pH 2.
Figure 3. HRMAS 1H NMR spectrum the humin material after extraction with base + urea (UHU) isolated from a well drained grassland surface soil that contain large amounts of mineral residue. The evidence presented here, and that of others show that aliphatic hydrocarbons, most probably derived from fatty acids, cutins, cutans and suberin are major components of humin. These materials are refractory and the humin that is formed from them is highly resistant to decomposition and makes up a large proportion of the SOM in the soils that we have studied. The evidence for lignin, peptide, and carbohydrate components might be explained by their intimate associations with clays, and trapped in the non-polar aliphatic hydrocarbon based matrix. There is a tendency to regard the dark-coloured material observed in the uppermost layers of centrifuged silt/ clay fractions as humin in association with the fine clay fraction. We have observed that this dark fraction can be composed of up to 40% organic matter. This material can certainly be classed as humin, but it is possible that is co-sedimented with the fine clay fraction. It is important to establish to what extent soil humin is associated with the soil clay.
Figure 2. CP/MAS (Top) and Bloch decay (Bottom) 13 C NMR spectra of the humin isolated in DMSO/H2SO4 from a grassland surface soil. Fig. 2 shows the CPMAS and Bloch decay spectra for the humin isolated in the DMSO (94%)/H2SO4 (6%) solvent system from a similar soil in long term grassland. It is clear that the spectra are dominated by aliphatic hydrocarbon functionality, but there is evidence also for carbohydrate and possibly alcohol/ether (on the 70-90 ppm resonance), but the evidence for aromatic functionality is weak. The carboxyl is likely to arise from acid/ester functionalities from long chain fatty acids. More than 90% of the residual material was recovered in the DMSO/acid system from the IHSS (Mollisol) soil standard, and >75% was recovered from other soils studied. The NMR spectra for the DMSO/acid product closely matched the spectra for the residual materials, and for the materials recovered when the residuals were demineralised with HF. The Bloch decay spectrum emphasizes the aliphatic hydrocarbon contribution and shows the presence of amorphous (30 ppm) and ordered (33 ppm) methylene groups. HRMAS and 1H-1H TOCSY data confirmed the minor contributions of carbohydrate and of peptide materials. The proton NMR spectrum of the same humin fraction (Fig 3) also provides clear evidence of the dominance of aliphatic (methyl and methylene) H in the humin fraction with only small amounts of aromatic, carbohydrate, carboxylic etc. moieties that are such prominent features of the HA and FA spectra
REFERENCES. (1) Piccolo,A. 2001. The supramolecular structure of humic substances. Soil Science 166, 810-832 (2) Kononova, M. M. 1966. Soil organic matter. Its nature, its role in soil formation and in soi fertility, Pergamon Press, Oxford, U.K. (3) Hedges, J.I. & Keil, R.G. 1995. Sedimentary organic-matter preservation: Assessment and speculative synthesis. Marine Chemistry 49, 81-115. (4) Stevenson, F.J. 1994. Humus Chemistry; Genesis, Composition, Reaction, New York, John Wiley and Sons (5) Rice, J.A. & MacCarthy, P. 1989. Isolation of humin by liquid-liquid partitioning. Science of the Total Environment 81, 61-69. (6) Song, G.X., Hayes, M.H.B., Novotny, E. H. & Simpson, A.J. 2011. Isolation and fractionation of soil humin materials using alkaline urea and DMSO plus sulphuric acid. Naturwissenschaften 98, 7-13.
Conclusions Much of the evidence that has been published for the compositions of humins is flawed because care was not taken to remove HSs, and the carbohydrate and peptide materials associated with the humin. Also some of the NMR spectra were obtained from samples
284
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Spectroscopic developments for study of soil organic matter in liquid extracts, with approach to its impact on metals behavior: Application to a soil amended with compost O. R. Mouloubou(a)*, P. Prudent(a), S. Mounier(b), F. Theraulaz(a) (a)
Aix-Marseille Université, CNRS, Laboratoire de Chimie de l'Environnement, FRE 3416, 3 pl. Victor Hugo, case 29, 13331 Marseille Cedex 3, France. (b) Université du Sud Toulon-Var, laboratoire PROTEE, EA 3819, 83987 La Garde, France. * Corresponding author e-mail: [email protected] Keywords: soil organic matter ; fractionation ; UV and fluorescence spectrophotometry ; metals. Abstract The characterization and the follow-up of Soil Organic Matter (SOM) evolution after compost spreading were studied through the use of a sequential extraction (Water Extractable OM, HCl, NaOH and pyrophosphate extracts), at 2 different depths and over one year after amendment. The SOM fate and behaviour show a difference between amended and non-amended soil. Especially the first month after spreading, it results a higher amount of Dissolved Organic Carbon and a decrease in the UV ratio E2/E3 characteristic of greater humification (aromaticity) and molecular size (more complex molecular structure) of the SOM. Similarly, we observed a decrease in fluorescence index on water and acid extracts, characteristic of a major contribution of organic compounds with terrestrial origin. The values of these indices tend to stabilize after the first month of experimentation. Copper, chromium, nickel are the most abundant metals with small variations between amended and non-amended soils. SOM in function of time is studied by the use of a sequential extraction of OM allowing to obtain different fractions (Water Extractable Organic Matter (WEOM), HCl extract, NaOH extract and pyrophosphate extract). An exploitation of spectral signals (UV spectrum and FEEM) of each liquid fraction (3), either by the study of different spectral indices or by PARAFAC treatment of FEEM, make possible study of amendment impact and of interactions and dynamic of SOM depending on its temporal evolution. In some liquid fractions, quantitative physicochemical measurements (Dissolved Organic Carbon (DOC), trace metal concentrations) are also realized. To attain these aims, an experimental survey has been realized on a plot of the French National Agronomic Research Institute (INRA) near Avignon (France), by spreading of urban compost (mixture of 65% Residual Urban Waste compost and 35% green waste compost) on a calco-silty-clayey soil. Compost has been applied with a ratio of 30t/ha. Soils samples were collected on amended plot (treated soil) and on non-amended one (control soil), at 2 depths (0-15cm, and 15-25 cm) and at different periods: 0, 0.5, 1, 2, 3, 6, 9 and 12 months after spreading. Soil samples were dried, sieved (2mm) and ground (<0.2mm), before carried out liquid sequential extractions and numerous analysis. A synthetic scheme of the applied methodology is presented on Figure 1.
Introduction The evolution of the European and French legislation regarding organic valorization of waste incites to the development of networks of valorization among which the composting is situated in the front line, account held among others by the economic and environmental interest of compost bound to the presence of humic substances conferring it a trade and sustainable capital gain. The sustainable use of soils which represent a not renewable resource needs amendments with Organic Matter (OM) which is usually based on various composts. OM is an important component of soil due to its physical, chemical and biological participation (1). The characterization and the follow-up of the Soil Organic Matter (SOM) evolution after compost spreading are thus important. An IHSS (International Humic Substance Society) procedure exists for the operational fractionation of organic matter and several techniques of analysis are available for its characterization (pyrolysis, isotopy, spectroscopy). Among the spectroscopic approaches, the absorption spectrum UV, and more particularly the fluorescence excitation emission matrix (FEEM) are nowadays a common and relevant analytical way due to its high sensitivity and its potentiality of OM discrimination coupled with simplicity and fast measurement (2). In this context, this research focus on the fate and behavior of organic matter, with natural and anthropogenic origin, within a soil submitted to the spreading of urban compost. Experimental Our works follow the study of SOM at 2 differents depths and over one year, about is OM origin, natural or anthropogenic. Impact of compost amendment on
285
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
extracted with pyrophosphate solution: between 35 and 45% of total Cu in soil solubilized by this extractant.
Figure 1: Synthetic scheme of the applied methodology
Analytic Jena multi N/C 2100S apparatus is used to COD determination, Agilent 8453 for UV spectroscopy (200-400 nm), Perkin Elmer instrument (λex/λem: 200-650 nm/220-700 nm) for fluorescence spectroscopy. Soils were mineralized (in triplicates) in a microwave mineralizer (Milestone Start D) using aqua regia (1/3 HNO3 + 2/3 HCl). A ratio of dry soil/ aqua regia solution corresponding to 1/20 w/v was used. The mineralization products were filtered with a 0.45 μm mesh and the metal levels were determined by ICP-AES (Jobin Yvon Horiba, Spectra 2000). Quality assurance–quality controls and accuracy were checked using standard soil reference materials (CRM049–050, from RTC-USA) with accuracies within 100 ± 10%.
[metals](mg/g)
Figure 2: Evolution of UV indices E2/E3 in the different liquid extracts = water (_E_) acid (_A_) soda (_S_) and pyrophosphate (_P_) in the soil “without compost” (control C, above) and with “addition of compost” (treated T, below). Box plot shows the distribution of the data around the median, whiskers delimit the non-outlier. 120,00
Cr
Treated soil
Cu
100,00
Ni
80,00
Pb
60,00
Zn
40,00 20,00
Results and Discussion The first results obtained show a significative influence of the compost amendment essentially the first month after spreading. An important increase of DOC in treated soil and specifically for pyrophosphate and NaOH extracts can be notice. Concerning fluorescence indices, a decrease in the biological index BIX (<1) characteristic of low biological activity (4) is observed in all fractions as a decrease in the fluorescence index (FI) (5) essentially in the WEOM and acid fractions, characteristic of a OM with terrestrial origin. A weak ratio E2/E3 (Abs250nm/Abs365nm), is characteristic of great humification (aromaticity) and high molecular size and weight(indicating more complex molecular structure of OM) (6). Major observation is an important decrease of this ratio during the first month after spreading in WEOM fraction of amended soil. However, we can notice a great variability and higher values of this index during all the experimentation time, either for non amended as for amended soil, in WEOM and HCl extracts (Figure 2). Values in WEOM tend to stabilization after 9 months. After analysis of total metal concentrations in soils, it appears that Copper (Cu), Chromium (Cr), Nickel (Ni), and Lead (Pb) are the most abundant metals in soils with globally low differences between control and amended soil. Only low evolutions are noticed according to time for total metal concentrations (Figure 3). Very low fractions of total metals are extracted by water in soils : for instance in the case of Cu, from 0.1 to 0.4 mg/kg, representing 0.01 to 0.5% of the total Cu in soil. The main proportions of Cu are
0,00
0
0,5
1 3 6 Time (month)
9
12
Figure 3: Evolution of total metal concentrations (mg/kg of dry weight) in amended soil according to time after spreading of compost (from 0 to 12 months).
Other information with PARAFAC treatment of FEEM and with the determination of chelating capacity of WEOM by coupled quenching of fluorescence and PARAFAC, are currently on development. REFERENCES
(1) Traversa, A.; D’Orazio, V.; Senesi, N. Forest Ecology and Management 2008, 256(12), 2018-2028. (2) Derrien, D.; Dignac, M.F.; Dudal Y. Biogeochem. 2011, 106, 1-4. (3) Hassouna, M..; Théraulaz, F.; Massiani, C. Geoderma,2012, 189-190, 404–414. (4) Para, J.; Coble, P.G.; Charrière, B.; Tedetti, M.; Fontana, C.; Sempéré, R. Biogeosciences 2010, 7(12), 4083–4103. (5) McKnight, D.M.; Boyer, E.W.; Westerhoff, P.K.; Doran, P.T.; Kulbe, T.; Andersen, D.T. Limnology and Oceanography, 2001, 46(1), 38–48. (6) Baduel, Voisin, Jaffrezo, Atmospheric Chemistry and Physics, 2009, 5949-5962.
Acknowledgments: The authors thank Laurent Vassalo and Carine Demelas for their analytical assistance in TMM measurements, and this study was initially funded by the French Research National Agency (ANR-08ECOT-004 CleanWast), and financially amended by the National Innovative Cluster on Risk Management.
286
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Scanning Transmission X-ray Microscopy and solid-state 13C NMR analysis of the chemistry and the composition of the humic components stabilized by smectite-illite clay minerals in soil Sofia A. Oufqir(a,b), Paul R. Bloom(b), Brandy M. Toner(b)
(a) Department of Chemistry, Faculty of Science, University Mohamed V-Agdal, Rabat, Morocco (b) Department of Soil, Water, and Climate, University of Minnesota, Saint Paul, MN, USA. * Corresponding author e-mail: [email protected] Keywords: Humic material, clay minerals, Stabilization, STXM, NMR Abstract The association of organic matter (OM) with clay minerals plays a key role in stabilizing and preserving OM against microbial biodegradation. In this study we determined the composition of organic C in direct contact with clay particles, described the spatial distribution of organic and black C (BC) associated with clay-size nanoparticles of a pedogenically young Minnesota soil in different subfractions obtained by particle size and density fractionation, and spatial correlation of C and N with the major phyllosilicate elements. Three types of analyzes were used, nano-scale scanning transmission x-ray microscopy (STXM), solid-state 13C nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction (XRD). The XRD results indicated a dominance of interstratified smectite-illite clays in the soil. The 13C-NMR and STXM results suggested that the smectite-illite sheets in our soil preferentially retain the peptide, lipid, and polysaccharide components of humified OM favoring the protection of these normally readily biodegradable fractions relative to the lignin-derived phenolic components. Oxidized BC constitutes a major component of the light fraction as a separate phase, and is likely associated with the lignin fraction. main goal of this study was to (i) elucidate the chemistry and the composition of the carbon associated with clay particles in different soil clay density fractions using solid-state 13C NMR spectroscopy combined with C STXM-NEXAFS nano-microspectroscopy; and (ii) distinguish between the BC and the lignin fractions of aromatics associated with these clay nanoparticles.
Introduction Understanding of soil function is crucial to environmental quality and agriculture. Clay particles are thought to interact with OM, favoring its protection from degradation and resulting in high quality and healthy soils capable of enhancing crop production for agricultural and ecosystem functions in natural or urban areas. Currently, the published literature presents differing results regarding the nature of OM associated with clay minerals; preferential binding of aromatic components of humified soil OM by clays [1] versus preferential binding of aliphatic components [2]. Aromatic components of soil C embody mainly degraded lignin and BC fractions. It is very important to distinguish between their chemistry in order to understand their fate in the soil. Recently, various studies showed compelling results about the preservation of lignin components by clay minerals in soil. Some scholars suggested that the content of degraded lignin in soil OM is related to clay minerals indicating the stabilization of lignin in clay mineral fractions by physico-chemical processes [3, 4]. Whereas others reported that lignin components are segregated from mineral-bound OM [5,6]. With regard to BC, there is some evidence that it is abundantly present in the light density fraction (LF) of soil and partially present in the middle and heavy fractions (MF, HF respectively) [7]. Moreover, a recent STXM-NEXAFS study showed association of the BC and non-BC OM with mineral oxide and hydroxides in soil [8]. However, little is known about the association with smectitic clays in soil. On this basis, we aimed to address these controversies about the nature of the carbon stabilized by clay particles of a clay soil with very low hydrous Fe and Al oxide content, using the powerful STXMNEXAFS nano-microspectroscopy technique. The
Experimental For this study, we collected a surface soil (0-15cm) from an agricultural field of a pedogenically young Mollisol rich in smectite-illite clays, located in southwestern Minnesota, USA, and characterized by pH 6.0, 32.5% clay, and 3.7% organic carbon. We prepared a whole soil clay sample of particle size <2 µm by dispersion using low energy sonication to disaggregate the soil in distilled water followed by sedimentation and siphoning, then we flocculated the siphoned aliquots with 0.5M MgCl2 prior to freezedrying. We additionally separated the whole soil clay sample using density fractionation in sodium polytungstate solutions at densities of 1.8 and 2.1g/cm3 to separate lower density fractions high in organic C and BC from the fractions of C bound to clay. The resulting samples LF, MF, and HF were freeze-dried and analyzed with the whole soil clay for C:N ratio. To successfully achieve our goals, we adopted a two-phase approach. Phase one consists of comparing our data to the published work by investigating the OM associated with the clay-size soil sample (<2μm). Phase two consists of identifying the humic components contained in the different density fractions, and their associations with each other and with the smectite-illite clay nanoparticles. We used XRD to determine the clay phase of the soil, cross polarization (CP) and direct polarization (DP) 13C
287
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
NMR spectroscopy to characterize the samples, and STXM-NEXAFS to map the spatial distribution of C forms associated with the clay nanoparticles. The STXM measurements were conducted at the Canadian Light Source and Advanced Light Source synchrotron facilities.
the lignin-derived phenolic components; (2) separate phase BC constitutes a major component of the light fraction, and is scarcely present in the MF and the HF; and (3) lignin components are embodied in the LF and are likely associated with BC.
Results and Discussion The XRD results indicated a dominance of interstratified smectite-illite clays in the soil. The C:N ratio exhibited the highest value (19.5) in the LF suggesting low peptide concentration in this fraction The solid-state CP-MAS 13C-NMR spectrum of the whole soil clay fraction revealed abundance of aliphatic C as compared to the aromatic C, with absence of phenolic C at 150-160ppm. In contrast, the DP-MAS 13C NMR data showed more intense bands of aromatic C than the aliphatic ones, with presence of a weak peak at 154ppm likely attributed to BC structure. Our data suggest that in addition to peptides, lipids, and polysaccharides, BC constitutes a major component of the whole clay fraction. The STXMNEXAFS results confirmed that the proteins are abundant in the whole soil clay fraction, associated with saccharides, and partially separate from lipids; and that the BC constitutes a distinct phase (Fig.1).
Figure 2. (a1 and b1) NEXAFS spectra of corresponding regions in (a2 and b2) showing the separate phase of the BC in each fraction. (a2 and b2) STXM maps of the density light and middle fractions of the whole soil clay describing the spatial distribution of C. The extracted x-ray absorption spectra are typical of oxidized BC [9].
REFERENCES
[1] Feng, X., A. J. Simpson, M. J. Simpson. Organic Geochemistry. 2005, 36, 1553. [2] Wang K, Xing B. 2005. J Environ Qual. 2005 Jan-Feb; 34 (1): 342-9. [3] Grünewald Grünewald, G., Kaiser, K., Jahn, R., Guggenberger, G., 2006. Organic Geochemistry, 37, 1573 e 1589. [4] Clemente J S, Simpson M J. 2013. Organic Geochemistry 58: 1-12 [5] Spielvogel, S., Prietzel, J., Kgel-Knabner, I., 2008. European Journal of Soil Science, 59,674e 692. [6] Kögel-Knabner , I., Guggenberger, G., Kleber, M., Kandeler, E., Kalbitz, K., Scheu, S., Eusterhues, K., Leinweber, P., 2008. Journal of Plant Nutrition and Soil Science 171,61e82. [7] Glaser B., Balashov E., Haumaier L., Guggenberger G., Zech W., 2000. Org Geochem 31:669-678 [8] Solomon, D., J. Lehmann, J. Wang, J. Kinyangi, K. Heymann, Y. Lu, S. Wirick, C. Jacobsen. Science of The Total Env., Vol 438, 1 November 2012, Pages 372-388 [9] Lehmann, J., B. Liang, D. Solomon, M. Lerotic, F. Luiza J. Kinyangi, T. Schafer, S. Wirick, and C. Jacobsen. Global Biogeochemical Cycles, Vol. 19, GB1013 0886-6236
Figure 1. STXM maps of the whole soil clay sample depicting C spatial distribution on smectite-illite nanoparticles. The images show different forms and associations of C manifested in different humic components associated with clay particles.
With regard to the density fractions, the 13C-NMR spectra showed that the LF has strong alkyl C-H bands characteristic of fatty acids plus strong C-O bands characteristic of polysaccharides, including the anomeric C band centered at 105 ppm. The aromatic band at 130 ppm and the phenolic C-O band at 150 pm are also pronounced indicating the presence of BC and/or lignin-derived components, contrary to the MF and the HF where the aromatic C is not abundant. However, smectite-illite nanoparticles are contained in all fractions. The STXM-NEXAFS results exhibited various components within the LF and MF. In the light fraction, C was found as separate phase BC (Fig.2a), aliphatic fractions of organic C associated with clay nanoparticles, and as separate aliphatic C. In the MF, we found aliphatic C bound to clay particles and as a well-defined structure and some separate phase BC with a particle size was about 300nm (Fig.2b). We conclude that (1) the smectite-illite sheets in our soil preferentially preserve peptides, lipids, and polysaccharides favoring the protection of these normally readily biodegradable fractions relative to
Acknowledgments: We thank Dr. Patrick Hatcher from Old Dominion University for providing us with the NMR data. The STXM-NEXAFS data were collected at the Canadian Light Source and the Advanced Light Source facilities at beamlines 10ID-1 and 5.3.2 respectively. We thank Dr. James Dynes, scientist at the Canadian SM beamline, for his analytical guidance.
288
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Genotoxicity induction by Multi-Walled Carbon Nanotubes interacting with Humic acids in cultured human lymphocytes M-S Vidali, E Bletsa, GC Skoutelis, Y Deligiannakis, D Vlastos*
Department of Environmental and Natural Resources Management, University of Patras Greece, 30100 Agrinio, Greece *E-mail: [email protected] Keywords genotoxicity; humic acid (HA); Humic-acid-like polycondensates (HALP); the cytokinesis block micronucleous (CBMN) assay; multi-walled carbon nanotubes (MWCNTs) Abstract Carbon nanotubes are fiber-shaped substances that consist of graphite hexagonal-mesh planes (graphene sheets) present as a single-layer or as multilayers with nest accumulation, and include single-walled carbon nanotubes and multi-walled carbon nanotubes (MWCNTs). Nonetheless, despite the already widespread use of nanomaterials in modern technology, there is a serious lack of available information on human health and environmental implications of manufactured nanomaterials. Humic Acids (HA) are the principal active components of soil organic matter. Humic-acid-like polycondensates (HALP) can be formed from polymerization of simple phenols and phenolic acids. The possible genotoxic effects of the mixtures of MWCNTs with HA and/or HALP were studied in cultured human lymphocytes applying the cytokinesis block micronucleous assay for the detection of micronuclei (MN) in the cytoplasm of interphase cells. Our study revealed a statistically significant induction of MN frequencies in cultured human lymphocytes treated with a mixtures of MWCNTs and HA as well as of MWCNTs and HALP. The use of cytochalasin-B, an inhibitor of actin polymerization, which prevents cytokinesis while permitting nuclear division leads to formation of binucleated (BN) cells which are scored for the presence of MN (OECD, 2010). The focus of this study is to compare the potential genotoxic effects in human lymphocytes in vitro of MWCNTs in combination with HA, one of the most common substances of soils as well as in combination with HALP, the synthetic HA.
Introduction Carbon nanotubes (CNTs) are an important new class of nanomaterials that have numerous useful applications. They are fiber-shaped substances that consist of graphite hexagonal-mesh planes (graphene sheets) present as a single-layer or as multilayers with nest accumulation, and include, among others, the multi-walled carbon nanotubes (MWCNTs). They are regarded as nanomaterials because of their nanoscale diameter (Ema et al., 2012). The widespread use of nanomaterials in modern technology has led to its significant presence in the environment and in the ecosystems. There is a serious lack of available information on the human health and environmental implications of manufactured nanomaterials (Hussain et al., 2005). Humic acids (HA) are the principal active components of soil organic matter. Humic-acid-like polycondensates (HALP) can be formed from copolymerization of simple phenols and phenolic acids under O2 (Giannakopoulos et al., 2009). There is accumulating evidence that HAs can solubilise CNT is waters under environmentally relevant conditions. However the ensuing effects of solubilisation of CNTs by HAs and/or HALP at the genotoxic level is under investigation from our research group (Vidali et al., 2013). Herein, the possible genotoxic effects of the mixtures of MWCNTs with HA and/or HALP were studied in cultured human lymphocytes applying the cytokinesis block micronucleous (CBMN) assay for the detection of micronuclei (MN) in the cytoplasm of interphase cells. MN may originate from acentric chromosome fragments or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The simplicity, rapidity and sensitivity of the CBMN assay make it a valuable tool for genotoxicity screening. The assay detects the potential clastogenic and aneugenic activity of chemicals in cells that have undergone cell division.
Experimental Blood samples were obtained from two non-smokers, healthy individuals (21 and 25 years old) not undergoing any drug treatment, who did not have any viral infection or X-ray exposure for over a year. Blood samples were kept under sterile conditions in heparinized tubes. Whole blood (0.5 ml) was added to 6.5 ml Ham’s F-10 medium (Gibco), 1.5 ml foetal bovine serum (Gibco) and 0.3 ml phytohaemagglutinin (Gibco) to stimulate cell division. The mixtures of MWCNTs+HA as well as of MWCNTs+HALP, were studied at different concentrations (5+20, 15+60, 25+100 μg ml-1) and (5+8, 15+25, 25+42, 30+50 μg ml-1) respectively. The reported results represent the pooled data from two independent experiments. 44 h after initiating cultures, 6 μg ml-1 Cytochalasin-B (Cyt-B) (Sigma) was added to the culture medium to block cell division. The use of cytochalasin-B, an inhibitor of actin polymerization which prevents cytokinesis while permitting nuclear division, leads to formation of binucleated (BN) cells which are scored for the presence of MN. Cultures were incubated at 37 o C in a humidified atmosphere of 5% CO2 for 72 h. 72 h after the initiation of culture, cells were harvested and collected by centrifugation. A mild hypotonic treatment with 3:1 solution of Ham’s medium and milli-q H2O was left for 3 min at room temperature which was followed by 10min fixation (for at least 3 times) with a fresh 5:1 solution of methanol/acetic acid. Cells were stained with 7% Giemsa (OECD, 1
289
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
2010). Standard criteria were used for scoring MN (Fenech et al., 2003). In order to determine possible cytotoxic effects, the Cytokinesis Block Proliferation Index (CBPI) was calculated by counting at least 500 cells for each experimental point. CBPI is given by the equation: CBPI = M1 +2M2 + 3(M3 + M4)/N where M1, M2, M3 and M4 correspond to the numbers of cells with one, two, three and four nuclei and N is the total number of cells (OECD, 2010). The statistical analysis of the MN data was conducted using the G-test for independence on 2x2 tables. The chi-square test (χ2test) was used for the analysis of CBPI among each treatment.
Figure 1.Induction of MN in human lymphocytes treated with MWCNTs+HA and MWCNTs+HALP mixtures (2000 BN cells scored per experimental point; * p<0.01, **p<0.001)
Results and Discussion The results obtained from human peripheral blood lymphocyte cultures treated with different concentrations of MWCNTs+HA and MWCNTs+HALP mixtures are shown in Figures 1 and 2. In the case of mixed MWCNTs+HA and MWCNTs+HALP treatments our results showed a remarkable three to five-fold increase and approximately three-fold increase, respectively, MNinduction in MWCNTs+HA and MWCNTs+HALP treated cultures. Our Attenuated Total Reflection FTIR study shows that HA solubilizes the MWCNT via specific interactions of the COO groups with the CNT’s surface. The formed HA/HALP-MWCNTs associates have the potential to penetrate not only the cell membrane but also the nucleus membrane inducing MN. Previous reports on the genotoxicity of MWCNTs have been reported, however for much higher concentrations (Gonzalez et al., 2011). Here, the enhanced genotoxic capacity, when in contact with HA as well as with HALP is documented for the first time herein. The cytotoxic effect of MWCNTs with HA or HALP mixtures was evaluated by the cytotoxic index (CBPI). Regarding this index, no statistically significant differences of the CBPI were observed between control and mixtures treated cultures. As can be seen in Figure 2, MWCNTs+HA mixture caused a drop of CBPI value, thus indicating that the formed MWCNTs+HA have the potential to affect the proliferation of the cells. In the case of mixed MWCNTs+HALP treatments, CBPI value was gradually increasing and reach the control value. By comparison of the tested mixtures, the MWCNTs+HA mixture showed higher genotoxic and cytotoxic potential than the MWCNTs+HALP mixture, in human lymphocytes treated cultures. A number of factors may influence the observed differences in the genotoxic and cytotoxic effects of the tested mixtures, such as the differential solubility of the MWCNTs between HA and HALP, mixtures absorption, rate and distribution of biotransportation, availability at the target site and cell permeability. In conclusion, our study revealed a statistically significant induction of MN frequencies in cultured human lymphocytes treated with a mixtures of MWCNTs and HA and/or HALP. A comparative analysis of the genotoxicity and cytotoxicity of the
Figure 2.Induction of CBPI values in human lymphocytes treated with MWCNTs+HA and MWCNTs+HALP mixtures (1000 BN cells scored per experimental point) selected mixtures in the tested concentrations points that the MWCNTs+HA mixture is more genotoxic and cytotoxic than the MWCNTs+HALP mixture. The present study showed the clear-cut genotoxic effect occurred in human lymphocyte cultures, after in vitro exposure to relevant concentrations of the tested mixtures. MN frequency observed in human lymphocytes, showed MWCNTs and HA or HALP mixtures ability to enhance genotoxic effects, while there was a first evidence of the potential genotoxic effects of the particular mixtures on human lymphocytes. Taking into account that the examined concentrations were very low, MWCNTs should be handled with great care, in order to minimize its environmental and human risk. REFERENCES
(1) Ema, M.; Imamura, T.; Suzuki, H.; Kobayashi, N.; Naya, M.; Nakanishi,J.Regul. Toxicol. Pharm.2012,63, 188– 195 (2) Fenech, M.; Chang, W.P.; Kirsch-Volders, M.; Holland, N.; Bonassi, S.; Zeiger, E.Mutat. Res.2003, 534, 65– 75 (3) Hussain, S.M.; Hess, K.L.; Gearhart,.JM.; Geiss, K.T.; Schlager, J.J.Toxicol. In Vitro2005, 19, 975–983 (4) Giannakopoulos, E.; Drosos, M.; Deligiannakis, Y.J. Coll. Interf. Sci.2009,336, 59-66 (5) Vidali, M.S.;Vlastos, D.; Bletsa, E.; Deligiannakis,Y.2013 In: Xu J et al. (eds.), Functions of Natural Organic Matter in Changing Environment, Zhejiang University Press and Springer Science+Business Media Dordrecht, 745-749 (6) OECD 2010,OECD Guideline for Testing of Chemicals No. 487. OECD, Paris. Available: http://www.oecd-ilibrary.org/environment/test-no-487-invitro-mammalian-cell-micronucleus-test (accessed: 29 June 2014)
290
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Can Humic Products Become Mainstream Amendments for Improving Crop Production? D.C.Olk (a) *, O.S. Yakimenko (b), W.R. Kussow (c), D.L. Dinnes(a)
(a) USDA-ARS, National Laboratory for Agriculture and the Environment, Ames, IA, 50011, USA (b) Department of Soil Science, Moscow State University, Moscow 119992, Russia. (c) Department of Soil Science, University of Wisconsin, Madison, WI, 53706, USA * Corresponding author e-mail: [email protected] Keywords: humic products, crop production Abstract Humic products have been used in cropland production for several decades but only by small numbers of farmers. Appreciable proportions of field studies demonstrate efficacy of the products for numerous crops, justifying their further evaluation. Their adoption by mainstream farmers could be accelerated by addressing critical knowledge gaps: (i) Thorough published reviews of field evaluations, including assessment of how product efficacy is affected by field factors including crop, soil type, management practices, weather patterns, and economic yield level; (ii) Multi-location, multi-year field evaluations published in leading journals that assess field factors and describe crop responses at key growth stages; (iii) Elucidation of the soil or plant processes responsible for crop responses; (iv) Identification of the compound(s) in humic products that promote crop growth; and (v) Improved quality control of humic products, including standard procedures for measuring their humic and fulvic acid contents and rapid bioassays for distinguishing effective products from inert frauds. Introduction Humic products are produced from any of multiple sources, including immature coal (lignite and leonardite) deposits, seaweed, compost, and sediments. They have been used for decades in production agriculture to promote crop growth and economic yield, yet evidence for their field efficacy is much sparser than desired for a crop input. Here we present a plan for a science-based evaluation of humic products, beginning with a review of existing literature on plant growth studies and leading to more rigorous, process-level field evaluations.
language literature. Most of the studies used humic products derived from lignite or leonardite, although details on the product sources or their application rates could not be gathered from all articles. Economic yield responded favourably in about two-thirds of the studies, but studies finding no yield response are admittedly less likely to be published. The positive responses occurred across wide ranges of crops and soil types. The authors concluded that field efficacy of humic products depends on a number of factors, including humic product quality, environmental conditions, and crop management practices. An overriding outcome from these reviews is that each field study was in most cases a controlled research trial having few site─years. No attempt was made by any single study to describe the effects of environmental factors and crop management practices on humic product efficacy, although the efficacy of other agricultural inputs will vary with soil type, landscape position, cropping history, and other field variables. In-season plant growth measurements were uncommon, and the acquired data did not allow any conclusions regarding mode of action. In a current field study of maize, Olk et al. (7) evaluated a humic product that was applied in test strips on farmers’ fields. The product caused increased grain weight in 70-80% of 30+ fields in each of three years, primarily due to reduced occurrence of short ears. In other replicated field treatments, increased leaf area indicated an early- to mid-season stimulation of plant growth. Later in the growing season, root growth increased, and crop senescence was delayed. Although the grain weight responded to the product across a range of soil types and locations, the response was less consistent in a droughty year. Very limited evidence suggested that maize on poorly drained, wet soil responded less to the product than on equivalent
Reviews of Greenhouse and Field Evaluations Major reviews of humic substances and plant growth have provided excellent descriptions of plant responses in growth, physiology, biochemistry and genetics, but in mostly greenhouse or growth chamber settings, for example Chen and Aviad (1), Nardi et al. (2), Verlander et al. (3), and Canellas and Olivares (4). More recently, Rose et al. (5) performed a metadata analysis on 81 published articles found through electronic literature searches using key terms. The reviewed literature included studies performed in greenhouses or in the field, and using humic substances derived from several source materials. Statistical analyses showed that humic substances had highly significant positive effects on plant shoot and root growth. This effect was modified by the humic substance source [immature coals, peat, soil, and compost (green waste versus manure)] and secondarily by environmental factors. Rose et al. also noted that the majority of studies followed plant growth for less than 3 months during solely vegetative growth and did not monitor economic yields. Olk et al. (6) reviewed solely field evaluations as published in U.S. university publications, recent on-line journals, and the Russian-
291
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
drained soil. An associated field study demonstrated a clear effect of landscape position (and hence soil type) on grain yield and the degree of yield response to the humic product (data not shown). These results question the value of greenhouse studies or single-year research station field trials as the sole means for evaluating humic products.
establishing the biological efficacy of a product. Consequently the consumer cannot ascertain for any product its concentration, source material, or variability among production batches. The consumer cannot discern between effective products and similarly appearing but much less expensive substances that are rumoured to be sold as humic products and are of questionable value to crop production, including molasses, lignosulphonates, and hard coal. Therefore sales of humic products typically occur through word of mouth or first-hand experience gained by the consumer, compelling a local approach to sales and marketing of humic products.
Knowledge Gaps Multiple and substantial knowledge gaps have suppressed widespread and informed use of humic products in cropland production. Their resolutions would accelerate growth of the humic product industry. These knowledge gaps include:
A Call for Future Action Validation of humic products is required for the industry to grow. We call for many local collaborations between industry and researchers to enable rigorous field evaluations, including adequate replication, formal statistical analyses, and in-season measurements that are often lacking in in-house trials performed by product vendors. Detailed descriptions of research site characteristics and crop development would help agronomists, crop physiologists, and soil scientists identify possible mechanistic explanations for product efficacy. Numerous local collaborations are needed to address the bewildering array of different products (varying in source material, extraction method, and post-extraction processing), crops, field management practices, and soil types. Dialogue among these local collaborations will help identify major trends in product efficacy. Should different types of products appear to promote different soil or plant processes, we call for multiple local investigations into identifying these processes. Collaboration between crop physiologists and soil scientists will be essential to determine whether the underlying mechanisms are soil- or instead plantbased. Finally, researchers should collaborate with industry to develop standard procedures for measuring the contents of humic products and developing commercialized rapid bio-assays for discerning effective products from inert frauds. Such procedures would give the consumer more confidence in the authenticity and reliability of marketed products.
When and where products promote crop growth No agricultural input increases economic yield in all cases, not even nitrogen fertilizer. Yet the current debate on humic product efficacy ranges from some vendors who guarantee specified crop yield boosts in all situations to researchers who argue no humic products ever promote crop growth based on limited studies in the greenhouse or on research stations. We need field studies having treatments and measurements that are uniform across years having different weather patterns and across multiple locations or soil types. In-season measurements of crop growth at key growth stages are needed to understand how the yield increase develops and expresses itself through yield components. Geographic information systems can be used to quantify soil and weather effects on product efficacy. The discussion also needs to consider those studies that found no benefit of humic product to crop growth. Mechanisms for stimulating crop growth Vendors describe a long list of soil and plant properties as possible causes for crop yield responses to their products, but their selection of soil properties is based only on known benefits of soil organic matter. In contrast, researchers have largely focused on plantbased mechanisms for stimulating growth. The true causes of increased yield must be identified for the validity of humic products to be recognized by the research community and those agricultural sectors that look to researchers for leadership—extension workers, crop consultants, government agencies, and some farmers. Industry has shown only modest interest in determining these mechanisms. This situation also describes the search for the compound(s) in humic products that provoke crop responses. The research community and its associates look for identification of the active ingredients before fully accepting humic products, while industry has shown little interest. Identification of the causal compound(s) could enable both better prediction of which crops and field settings are most suitable for humic products and also more effective products.
REFERENCES
(1) Chen, Y.: Aviad, T. In: MacCarthy, P. et al. (eds.) Humic substances in soil and crop sciences: Selected readings. 1990. Am. Soc. Agron., Madison, WI. pp. 161186. (2) Nardi, S.; Pizzeghello, D.; Muscolo, A.; Vianello, A. Soil Biol. Biochem. 2002, 34, 1527-1536. (3) Verlander, G.; Pycke, B.; Mertens, J.; Debersaques, F.; Verheyen, K.; Baert, G.; Bries, J.; Haesaert, G. J. Plant Nutr. 2009, 32, 1407-1426. (4) Canellas, L.P.; Olivares, F.L. Chem. Biol. Tech. Agric 2014, 1. http://www.chembioagro.com/content/1/1/3. (5) Rose, M.T.; Patti, A.F.; Little, K.R.; Brown, A.L.; Jackson, W.R.; Cavagnaro, T.R. Adv. Agronomy 2014, 124, 37-89. (6) Olk, D.C.; Yakimenko, O.S.; Kussow, W.R.; Dinnes, D.L. Agron. J. (in review). (7) Olk, D.C.; Dinnes, D.L.; Callaway, C.; Raske, M. In: Xu, J. et al. (eds.) Functions of natural organic matter in changing environment. 2013. Zhejiang University Press and Springer, Dordrecht. pp. 1047-1050.
Improved quality control of humic products The humic product market is wholly unregulated. It is lacking universally recognized standard procedures for measuring the humic acid and fulvic acid contents of commercial products and also any rapid assay for
292
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Compositional Studies of Humic Substances and Humin Associated with Sediments from an Estuarine Environment Rosaleen Mylotte and Michael H. B. Hayes Dept. Chemical and Environmental Science, University of Limerick, Ireland. Keywords: Humic Acid (HA), Fulvic Acid (FA), Humin, NMR Spectroscopy, Sediments. Abstract Humic substances and Humin from sediment cores in an estuarine environment were isolated and characterised. The study showed that the organic matter (OM) associated with fossil sediments (ca. 9000 years BP) was dominated by terrestrial OM signals. This suggests that this coastal region was a terrestrial environment prior to sea level rise. The study also found significant contributions from peptides to the organic compositions. Microbial necromass is the most likely source. It is suggested that labile organic components are protected from mineralisation through interactions with hydrophobic aliphatic hydrocarbons and with clay minerals. Using a novel NMR probe the humin was swollen in DMSO-d6 and the true solid species were identified. These components represent the truly recalcitrant organic species that are sequestered in the environment. 14’15.44’N, 09° 05’21.54’ W) in order to assess compositional differences in the organic materials (Fig. 1). Cumulative samples were taken from the top 100 cm of sediment from Core 1 (representing ca. 2260 years before present (BP)), the bottom 100 cm of Core 1 (representing ca. 9770 years BP), the top 100 cm of Core 4 (representing ca. 9200 years BP), and the bottom 100 cm of Core 4 (representing ca. 9450 years BP). Samples studied with NMR spectroscopy include: humic acid (HA); fulvic acid (FA); and humin (HU). HAs and FAs were extracted using 0.1 M NaOH, and 0.1 M NaOH + 6 M urea, then fractionated using XAD-8 resin [2]. HAs and FAs were not isolatable from the fossilied sediments from Core 1. Clay from the sediment (following HA and FA extraction) was isolated for HU extraction. The HU was isolated using DMSO + H2SO4 (SHU) [2]. Sugar analysis was carried out following the method of Hayes (3). Solid-state, solution-state, and swollen-state NMR experiments were carried out on the organic samples using a Bruker 500 Avance III spectrometer.
1. Introduction An estuary is a semi-enclosed body of water with riverine inputs and open access to the ocean. Galway Bay is a mixed aquatic environment on the West of Ireland where the Atlantic Ocean receives a fresh water input from the River Corrib. This organic matter (OM) associated with the sediments in this environment represents inputs from both the terrestrial and marine environment. The major focus has been on humic substances (HSs) in soils and aquatic environments. Sediment cores (6 m) offer a unique opportunity to study HSs and humin (HU) from a mixed environment. These cores can provide an archaeological record of environmental changes. The estuarine environment can have high organic carbon retention due to higher primary productivity (compared to the open ocean) and there is continuous organic input from riverine sources. Preservation of OM is especially high close to river mouths [1] and these retained components represent a valuable carbon sink. There is currently no standard approach for the isolation of organic components from sediments. Therefore, using methods developed for soils [2] the organic compositions were extracted from the sediments. Radiocarbon dating of the sediments provides indications of time when the organic components were laid down and can help make inferences about the environment. Nuclear magnetic resonance (NMR) spectrometry is a powerful analytical technique and it was used for indepth characterisation of the organic compositions. Data acquired provide details on the organic components retained in estuarine sediments and indicate which components are concentrated in fossilised sediments.
Figure 1. Map of the coring positions in Galway Bay, Ireland.
3. Results
2. Experimental
There was a focus on the HU from the top of Core 1. Most interesting was the strong protein resonances identified using a number of NMR techniques. To confirm these protein compositions, a 1H diffusion edited (DE) spectrum of the humin is compared to a 1H spectrum of albumin (Fig. 2). Sugar analyses were carried out on the HAs. The [MAN+GAL]/[XYL+ ARA] ratios for these samples from Core 1 and Core 4 are ca. 2
Four sediment cores (up to 6 m in depth) were taken to track the outflow of the River Corrib into the Bay. The Nuclear Magnetic Resonance (NMR) spectroscopy study focuses primarily on Core 1 (the core nearest the freshwater outflow; 53° 15’22.21’N, 09° 02’14.4’W), and Core 4 (the furthest from the freshwater outflow; 53°
293
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
or greater, suggesting that microbial sugars dominate over plant sources.
the solvent. and confer recalcitrance to the organic materials as they impede microbial degradations.
Figure 2. 1H NMR spectrum of albumin, and DE 1H NMR spectrum of SHU from Core 1 top. Assignments are (1) amide in peptides, (2) aromatic amino acids ( denotes phenylalanine, denotes tyrosine), (3) α-proton (peptides), (4) O-Aromatics (methoxyl signal in humin), (5) DMSO (solvent), (6) methylene adjacent to a carbonyl (R2-OCO-CH2-R1, some may be in the form of lipoprotein, thus R2 would be a protein), (7) aliphatic methylene units γ to an acid or ester, (8) amino acid side chains, (9) aliphatic methylene (CH2)n, (10) CH3 [4].
Figure 4. 13C CPMAS NMR (swollen-state) of Humin from Core 1.
Protein and carbohydrates are considered to be labile OM as these provide an important energy source to organisms in the environment. However, these ‘labile’ components were identified in the recalcitrant HUs retained in the sediments. The highly ordered aliphatic hydrocarbon components identified (Fig. 4) can provide hydrophobic domains for the encapsulation of these labile organic components. The highly ordered (CH2)n and cellulose are inherently recalcitrant due to their structure, and therefore, are retained over long time scales. Clays also provide stability to OM in the environment. Fe, Al and Ca were present at high concentrations in the clay-sized fraction of the sediments. These are the most important inorganic species associated with C sequestration in the environment.
Humin from Core 1 bottom and the organic isolates from Core 4 (Fig. 3) have strong resonances in the aromatic region (ca. 130 ppm), relative to those in the Core 1 top isolates. These aromatic structures are associated with higher plants e.g. phenolic units in lignin, cutin, suberin, and tannins. Lignin resonances were strongest in the HA urea and the FA isolate from the base of Core 4, and these isolates also had a C/N ratio of 13 and 15, respectively. Aliphatic resonances, especially the ordered (CH2)n (33 ppm), ester resonances (ca. 170 ppm), and alkyl-OH (ca. 70 ppm) resonances may have contributions from cutan. The NMR data, coupled with chemical analyses, suggest that there were significant terrestrial organic inputs to this environment 9,000 years BP. It was expected that Core 4 would have less terrestrial influence due to the coring location (Fig. 1).
4. Conclusions NMR studies have shown the organic materials associated with the sediments to be composed mainly of aliphatic materials (ordered and amorphous), and small amounts of protein, and carbohydrate. The sources of these biomolecules are from the recalcitrant components of higher plants and from microbial necromass. Protection to the OM is provided from hydrophobic domains in the recalcitrant OM and from clay minerals. Acknowledgements We thank Prof. Andre Simpson for assistance in the NMR studies, and the IHSS for a training award to study in the laboratory of Prof. Simpson, References [1] Berner R. A. (1982) Burial of organic carbon and pyrite sulfur in the modern ocean: geochemical and environmental significance. Am. J. Sci. 282, 451-73. [2] Song G., Hayes M. H. B. Novotny E. H. and Simpson A. J. (2011) Isolation and fractionation of soil humin using alkaline urea and dimethylsulphoxide plus sulfuric acid. Naturwissenschaften 98(1), 7-13. [3] Hayes, D.J.M. (2012) Development of NIR spectroscopy models for quantitative predictions of lignocellulose components in wet Miscanthus. Bioresource Technology 142, 591-602 [4] Simpson A. J., McNally D. J. and Simpson M. J. (2011) NMR spectroscopy in environmental research: From molecular interactions to global processes. Prog. Nucl. Magn. Reson. Spectros. 58, 97-175.
Figure 3. 13C CPMAS NMR of humic substances (HSs) extracted from Core 4 (HA = humic acid, FA = fulvic acid, HA urea refers to the HAs extracted in 0.1 M NaOH + 6 M urea). 13
C NMR data were obtained for Core 1 top humin following the addition of DMSO-d6 (Fig. 4). This NMR experiment only detects the truly rigid components of the sample. Molecules that solubilise/swell will gain mobility and cannot be detected in the solid-state. This spectrum indicates that a portion of the aliphatic hydrocarbons, especially those with an ordered structure (33 ppm) are inaccessible to the solvent. Additionally the strong resonance at 70 ppm suggests that there may be large carbohydrate domains, or cellulose, in the humin composition. These components may be inaccessible to
294
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Chemical composition of soil humic acids after long-term swine manure applications C.R. Lourenzi(a)*, V. D’Orazio(b), C.A. Ceretta(a), D.P. Dick(c), A. Traversa(b), A. Cancian(a), T. Miano(b). (a)
Departamento de Solos, Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, 97105-900, Santa Maria, Brazil. (b) Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti - DiSSPA, Università degli Studi di Bari "Aldo Moro", Via Amendola, 165/A, 70126, Bari, Italy. (c) Departamento de Físico-Química, Instituto de Química, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves, 9500, 91501-970, Porto Alegre, RS, Brazil. * Corresponding author e-mail: [email protected] Keywords: Elemental analysis, FTIR, E4/E6 ratio, humic acids, swine manure. Abstract Successive applications of swine manure (SM) in soil can cause qualitative changes in soil organic matter (SOM). The study aimed to evaluate the impact of successive applications of SM on the chemical composition of humic acids (HA) isolated from the surface layer (0-4 cm) of two subtropical soils with distinct textural characteristics. In detail, the selected soils are: i) an Hapludalf soil (170 g kg-1 clay), treated for a long time with 19 applications rates of 0, 40 and 80 m3 ha-1 of SM; and ii) an Hapludox soil (834 g kg-1 clay), that received 6 applications of NPK and rates of 0, 8 and 16 Mg ha-1 of a mixture of compost shavings and SM. HA were characterized by FTIR spectroscopy, elemental analysis and E4/E6 ratio. Data obtained indicate significant correlations between manure applications rates and soil properties and HA elemental composition and spectroscopic features. of swine manure (SM), and an Hapludox soil (834 g kg-1 of clay), that received six applications of NPK and rates of 0, 8 and 16 Mg ha-1 compost shavings with SM. The extraction of HA were performed according to the methodology described by Almeida et al (6). The HAs were then characterized by elemental analysis, Fourier transform infrared (FTIR) and UV spectroscopies. In detail, the contents of C, N, H and S were determined in triplicate using a Fisons Elemental Analyzer model EA 1108 Instrument. All data were corrected for the ash content, previously determined gravimetrically after heating HA in an oven at 550 °C for 4 h. The O content was calculated as follows: O% = 100 - (C + N + S + H) %. FT IR spectra were recorded on pellets obtained by pressing under reduced pressure a mixture of 1 mg of HA and 400 mg of dried KBr, spectrometry grade, using a Nicolet Nexus spectrophotometer. The E4/E6 ratio was calculated as the ratio of the absorbances measured at 465 and 665 nm of a 0.05 N NaHCO3 solution containing 300 mg L-1 HA at pH 8-9, by using a UVVis spectrophotometer Perkin Elmer, model Lambda 15.
Introduction The Southern Region of Brazil is responsible for as much as 54% of the national swine production (1). The manure is used in composting processes and later on is distributed on the top soils cultivated with annual crops or pastures. However, as agronomic and environmental issues affect the soil's ability to retain C, it is important that changes in soil characteristics, especially those related to organic matter, are evaluated quantitatively and qualitatively. From a qualitative point of view, the soil organic matter (SOM) composition is conditioned mainly by environmental parameters, vegetation, land use and anthropogenic activity (2), among which the use of manure is crucial. In fact, repeated applications of swine manure (SM) to soil can alter the chemical composition of SOM, due to the presence of extremely high amounts of dissolved organic matter particularly rich in aliphatic moieties (3, 4). Anyway, several studies on the effects of SM application on the chemical characteristics of soil fulvic acids underlined that the manure has limited effects on the composition of soil humic substances (5). This study aimed to evaluate the impact of successive applications of SM on the chemical composition of humic acids (HA) in two subtropical soils with distinct textural characteristics.
Results and Discussion As expected, SM applications affected the chemical composition of HA. In particular, a significant increase of N content is measured in the HA from both soils treated with the higher SM doses, whereas no relevant effect is measured at the lower ones (Table 1). This result is strictly related to the large amount of Ncontaining compounds in the raw material applied (7).
Experimental The humic acids (HA) were isolated from the surface (0-4 cm) soil samples (air-dried and 2 mm sieved) of an Hapludalf soil (170 g kg-1 of clay), that received 19 applications rates of 0, 40 and 80 m3 ha-1
295
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
With respect to the other element contents, no significant changes are evident in HA from Hapludalf soil (Table 1). On the contrary, Hapludox soil HA show a significant increase of C content and a corresponding decrease of H and O contents at both treatment doses (Table 1). No changes are showed by C/N ratios in all HAs from both soils with respect to their corresponding control. On the contrary, even though H/C, O/C and (O+N)/C ratios do not show changes in the HA from Hapludalf soil, they are characterized by a significant
decrease in the HA samples from Hapludox soil, in general proportionally to the application dose (Table 1). In particular, the decrease of H/C ratios measured apparently could be associated with an increase of the aromatic character of the humic molecules (7). The lower H/O ratio suggests a larger amount of oxygenated groups, and, finally, the decrease of (O+N)/C ratio likely suggests a more hydrophobic character, following the SM treatment and independently from the application rate (Table 1).
Table 1. Elemental analysis, atomic ratios and E4/E6 ratio of HA isolated from untreated and treated Hapludalf and Hapludox soils. (O+N)/C
E4/E6
24.15 b 24.31 b 20.63 b
0.46 d 0.47 d 0.47 d
1.21 a 1.24 a 1.23 a
0.50 c 0.51 c 0.52 c
3.20 3.40 4.03
20.76 c 18.58 c 16.12 c 20.76 c
0.56 b 0.50 c 0.46 c 0.46 c
1.38 b 1.26 c 1.22 c 1.30 c
0.61 0.55 0.52 0.51
5.80 5.69 6.18 6.13
SM Control
a b b b
(a)
4000
Control
(b)
3500
3000
3500
3000
2500
2000
2500
2000
1500
1030
40 m3 ha-1 80 m3 ha-1
1259
These results are confirmed by the E4/E6 ratio values, which in general for all HA samples from treated soils are higher with respect to those of the corresponding control (Table 1). On the whole, E4/E6 ratios of HA from Hapludalf soil are lower than those measured for HAs from Hapludox soil. It is well known that E4/E6 ratio is inversely related to C content, aromaticity, degree of polycondensation, and molecular weight of organic molecules, whereas it is directly correlated to aliphaticity, O, and acidic groups contents. In our HA samples the increased E4/E6 ratios can be ascribed to a greater aliphatic character, a smaller molecular weight and larger acidic functional groups content, likely produced by SM application. The FTIR spectra of Hapludalf and Hapludox soil HA are showed, respectively, in Fig. 1a and 1b. On the whole, the spectra are characterized by common absorption bands typical of soil HA. The main features of these spectra and their corresponding assignments are: 2925-2846 cm-1, aliphatic C-H stretching; 1720 cm-1, C=O stretching of carboxylic groups; 1660-1600 cm-1, aromatic C=C skeletal vibrations; 1458 cm-1, aliphatic C-H stretching; 1260 cm-1, CO stretching and OH deformation of carboxylic groups; 1030 cm-1, OH stretch of polysaccharides. The main changes in the relative absorption bands observed in the FTIR spectra of treated soils HA are: i) the increase of the bands in between 2925-2846 cm-1; ii) the increase of carboxylic bands, especially in the HA from Hapludalf soil; and iii) the increase of the band at 1458 cm-1. In general, the results obtained by FTIR spectroscopy confirm those of elemental analysis and UV spectroscopy, also providing additional information and showing a direct correlation between application rates and aliphatic components of HA macromolecules. In conclusion, the approach utilized in this study proved to be a powerful tool to monitor the SOM quality after SM application, but additional study are necessary to clarify further aspects appearing critical for the prediction of SOM stocks and stability in soils.
H/C
1259
0-4 0-4 0-4 0-4
O/C
1000
500
8 Mg ha-1 16 Mg ha-1
NPK
4000
W avenu mb er, cm-1
1500
1034
Control 8 Mg ha-1 16 Mg ha-1 NPK
C/N
1725 1605 1458
0-4 0-4 0-4
H N O ------------------%----------------Hapludalf soil 56.50 a1 5.70 a 2.74 b 34.74 d 2.70 b 35.20 d 56.29 a 5.80 a 3.16 a 35.27 d 55.83 a 5.74 a Hapludox soil 52.04 b 6.00 c 2.93 b 39.04 a 54.69 a 5.75 d 3.43 b 36.12 b 55.82 a 5.70 d 4.04 a 34.44 b 56.12 a 6.07 c 3.16 b 34.66 b
1604 1466
Control 40 m3 ha-1 80 m3 ha-1
C
2922 2852
Depth, cm
2921 2846
Treatments
1000
500
Figure 1. FTIR spectra of the HA samples isolated from the surface layer of untreated and treated Hapludalf (a) and Hapludox (b) soils.
REFERENCES
(1) Abipecs 2011. Available in: http://www.abipecs.org.br Access in 08/04/2012. (2) Dick, D.P.; Silva, L.B.; Inda Jr.; A.V.; Knicker, H. R. Bras. Ci. Solo 2008, 32, 2289-2296. (3) Hsu, J.H.; Lo, S.L. Environ. Pollut. 1999, 104, 189196. (4) Hernández, D.; Plaza, C.; Senesi, N.; Polo, A. Environ. Pollut. 2006, 143, 212-220. (5) Hernández, D.; Plaza, C.; Senesi, N.; Polo, A. Eur. J. Soil Sci. 2007, 58, 900-908. (6) Almeida, H.C.; Dick, D.P.; Bertotto, F.L.; Chitarra, G.S. Quim. Nova. 2012, 35, 1329-1335. (7) Canellas, L.P.; Santos, G.A.; Moraes, A.A.; Rumjanek, V.M.; Olivares, F.L. R. Bras. Ci. Solo, 2000, 24, 741-750.
296
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Biochar Colonization by Soil Microorganisms L. Piscitelli(a)*, A. Shaaban(b), D. Mondelli(a), G.N. Mezzapesa(b), T.M. Miano(a), S. Dumontet(c) (a) Università degli Studi di Bari Aldo Moro, Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Italy (b) CIHEAM – IAMB Istituto Agronomico Mediterraneo di Bari, Valenzano (BA), Italy (c) Università degli Studi di Napoli “Parthenope”, Dipartimento di Scienze e Tecnologie, Italy * Corresponding author e-mail: [email protected] Keywords: biochar, soil microbial colonization, soil microflora, olive pomace, microorganism carrier Abstract Soils with low organic matter and modest biological activity portray a reduced capacity to produce goods and services. In many cases the decrease of the organic matter contents is due to improper use of soil resources, but sometimes it is a function of climatic factors as in the case of Mediterranean region. The use of biochar as a soil amendment has been suggested to improve fertility through is positive effect on soil physical and chemical characteristics. In addition, biochar also shows a positive effect on soil microbial community being a structural support giving an ideal habitat for microorganisms. This study investigates the capacity of biochar to play a role of microbial biomass carrier for the inoculation of low fertility soils. Introduction Offering enough food to an ever increasing population, without raising the greenhouse gases emission, is one of the main challenges that soil scientists have to face. Sustainable agricultural practices are focused on keeping soil quality and enhancing its fertility without detrimentally affecting physical, chemical and biological soil properties. The most common practices used to maintain and improve soil fertility consist of adding exogenous organic matter as compost and manure. The limited availability of animal manure and the high cost of good quality compost make difficult for farmers such a soil management. Therefore, it is important to find alternative organic soil amendments and more flexible strategies able to sustain crop productivity and to maintain, and even enhance, soil quality. Biochar is becoming today very popular as soil organic amendment. Biochar couples in one product two important features: a good soil amendment and a good strategy for managing organic waste. It is defined as the carbonaceous product of the thermo chemical conversation of biomass under limited concentration of oxygen. Its characteristics largely depend on the biomass used and the process parameters. The porous structure of biochar allows to absorb soluble organic matter and inorganic nutrients, which are likely to offer and provide suitable habitat for soil microorganisms to grow and reproduce (1). Biochar can be, then, a habitat, or a shelter, for soil microorganisms. However the mechanism, the effects and the extent of microbial colonization are not completely investigated. These information are of pivotal importance for the use biochar as a microbial biomass carrier for the microbial seeding of low fertility soils. This study investigates the effect of biochar produced from olive pomace as a carrier of a microbial community to be inoculated into a low fertility soil.
Experimental Biochar was produced from olive pomace at the Mediterranean Agronomic Institute of Bari by a slow pyrolysis process (450°C for 1h) using an experimental reactor under a moderate nitrogen flow (1L∙min-1). The microstructure of the produced biochar was checked by Scanning Electron Microscopy A fertile soil (soil F), with high organic matter content, and a sandy agricultural soil (soil S) were characterized for their physical and chemical and biological parameters (texture, pH, EC, organic C, total N and microbial content). The microbial biomass of the fertile soil and of commercial compost were extracted by means of a solution made by 0.9 % of NaCl, 1% of glycerol and 0.1% of sodium pyrophosphate. The extracted microflora was incubated in 1/10 strength Tryptic Soy Broth (TSB). Biochar was then added to TSB in order to allow the microbial colonization of the carrier. The inoculated biochar was incorporated into the sandy soil at two different doses (10 and 30 Mg ha-1). The increase of microbial biomass in the sandy soil was measured both with plate count and the fumigation-extraction method. Results and Discussion Biochar characterization. A biochar characterized by a carbon content of more than 50% is considered a stable product in the soil environment. A O:C ratio below 0.3 indicates a high degree of aromaticity (2), and the molar H:C ratio value between <0.6 and >0.1 also indicates a high level of carbonisation. According to these parameters, the biochar produced from olive pomace [Tab. 1] is a product characterized by a high degree of stability in the soil environment ans a high degree of aromaticity. Biochar produced in this way has a large porosity as shown by the SEM photo [Fig. 1].
297
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
kg-1 soil. The other biomass C values were not statistically different from each other. The mean of all the inoculated biochar amendments (irrespective of the dose applied) gives a microbial biomass C value 2.2 times higher than that of the non-inoculated soil S. Despite the short time incubation of microbial charged biochar in soil S, the microbial biomass C increased sensibly, pointing out a beneficial effect of this amendment.
Biochar pH 1:5
10.4±0.1
EC 1:5
dS/m
1.7±0.0 77.5±1.6
C N
0.9±0.2
%
H
2.6±0.1
O
7.6±0.4
Atomic O:C
0.07
Atomic H:C
0.4
Table 1. Properties of olive pomace biochar Microbial plate count. Soil F, characterized by a total organic C content of nearly 7.5%, shown a microbial content 2.4 times less than soil S, which has much lower total organic C (0.5%). Compost, instead, revealed as expected to harbor a high number of microbial cells, being the result of plate count 9.01 109 cfu g-1 [Tab. 3]. Biochar inoculated with microflora of soil F (IBF) and compost (IBC) shown a concentration of soil microbes higher than soil F and soil S, confirming the successful colonization of this substrate. The inoculation of soil S with biochar seems to have no effect on the total aerobic heterotrophic microflora evaluated with the plate count method. No differences were observed both for the biochar inoculated with soil F and for biochar inoculated with compost microflora. No dose effect was also observed [Tab. 3]. Mean cfu g-1 Soil F Soil S
Soil F
Compost Compost Biochar inoculated IBF Soil S
microflora Microflora 10 Mg ha-1 -1
30 Mg ha
-1
IBC
10 Mg ha
-1
30 Mg ha
s.d.
Soil F
2,183d
149
Soil S
185a
36
Compost
17,858e
1,784
530c
114
479b,c
89
10 Mg ha-1
318b
22
30 Mg ha-1
324b
17
IBF Soil S IBC
10 Mg ha-1 30 Mg ha
-1
Table 4. Microbial biomass C
s.d.
5.63 106
5.45 105
1.35 107
1.61 106
9
3.91 109
3.53 108
1.84 107
1.29 108
1.55 107
2.19 107
9.86 106
1.82 10
7
6.67 106
1.64 10
7
7.49 106
1.87 10
7
5.62 106
9.01 10
μg g-1
Figure 1. SEM picture of the produced biochar REFERENCES
Table 3. Microbial plate counts
(1) Lehmann J.; Skjemstad JO.; Sohi S.; Carter J.; Barson M.; Falloon P.; Coleman K.; Woodbury P.; Krull E. Nature Geoscience, 2008, 1, 832– 835. (2) Baldock, J.A.; Smernik, R.J. Org. Geochem 2002,33,1093-1109.
Microbial biomass C. The microbial biomass C values [Tab. 4] shown figures largely different from that revealed by the plate count method for both soil S and F. The fertile (F) soil shown a microbial biomass C 11.8 times higher that sandy (S) soil. Compost shown the highest value of biomass C (17,858 μg g-1 dry soil). In term of microbial biomass C, soil amendments with different doses of biochar, inoculated with the different microflora extracted for soil F and compost, point out slight differences among samples, which are independent from the quantity of biochar added. All the soil S samples inoculated with biochar were significantly richer in biomass C than the un-amended soil. The statistically highest value was shown by the sample amended with biochar, inoculated with soil F microflora, at the dose of 10 Mg
Acknowledgments: We gratefully acknowledge support by Prof. Francesco Porcelli of the University of Bari for the SEM analysis
298
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Effect of Humic acid on growth and yield of two cowpea varieties infected with Cowpea mild mottle virus Adu A.A.a, Adesanwo J.K.b and Odu B.O.c* aInstitute
of Ecology, Obafemi Awolowo University, Ile Ife 220005, Nigeria. bDepartment of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile Ife 220005, Nigeria. cDepartment of Crop Production and Protection, Faculty of Agriculture, Obafemi Awolowo University, Ile Ife 220005, Nigeria. *Corresponding Author: [email protected] Abstract: The antiviral activity of Humic acid isolated from soils at Ile-Ife was evaluated. Two cowpea varieties (Ife Brown and TVu 76) were used as test plants in a RCBD design with 6 treatments which included the control experiment. The results suggest that the humic acid did not in any way contribute to the growth parameters of the plants. There were significant differences for all the yield parameter in varieties by treatment interaction, while variety show significance for number of pods per plant and average pod length. Treatment was significant for all the yield parameters except for seed weight per plant. This suggests that humic acid was able to suppress the effect of the virus on the cowpea plant for ultimate increase in yield. Introduction Humic substances have been found to be useful in agriculture, environmental by significantly influencing quality and productivity of soil. Currently, humic substances are used as additives in fertilizers. These substances can be divided into humic acid, fulvic acid and humin on the basis of the solubility in water as a function of pH. Humic acid enhances the root growth of maize seedling in conjunction with marked proliferation of sites of lateral root emergence, and stimulate the plasma membrane H+-ATPase activity (Canellas et al. 2002). There is therefore the need to investigate possible effect of the humic acid on virus-infected cowpea plants, hence this study. Materials and Methods Soil samples were collected randomly from five sampling points to form composite samples with a sampling Auger at a depth of 0-15cm. The soil samples were air dried, sieved and humic acid was isolated by alkaline extraction. Two cowpea varieties (Ife Brown and TVu 76) were sown into plastic pots filled with sterilized soil in the screenhouse. Five seeds were sown and later thinned to one seedling per pot prior to treatments application. Nine days after planting the cowpea varieties, the following six treatments were applied to each of the varieties: application of solubilised humic acid alone, inoculation of virus (Cowpea mild mottle virus (CMMV)) alone, humic acid two days before virus inoculation, virus inoculation two days before humic acid, humic acid and virus inoculation simultaneously, no application (control) in four replicates. There were 24 pots of each cowpea varieties. The cowpea varieties were dusted with Caborundum (600 mesh) to create wound on them prior to inoculation, and thereafter inoculated with the virus by grinding the inoculum in a mortar with pestle and diluting with 0.01M Phosphate buffer saline solution pH 7.7. Humic acid was applied to the soil close to the plant roots. The cowpea seedlings were observed for symptom (such as mottle and mosaic) development starting from five days after application of treatments. The followings were collected on the test plants: plant height, number of leaves per plant, number of pods per plant, number of seeds per pod, weight of seeds per pod
299
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
and weight of seed per plant. All data collected were subjected to statistical analysis using SAS (2000). Results and Discussion Tables 1 and 2 show the means square values of the plant height and number of leaves of the two varieties of cowpea used in this study respectively with the data collected on weekly basis. Of all the sources of variation, there was only variety shows significant differences. This indicates that the treatments did not in any way contribute to the growth parameters of the plants. Table 3 shows the effect of humic acid on the yield parameters of the cowpea varieties. Varieties by treatment interaction showed significant differences in all the yield parameter, while variety was significant for number of pods per plant and average pod length. Treatment was significant for all the yield parameters except for seed weight per plant. This suggests that humic acid was able to suppress the effect of the virus on the cowpea plant for ultimate increase in yield. References Canellas LP, Olivares FL, Okorokova-Facanha AL and Facanha AR (2002). Plant Physiology 130:1951-1957. Table 1. Mean Square of effect of humic acid on the Plant height of two cowpea varieties infected with Cowpea mild mottle virus Source VAR TRT Rep VAR*TRT
DF 1 5 3 5
wk2 303.01* 13.42 5.13 5.76
wk3 227.51* 9.21 8.79 14.29
wk4 6.02 51.5 57.18 16.67
wk5 5.13 70.18 98.05 26.79
wk6 168.75 22.7 130.53 34.6
wk7 945.19* 64.67 216.35 8.63
wk8 1474.08* 89.23 293.39 6.78
wk9 736.33* 83.68 35.94 31.38
Table 2. Table 1. Mean Square of effect of humic acid on the number of leaves of two cowpea varieties infected with Cowpea mild mottle virus Source VAR TRT Rep VAR*TRT
DF 1 5 3 5
wk2 0 3 2.75 7.65
wk3 11.02 2.14 0.58 5.67
wk4 0 4.53 4.14 2.25
wk5 38.52 6.74 5.69 3.27
wk6 140.08 10.33 19.5 14.58
wk7 154.08 20.65 25.92 10.18
wk8 736.33* 83.68 35.94 31.38
wk9 768* 45.4 13.61 14.1
Table 3. Mean Square of effect of humic acid on the yield parameters of two cowpea varieties infected with Cowpea mild mottle virus Pod weight/ No of pod/ Seed weight/ Average Pod Source DF VAR TRT Rep VAR*TRT
1 5 3 5
plant (g)
1.11 1.36* 0.62 1.65*
plant
130.02* 55.27* 24.58 41.87*
300
plant (g)
1.08 0.66 0.17 1.95*
length (cm)
184.08* 40.18* 4.41 61.80*
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Chemical characteristics of NOM from different soil profiles derived from an Amazonian Spodosol B.S. De Paula (a,b)*, D.M.B.P. Milori(b), C.R. Montes(c), Y. Lucas(d), W.T.L. Da Silva(b) (a) Institute
of Chemistry of São Carlos (IQSC) – University of São Paulo, São Carlos, SP, Brazil Agricultural Instrumentation Center, São Carlos, SP, Brazil. (c) NUPEGEL, CENA, University of São Paulo, Piracicaba, SP, Brazil. (d) Lab PRISME, University of Orleans, IUT Bourges, Orleans, France * Corresponding author e-mail: [email protected] (b) Embrapa
Keywords: spodosol, amazon rainforest, soil organic matter, laser induced fluorescence, 13C RMN Abstract The Amazon rainforest is one of the largest carbon sinks in the World. Recent studies have found that Black River basin constitutes a vast area of Spodosols, which has capacity to store carbon until great depths. The objective of this study was to assess the humification degree of this stored carbon at different depths, as well as understand their structural differences in order to ascertain its stability and lability. Laser Induced Fluorescence spectroscopy (LIFS) was used to select soil samples for the humic acids extraction. Humic acids were analyzed by elemental analysis, FTIR and CP-MAS 13C NMR. The results showed that the surface humic acids samples have characteristics of fresh and poorly processed material. For samples from Bh horizon and deeper, there is a pronounced structural similarity among them, with a higher proportion of phenolic and aromatic carbon, comparing with surface ones. Introduction A complete understanding of the global C budget (pools and fluxes) is required to identify sources and sinks of C and develop strategies for mitigation the risks of climate change(1). Despite the expansion of the deforestation area, the Brazilian Amazon still represents about 40% of the World's remaining tropical forests and plays a vital role in maintaining biodiversity, climate and hydrology, as well as, the maintenance of the terrestrial carbon stock(2). Studies in the Amazon, estimated that carbon stocks in soils are around 6 to 9.4 kg C m-2, considering the first 50 cm of soil. Montes et al (3) has shown that in some regions of Spodosols, the reserves of total organic carbon (TOC) may exceed the value of 66.7 kg C m-2, considering the deep spodic horizons. Thus, the carbon stored in these horizons should not be overlooked in the application of models that aim to estimate CO2 emissions in tropical forest, in a context of climate change. The total carbon stock in the Rio Negro Basin may considerably exceed the current determinations, which consider only organic matter stored in the form of plant above ground biomass and in the most superficial soil horizons. This stock is susceptible to climate change, due to variations in temperature and rainfall in the Rio Negro basin (3). The Spodosols or podzols, are characterized by a typical horizon sequence: an organic surface (H or O horizon) followed by an A mineral horizon, a weathered grey eluvial E horizon, and a dark brownish–reddish illuvial B horizon (Bh, Bhs or Bs), which contains an accumulation of Al and/or Fe with varying amounts of organic matter. It has generally been accepted that the downward transport of aluminium and iron as organic complexes is the dominant mechanism of eluviation in podzols(4).
Therefore, this study aimed to understand the chemical structure of soil organic matter present in Amazonian spodosol by crossing information of different Spectroscopic techniques. Experimental There were collected 127 samples by probe from 9 soil profiles (coded from P1 to P9), selected at the Spodosol area on the left bank of the River Marié, Amazonas state, Brazil. Elemental analysis and Laser Induced Fluorescence Spectroscopy was performed in the whole soil, in order to investigate different degrees of humification and then selecting representative samples arisen from different depths, based on HFIL index (5). Once the selection was made, humic acids extraction from different horizons of two different profiles, was conducted according to the protocols of IHSS. The HA extracted from the selected samples were analyzed (13C-NMR, FTIR and CHN) in order to elucidate the general chemical structure and characteristics of these unexplored samples. Results and Discussion Whole Soil Analysis: The elemental analysis showed an expressive variation in the amount of carbon with depth, as it was expected for a Spodosol soil. Analyses by LIFS were carried out for all points in order to obtain the HFIL index, which indicates a general estimation of the degree of humification for each sample. To choose the samples for HÁ extraction, the parameters taken into account were the carbon content and the HFIL index, according to its variations. With both results for all collection points, we chose to work with P4 and P5 samples, because they represent two different sub-regions, with deeper sampling and
301
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
profiles with interesting variation of carbon content and HFIL. Humic Acids Analysis: The HA extracted from selected samples were first analyzed for carbon, and nitrogen [Table 1]. Table 1- C/N relation of extracted humic acids
P4
For the surface profile, it is also possible to notice a remarkable line around 56ppm, due to the presence of metoxilic carbon probably from lignin and also due to amine carbon related to proteins. Between 60 to 110ppm, the observed lines are from C-O bond of polysaccharides, which are pronounced only in the surface samples for both profiles. In depth, there was an increase in the amount of aromatics, phenols, carboxyl and carbonyl, due to the larger area of such regions carbons (110-160 ppm). Ussiri and Johnson (7) extracted HA from Bh horizons of a Spodosol under forest in New Hampshire and the results show different characteristics compared with HA extracted from the rainforest. The NOM extracted from the Boreal Forest showed a large increase for carbon, even in deeper profile, and small aromatic carbon proportion. Our study shows a reduction of aliphatic carbon and a more pronounced rise of aromaticity in depth. These structural differences is probably related to climate and weathering processes inherent to the Amazon region and the constant water regime, which ensures that a wide diversity of microorganisms consume the aliphatic carbon, and thus leaving more pronounced aromatic part of organic matter. From the intersection of analytical techniques, we found that the samples from humic deep profiles of Amazonian Spodosols differ few structurally. Thus, it is possible to separate the samples into two groups with distinct structural features. The samples 4A, 4B, 5A and 5B (surface), indicated the presence of polysaccharides, lignin, protein, oils and waxes. To the samples extracted below the surface layer, the spectra showed an abrupt change in structural profile, indicating that the beginning of the transition is not simply a deposit of leached surface material. These deeper samples showed high structural similarity, low incidence of polysaccharides and proteins and more pronounced signs for phenols and aromatic structures, probably because this material was fairly transformed with redox, water and microbial conditions inherent in spodosol greater depth.
P5
Depth(cm)
C/N
Depth(cm)
C/N
0-25
23,2
0-15
21,8
60-90
28,2
60-85
24,7
185-195
68,6
100-105
55,9
210-230
70,8
170-190
40,0
250-280
69,5
275-330
51,8
450-490
70,5
340-380
44,5
These results suggest that organic nitrogen is consumed with depth, where N is lost because of the inherent transformations of anaerobic environment. This is an expected result for flooded soils where organic nitrogen tends to be transformed to ammonium, which is soluble and is lost easily (6). The FTIR spectra (not shown), corroborates the information inferred by the VACP/MAS 13C NMR spectra, which is played below [Figures 1 and 2].
Figure 1. RMN spectra of humic acids from P4 sampling
REFERENCES
(1) Lal, R. Geoderma, 2004, 123, 1 –22. (2) Laurance, W.F., Albernaz, A.K.M., Da Costa, C. Environmental Conservation, 2001, v. 28, n. 4, p. 305-311. (3) Montes, C. R., Lucas, Y., Pereira, O. J. R., Achard, R., Grimaldi, M., Melfi, A. J. Biogeosciences, 2011, 8, 113–120. (4) Lundström, U.S., Van Breemen, N., Bain, D., Geoderma, 2000, 94, 91–107. (5) Milori, D.M.B.P., Galeti, H.V.A., Martin-Neto, L., Dieckow, J., González-Pérez, M., Bayer, C., Salton, J.Soil Sci. Soc. Am. J, 2006, 70, 57–63. (6) Buresh, R.J.,Reddy, K. R.,van Kessel,C. Nitrogen Transformations in Submerged Soils. Nitrogen in Agricultural Systems, p 401-436, 2008. Soil Sci. Soc. of Am. (7) Ussiri, D.A.N., Johnson, C.E. Geoderma, 2003, 111, 123–149.
Figure 2. RMN spectra of humic acids from P5 sampling
The line close to 35 ppm refers to primary and secondary alkyl carbons (probably originating from plants residues such as cutin and suberin) of the two surface profiles occur with greater intensity and definition than the deeper samples, which have lower resolution in this region because of their more complex and non-uniform structures.
Acknowledgments: Embrapa Instrumentation Center, IQSC/USP and NUPEGEL/ESALQ/USP by the supporting and possibility of investigating these samples. Authors also thanks to CAPES for the scholarship grant and to Fapesp for financial support (2011/03250-2).
302
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Biochar from babassu residues: chemical characterization and thermo gravimetric analysis C. M. B. F. Maia(a) *, M. Guitoku(a), C. C.M. Rauen(b), Diana Signor(c)
(a) Embrapa Forestry, Estrada da Ribeira km 111, Colombo, PR 83411-000, Brazil (b) UFPR, Setor de Ciências Agrárias, Rua dos Funcionários, 1540, Curitiba, PR 80035-050, Brazil (c) Embrapa Semi- Arid, Av. São Luís Rei de França, nº 4, Q11, São Luís, MA 65065-470, Brazil * Corresponding author e-mail: [email protected] Keywords: biochar, TGA, slow pyrolysis Abstract Babassu is Brazilian palm tree considered the largest native oil resource worldwide. In this work we analyse the lignin, extractives, and holocellulose in the babassu fruit residues (endocarp) and the thermo gravimetric behaviour of the in natura residual biomass, its charcoal and its lignin. Thermogravimetric analysis showed that the removal of extractives and lignin compounds affect the thermal stability of the materials. The biochar from babassu residue is thermally stable and it does not shows functionalized groups in their structure. Besides its potential as C sequester was also demonstated fixed Carbon and thermal stability. Introduction The transition region between the Brazilian Amazon forest and the other surrounding biomes is characterized by a vast area (~18 million ha) of native palm trees, whose main species is the babassu (Attalea speciosa and Orbignya spp.). The babassu is a large palm, highly productive in fruits (drupe), weighing about 150 g each (Teixeira, 2002). The fruit consists of epicarp, mesocarp, endocarp and nuts. The nuts are oil rich (about 7% of fruit weight), which has an important value as biofuel. The Brazilian production of babassu kernels is estimated in 100 kt by year ((Teixeira, 2002). The babassu collecting is a socially important activity because it involves the work of women of about 450 thousand Brazilian rural families (Teixeira, 2008). It is also environmentally relevant because it produces around 93 kt in lignocellulosic wastes (Protásio et al., 2014). The aims of this work were (a) to analyse the lignin, extractives, and holocellulose in the babassu fruit residues (endocarp) and (b) the thermo gravimetric behaviour of the in natura residual biomass, its charcoal and its lignin.
Results and Discussion Babassu endocarp and charcoal analysis: Chemical analysis of babassu endocarp showed extractives and lignin values lower than the one found to babassu residues (epicarp, endocarp, mesocarp all together), which were 5,59% and 31,03% respectively and a higher value to holocellulose (Protásio et al., 2014). The charcoal showed a high value in fixed C and 5,38% of ash content, which probably reflects not only plant ashes but mineral from soil contamination. Table 1. Chemical analysis of babassu endocarp and proximate analysis of babassu charcoal. Property (%) Extractives Lignin Holocellulose Moisture Ash content Volatile matter Fixed carbon
Experimental Babassu endocarp was obtained after processing at local company in Maranhão, Brazil. Babassu residues were dry at 60° degree, ground and sieved to 2 mm for chemical analysis. Moisture, ash, extractives and lignin were determined. Charcoal from babassu fruit wastes were prepared using local traditional oven (slow pyrolysis). The proximate analysis of this charcoal was performed by using standard methods (ASTM D-3172–D-3175). The lignin and extractives experiments were performed according to NBR 7989 and NBR 14853, respectively standards. The thermal degradation studies were conducted on Shimadzu DTA-50 analyser, with heating rate of 10°C.min-1 from room temperature to 900°C under N 2 atmosphere (20 ml.min-1).
Endocarp 4,27 21,88 71,63 4,63 2,22 -
Charcoal 5,58 5,38 11.19 83,43
Thermo gravimetric analysis According to Figure 1, the first degradation stage (up to 120°C) is related to water loss for all samples. From 200°C starts the second degradation stage, which corresponds to the decomposition of cellulose and hemicellulose molecules. This thermal event takes place simultaneously and presents maximum degradation speed close to 290ºC for in natura and extracted samples, with mass loss of 52.5 and 40.5 %, respectively. The third degradation stage is associated with the lignin decomposition and reaches a maximum degradation speed above 450°C (Dollimore & Wu, 1998). The behaviour of thermal degradation for in natura and extracted samples are similar.
303
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Acknowledgments: Authors thank to Embrapa Cocais for collecting babassu feedstock and charcoal.
110
In natura
Mass loss (%)
100
Extracted Lignin Babassu biochar
90 80 70 60 50 40 30 20 10 0 100
200
300
400
500
600
700
800
900
Temperature (°C)
Figure 1. TG curves under nitrogen atmosphere for samples of babassu endocarp:(──) in natura, (──) extracted, (──) lignin and (──) Babassu biochar.
There are cellulose traces in the extracted lignin from babassu, however this material is more thermally stable than the in natura one and their maximum degradation speed is close to 343°C. Besides, lignin decomposes completely at higher temperatures than the in natura and extracted samples, with its maximum degradation rate at 590°C with 68% mass loss. Biochar from babassu residues has different thermal degradation profile. In the absence of functionalized molecules, it has one more stage of decomposition in addition of water loss, which has the maximum degradation rate at 640°C with weight loss of 87%. CONCLUSIONS Besides its potential for energy, babassu endocarp showed a significant potential as biochar for C sequestration, due to their high fixed carbon and thermal stability. The results of thermogravimetric analysis suggested that the removal of extractives and lignin compounds affect the thermal stability of the materials, probably by the increasing porosity and changes in the chemical structure thereof. The biochar from babassu residue is thermally stable and it does not shows functionalized groups in their structure. REFERENCES Dollimore, D., & Wu, Y. (1998). Kinetic studies of thermal degradation of natural cellulosic materials. Thermochimica Acta, 324, 49–57. Protásio, T. de P., Trugilho, P. F., César, A. A. da S., Napoli, A., Melo, I. C. N. A. de, & Silva, M. G. da. (2014). Babassu nut residues: potential for bioenergy use in the North and Northeast of Brazil. SpringerPlus, 3, 1– 14. doi:10.1186/2193-1801-3-124 Teixeira, M. A. (2002). Biomassa de babaçu no Brasil. In 4th Encontro de Energia no Meio Rural (p. 4). Campinas. Retrieved from http://www.proceedings.scielo.br/scielo.php?script=sc i_arttext&pid=MSC0000000022002000100032&lng= en&nrm=abn Teixeira, M. A. (2008). Babassu—A new approach for an ancient Brazilian biomass. Biomass and Bioenergy, 32(9), 857–864. doi:10.1016/j.biombioe.2007.12.016
304
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Contribution of the extracted humin soil in environmental studies: fluorescence investigation A. M. Tadini (a,b)*, G. Nicolodelli (a), W. T. L. Silva(a), C. R. Montes(c), D. M. B. P. Milori(a) (a) Embrapa Agricultural Instrumentation, São Carlos, SP, Brazil. (b) Institute of Chemistry of São Carlos, University of São Paulo, São Carlos, SP, Brazil. (c) Centro de Energia Nuclear na Agricultura and NUPEGEL, University of São Paulo, Piracicaba, SP, Brazil. * Corresponding author e-mail: [email protected] Keywords: humin, soil, humification, fluorescence Abstract Humin represents around 50-60% of humic substances in soil. However, there are few studies that discuss chemical characteristics of Humin. The present study shows that Laser Induced Fluorescence Spectroscopy (LIFS) and excitation-emission matrix (EEM) fluorescence spectroscopy combined to parallel factor analysis (PARAFAC) allow assessing some structural characteristics of soil humin. The LIFS results showed an increasing of cycled conjugated systems at depth, related to the increasing of humification index of humin. Through fluorescence excitation-emission matrix and PARAFAC, it was verified the contribution two similar fluorophores structures both in humin as whole soil. The results indicate that soil fluorescence is mainly determined by the humin fluorescence. Introduction The soil organic matter (SOM) has an important role in environmental sustainability, working mainly in the carbon cycle in soil, which has been attracting considerable interest due to the aspect of using soils as carbon dioxide (CO2) released to the atmosphere by human activity, such as through agricultural expansion in areas under natural vegetation. The main fluorophores SOM are the humic substances (HS), which have physical and chemical characteristics defined, and are fractionated according to their solubility, in humic acids (HA), fulvic acids (FA) and humin (HU)1. Recently, there are few studies that discuss the characteristics of HU, which is defined as the portion of humus that was not extracted from soil, which remains insoluble in aqueous solution at any pH, and is operationally treated as residue remaining from the soil organic matter1. The determination of the optical properties of organic matter is an important tool for structural understanding of its fractions. Laser Induced Fluorescence Spectroscopy Fluorescence technique (LIFS) applied to soils is a recent technique that has been shown to be effective in the analysis of SOM providing results fast, clean, and in conditions of natural2,3. The use of LIFS is possible to determine the content of humus, which is related to the concentration carbon in the rigid structures containing quinone groups and aromatic rings present in the sample. The fluorescence emission-excitation (EEM) is another fast, selective and sensitive spectroscopic technique, whose spectra allow verify information on the composition and configuration of organic matter and humic substances of various origins4. Moreover, the spectrum of EEM can be used for the qualitative and quantitative characterization of an OM when combined with quantitative techniques such as the use of parallel analysis of factors (PARAFAC). Thus, the main objective of this study was to characterize the extracted humin soil in the Amazon region using the LIFS and combination of EEM with PARAFAC.
305
Material and Methods Soil and Humin samples: The samples were collected from different soil profiles in the Barcelos region (Amazonas) which ranged from 0-350 cm depth. Sampling procedures, preservation, preparation of the samples for the extraction and purification of humin followed the recommendations of official methods 5,6. The pellets whole soil and humin containing 30% boric acid were prepared for the analysis of LIFS and EEM. Elemental Analysis: The determination of carbon in the samples of whole soil and humin, were performed using an elemental analyzer Perkin Elmer model 2400. Laser Induced Fluorescence Spectroscopy: Fluorescence measurements in the LIFS portable system were performed using the pellets of whole soil and humin. The system constitute a diode laser exciting at 405 nm with 20 mW power, resolution of 10 nm and a detection system in the region 420-800 nm. Based on the fluorescence spectra obtained was estimated humification index (HLIFS) as the ratio between the fluorescence curve area and the total carbon presented in the samples2. Fluorescence Spectroscopy: Spectra were collected on a Perkin Elmer LS50B spectrofluorimeter. Spectra were obtained using pellets whole soil and humin. It was measured emission spectrum with excitation at 465 nm in the scan range 480-700 nm using an open filter2. Fluorescence in EEM mode was performed employing a Perkin Elmer Spectrometer model LS50B. The measurement was obtained using pellets of whole soil and humin. The spectral scan range was 240-700 nm for emission and 220-510 nm for excitation. The measurements were done with filter at 290 nm, with increment wavelength of 10 nm and number of scans 30. The spectra were treated using the mathematical method known as parallel factor analysis (PARAFAC). Results and Discussion The humification index (HFILS) was determined according to Milori et al (2006). Figure 1, shows humification index determined by LIFS in the samples
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
6
3,5x1 0
6
3,0x1 0
Humification index of Humin Humification index of Whole soil
1,2x10
5
6
1,0x10
5
2,5x1 0
6
8,0x10
4
2,0x1 0
6
1,5x1 0
6
1,0x1 0
6
5,0x1 0
5
0,0
6,0x10
4
4,0x10
4
2,0x10
4
0,0
0-15
15-30
40-50
Depth (cm)
260
350
Figure 1 shows an increasing of humification index at depth, which may be related to percolation of humified organic acids. Furthermore, high correlation was observed between whole soil and humin (R = 0.82). It can be observed that the humification index to whole soil had higher value at 260 cm, which refer to the presence of Bh horizon. So, probably there is a greater presence of others humic fractions more humified in the organic matter of this soil profile. Therefore, the observed behavior of humification index can be attributed to two factors: contribution of fresh organic matter from the soil surface, causing a dilution effect of the more humified organic matter, or associated to natural percolation of soluble humic substances, causing the accumulation of humified material in the depth. The fluorescence spectra (EEM) to the humin and whole soil were treated by mathematical method PARAFAC. This mathematical analysis allowed to identify the presence of two fluorophores (Figure 2 a and b) and had a core consistency diagnostic (concordia) of 96.95% and 98.34% for the whole soil and humin, respectively.
Intensity of the Fluorophore 1
Figure 1: HFIL values obtained for samples of whole soil and extracted humin soil different profiles.
Whole soil Humin
0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0
0-15 15-30 40-50
260
Depth(cm)
350
Intensity of the Fluorophore 2
4,0x1 0
contribution of fresh plant residue in the structure of extracted humin soils in the Amazon region. Thus demonstrating the importance to the study of this fraction of humic substances that currently shows little environmental interest. Figure 3 displays two graphical with the contributions of the two fluorophores in the depth: a) fluorophore 1 and b) fluorophore 2. It can be observed that the surface (0-15 cm) both fluorophores 1 and 2 exert greater contribution to the whole soil samples. In deeper profile (350 cm) occurs inversion of the contribution of Fluorophore when the compared with the surface. Thus, it possible to observe a largest increase of the Fluorophore 2 to extracted humin soil demonstrating that there is a higher concentration the humified material in this profile. In profile 260 cm, referring to the Bh horizon, it is observed that there is a larger contribution of the fluorophore 1 to humin extracted soil, whereas in the whole soil is observed a greater contribution of the fluorophore 2. The results presented in Figure 3 corroborate those obtained in Figure 1, where a higher humification index was obtained for the whole soil that was observed in profile (260 cm), indicating a greater contribution of Fluorophore 2, which refers 0,7to a more complex and humified structure.
HLIFS (Whole soil)
HLIFS (Humin)
of whole soil and humin at different depths. For humin, it was observed a broad band (650-750 nm), with peak at 690 nm. This peak was observed only at the surface. It was identified as chlorophyll fluorescence, present in the compounds of vegetable origin7.
Whole soil Humin
0,6 0,5 0,4 0,3 0,2 0,1 0,0
0-15
15-30 40-50
Depth (cm)
260
350
(b) (a) Figure 3: Contribution of Fluorophores 1 (a) and 2 (b) whole soil and extracted humin soil of different profiles analyzed.
This study concluded that humin extracted from Amazonian soil has in its structure characteristics fluorophores from humified and non-humified organic matter. It is possible to observe the contribution of compounds with characteristics of vegetable origin, such as the presence of chlorophyll. Furthermore, soil fluorescence is mainly determined by the humin fluorescence. References
(b) Fluorophore 2 (a) Fluorophore 1 Figure 2: Representation of the Fluorophores: 1 (a) and 2 (b) present in the whole soil and extracted humin soil.
The Fluorophore 1 (λexc / λem: 350/400 nm) is typical in simpler structures present in SH terrestrial environments. The Fluorophore 2 (λexc / λem: 450/510 nm) are typical of terrestrial HA and lignin derivatives, which are associated more complex and humidified structures, with the presence of fused aromatic rings4,8. According to Matthews et al. (1996)8, humic acid extracted from soils are predominantly derived from lignin precursors, which have in its structure components of terrestrial plants. The results obtained by using the LIFS and EEM in combination with PARAFAC technique, showed that there was the
306
1-Stevenson, F.J. New York: Wiley Interscience 1994, 443. 2-Milori,D.M.B.P; Galeti,H.V.A; Martin-Neto,L; Dieckow, J; GonzálezPérez, M; Bayer, C; Salton, J. Soil Sci. Soc. Am. J. 2006, 70, 57-63. 3-Martins, T; Saab, S.C; Milori, D.M.B.P; Brinatti, A.M; Rosa, J.A; Cassaro, F.A.M; Pires, L.F. Soil Till. Res. 2011, 111, 231-235. 4-Coble, P.G. Mar. Chem. 1996, 51, 325–346. 5-Swift, R.S. Soil Sci. Soc. Am. Madison, 1996. 6-Rice, J; MacCarthy, P. Sci. Total Environ. 1989, 81/82, 61-69. 7-Lichtenthaler, H; Wenzel, O; Buschmann, C; Gittelson A. Annals New York Academy of Sciences, 1999, 271-285. 8-Matthews, B.J.H; Jones, A.C; Theodorou, N.K; Tudhope, A.W. Mar. Chem. 1996, 55, 317-332.
Acknowledgments:
The authors acknowledge the financial support for this work from the of São Paulo Research Foundation (FAPESP) by scholarship (Process 2013/13013-3). The Embrapa Agricultural Instrumentation for supplying the structure the development of research.
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Humic substances of residues from industrial sugarcane processing: complexation study W.G. Botero(a)*, O.S. Santos(a), L.C. Oliveira (b), J.S. Jacundino(b), S.O. Souza(a)
Federal University of Alagoas (UFAL), Campus Arapiraca, 57309-005-, Arapiraca, AL, Brazil. Federal University of São Carlos, Campus Sorocaba, 8005-139 Sorocaba, Brazil. * Corresponding author e-mail: [email protected] (a)
(b)
Keywords: humic substances, filter cake, complexation capacities, metals. Abstract The study the complexation of humic substances from filter cake (residues from Brazilian industrial sugarcane processing) with Cu2+ , Cd2+ , Cr3+ , Ni2+ and Pb2+ by tangential ultrafiltration system was evaluated. By the results (Table 1) can be described by the following order of increasing affinity of HS from FC by metals: HSFCAL – Cr3+
contamination and availability of these species in different environmental systems (5). The objective of this work is to study the complexation of humic substances extracted from samples of filter cake collected in different regions of Brazil by ions Cu2+, Cd2+ , Cr3+ , Ni2+ , and Pb2+ , evaluating the influence of this material on the complexation of metals. Experimental Samples of filter cake were obtained from sugar production plant located in the city of Maceió, Alagoas State, Brazil (FCAL) and Jaboticabal city, the São Paulo State, Brazil (FCSP). The humic substances (HS) were extracted following the procedure recommended by the IHSS (6). The analytical procedure proposed by Burba (7), based on ultrafiltration, was used for studies of the interactions between humic substances from FC AL sample (HSFC AL) and FC SP sample (HSFC sp) and Cu2+, Cd2+, Cr3+, Ni2+ and Pb2+. This technique utilizes a tangential ultrafiltration system (Sartorius Ultrasart X), fitted with a 1 kDa membrane (Gelman Pall-Filtron OMEGA), that permits separation (from the free metals) of HSFC-metals complexes having molecular size greater than 1 kDa. In this study were determined the complexation capacity of HSFCAL and HSFCSP for metals. The concentrations of metals were determined by Atomic Emission Spectrometry with induced argon plasma (ICP-OES), Thermo Jarrell Ash. Results and Discussion According to Oliveira [8], complexes with organic matter tend to stabilize as a function of time, due to inter- and/or intramolecular rearrangements and transfer of complexing species to complexation sites deeper within the molecules.
307
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
[Cufree / (mg L-1)
Complexation equilibrium between HS extracted from different matrices, with potentially toxic inorganic elements around 30 minutes (4). The complexation capacity (CC) was determined by plotting the free metal concentration (mg L-1) as a function of the total metal concentration (mg L-1) (Figure 1) (9).
values of complexation capacities from HS from peat determined by our group for the micronutrient copper and potentially toxic metal lead that presented the greatest affinities with the HS (4). The complexation capacities for ions Cd2+ and Ni2+ in the samples (HSFCAL and HSFCSP ) were similar, indicating that the affinity of metals by HS is not influenced by the characteristics of the material for these metals. However, the affinity of HSFCAL were superior for Pb2+ compared with another sample. The HS from FC showed high affinity for potentially toxic metals such as Pb2+ , and have potential application in polluted aquatic systems. Studies in situ and microcosms are being made to evaluate the applicability of this material as environmental remediation.
16 14 12 10 8 6 4 2 0 -2
REFERENCES
-2
0
2
4
6
8
10
12
14
16
18
(1) Almeida, J.R. Brasil Açucareiro 1994, 24: 91-3. (2) Dematte, J.A.M.V R. Brás. Ciên. Solo 2005, 29:317326, 2005. (3) Rossetto, R.; Dias, F.L.F. Inform. Agron. 2005, 110:6-11. (4) Botero, W.G.; Oliveira, L.C.; Rocha, J.C.; Rosa, A.H.; Santos, A. J. Haz. Mat. 2010, 177:307-311. (5) Rocha, G.N.; Gonçalves, J.L.M.; Moura, I.M. R. Bras. Ci. Solo 2004, 28:623-639. (6) Thurman, E.M.; Malcolm, R.L. Environ. Sci. Technol. 1981, 15:463-466 (7) Burba, P.; Van den Bergh, J.; Klockow, D. Fresenius J. Anal. Chem. 2001, 371: 660-667 (8) Oliveira, L.C., Botero, W.G.; Santos, F.A.; Sargentini Jr, E.; Rocha, J.C.; Santos, A. J. Braz. Chem. Soc. 2012, 23: 1711-1718. (9) Romão, L.P.C.; Castro, G.R.; Rosa, A. H.; Rocha, J.C.; Padilha, P.M.; Silva, H.C.; Anal. Bioanal. Chem. 2003, 375: 1097-1103. (10) Santos, A.; Botero, W.G.; Bellin, I.C.; Oliveira, L.C.; Rocha, J.C.; Mendonça, A.G.R; Godinho, A.F. J. Braz. Chem. Soc. 2007, 18: 824-3829.
20
[Cutotal] / (mg L-1) Figure 1. Determination of the complexation capacity (CC) of humic substances from FCAL (HSFCAL) and FCSP (HSFCSP) by Cu2+.
Table 1 presents complexation capacities of humic substances from FCAL (HSFCAL) and humic substances from FCSP (HSFCSP) for Cu2+ , Cd2+ , Cr3+ , Ni2+ and Pb2+ . Table 1. Complexation capacities (mg metal g-1 HS) of humic substances from FC AL (HSFCAL) and FC SP
(HSFCSP) for Cu2+, Cd2+, Cr3+, Ni2+ and Pb2+. Samples/ metals
SHFCAL mg g-1 HS
SHFCSP mg g-1 HS
2+
21.20
24.30
Cr3+
13.00
23.60
2+
Pb
39.20
26.11
Ni2+
15.40
16.00
Cd2+ Cu
16.90
15.50
Acknowledgments: The authors thank the Coordenadoria
de Aperfeiçoamento do Pessoal de Nivel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for financial support.
Humic substances are the main form of organic matter and influence the bioavailability, toxicity, transport, accumulation and concentration of metals and depending on the environment, have redox characteristics influencing the metallic species of the environment, forming complexes with different labilities. From the values of the complexation capacity (Table 1), it can be observed the importance of the organic matter in the metal complexing species. By the results (Table 1) can be described by the following order of increasing affinity of HS from FC by metals: HSFCAL – Cr3+
308
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Developing a new methodology to study metal ion binding by humic matter using AGNES and SSCP W.G. Botero(a)*, L.S. Rocha(b), M.F. Pineau (c), J.P. Pinheiro(b)
Federal University of Alagoas (UFAL), Campus Arapiraca, 57309-005-, Arapiraca, AL, Brazil. IBB/CBME, DQF/FCT, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal (c) Univ Paris Diderot, Sorbonne Paris Cité, UFR de Chimie, 75013, Paris, France. * Corresponding author e-mail: [email protected] (a)
(b)
Keywords: humic matter, metal ions, AGNES, SSCP, heterogeneity. Abstract A new methodology to study the interaction of humic matter (fulvic acid - LFA and humic acid - RPHA) with Cd(II) and Zn(II) using SSCP and AGNES is presented. It consists on titrating a fixed amount of humic matter with the metal ions and following the free metal ion by AGNES, followed by an SSCP analysis on a selected point of the titration. The analysis of the M/L titration by AGNES provides both the bulk thermodynamic stability constants and the heterogeneity behavior which can be compared with the heterogeneity information obtained from the slope of the SSCP curve. Dynamic information can also be obtained from both the limiting transition time of the SSCP curve and the slope behavior. Experiments were performed using Cd(II) and Zn(II) in presence of LFA and RPHA in order to test this methodology. Introduction The environmental contamination level by toxic metals has increased as a result of the industrial and population growth and poor control of emissions. In most natural aquatic systems metal ions form stable complexes with a large variety of dissolved inorganic and organic ligands, of which the larger component is the natural organic matter (NOM). The main components of the NOM present in soil, sediments and waters are humic substances (HS), which are operationally divided in humic acids (HA) and fulvic acids (FA) (1). Metal interactions with humic matter show both polyfunctional and polyelectrolytic characteristics and therefore cover a broad range of free energy of complex formation and a corresponding range of dynamic behaviour, thus controlling the bioavailability and mobility of the metal ions (2). Due to their sensitivity voltammetric techniques, especially the stripping modes are particularly suited for trace metal complexation studies (3). The interpretation of the results obtained by stripping voltammetric techniques is not difficult for they are affected by the dynamic nature of the system (lability of the complexes), the heterogeneous nature of the ligands and the adsorption of organic matter at the electrode surface. (1,3). Stripping chronopotentiometry at scanned deposition potential (SSCP) is particularly suited for trace metal complexation studies due to its sensitivity, capacity to provide information about the dynamic nature of the complexes, even in presence of heterogeneous metal complexes and not being affected by the adsorption of organic species at the electrode surface (4, 5). The absence of gradients and Nernstian equilibrium stripping (AGNES) is a stripping technique for the determination of the free metal ion
concentration. During the deposition step the amalgamation of the metal in the mercury electrode is allowed to proceed until reaching Nernstian equilibrium. The concentration of amalgamated metal and thus the measured faradaic stripping current are proportional to the free metal ion concentration in the bulk, with the advantage of having a lower detection limit when com- pared to commercial ISEs (1). The objective of this work is to develop a new methodology to study the complexation of humic matter with Cd(II) and Zn(II) by using the excellent characteristic of SSCP and AGNES. Experimental Peat samples were collected in the Mogi river region of the municipality of Ribeirão Preto, São Paulo State, Brazil. The humic acid (RPHA) were extracted following the procedure recommended by the IHSS (6). The Laurentian fulvic acid (LFA) was obtained from C. H. Langford. It is extracted from a podsol collected in the Laurentian Forest Preserve of Laval University, Quebec, Canada. Physicochemical characterization of the sample has been reported elsewhere (7). The polarographic experiments were performed using an Autolab System attached to a Metrohm 663 VA stand and to a personal computer using GPES 4.9 software (Eco Chemie). The electrodes were a mercury film electrodes (TMFE), an Ag/AgCl reference electrode with a salt bridge and a platinum wire counter electrode. Each SSCP and AGNES experiments (which were performed in the same electrochemical cell and one technique followed by the other) included a calibration plot measured at a given concentration (5x10-8M) of total Cd(II) or Zn(II). The ligand (LFA and RPHA) was then added directly to the calibration solution, together with a pH
309
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
buffer (MES or MOPS). The pH was adjusted AGNES data were acquired after the addition of metal concentrations (1x10-7, 2 x10-7, 3 x10-7, 6 x10-7, 1x10-6, 2x10-6, 3x10-6 and 5 x10-6 M). The SSCP data were obtained for the concentration 6 x10-7 M of metals. The experiments were done at pH 6, 7, and 8 and ionic strength 0.01 M.
A
Results and Discussion The Figure 1 (1A) shows Cd bound to organic matter (HA and LFA) as function of free Cd obtained by AGNES at pH 6.0, 25◦C and (1B) shows Cd bound to organic matter (RPHA and LFA) as function of free Cd obtained by AGNES at pH 6.0, 25◦C and.
A B
B
Figure 2. (A) SSCP curve with Cd(II) and RPHA and LFA at pH 6.0, 250 C and (B) SSCP curve with Zn(II) and RPHA and LFA at pH 6.0, 250 C. Figure 1. (A) Cd bound to RPHA and LFA as function of free Cd obtained by AGNES at pH 6.0, 250 C and (B) Zn bound to RPHA and LFA as function of free Zn obtained by AGNES at pH 6.0, 250 C.
We plan to continue this work with Pb(II) binding and also by performing NICA-Donnan modelling of the results and to develop a fitting procedure to obtain the NICA-Donnan parameters from the SSCP curve.
Binding of metal ions to humics is assumed to occur through specific interactions between cations and the “surface” functional groups, and by non-specific binding to any residual negative charge (8). The isotherms (Figure 1A and B) shows Cd binding to RPHA is significantly larger than to the LFA while Zn binding is similar. Both are significantly smaller than Pb binding to LFA as reported by Domingos et al (8) The Figure 2 A and B shows SSCP curve obtained with Cd(II) and RPHA and LFA at pH 6.0 (A) and Zn(II) and RPHA and LFA at pH 6.0 (B). In SSCP we observe that the slopes Cd-LFA and CdRPHA are similar showing a small degree of heterogeinity as expected for the electrostatic nature of the binding of Cd with humic matter, while the slopes of Zn(II) are slightly smaller than for Cd(II) indicating a larger heterogeneity of this metal regarding humic matter. The results obtained in this study show promise in obtaining relevant information for assessing the heterogeneity of the complex formed between LFA and HA and Cd(II) and Zn(II).
REFERENCES
(1) Malcolm, R.L.; MacCarthy, P. E Environ. Sci. Technol. 1986, 20: 904-911. (2) Buffle, Complexation Reactions in Aquatic Systems:
an Analytical Approach, Ellis Horwood, Chichester, 1998. (3) Mota, A.M.; Correia dos Santos, M.M., in A. Tessier, D. Turner, Metal speciation and bioavailability, John Wiley & Sons, New York, 1995: chapter 5. (4) Van Leeuwen, H.P.; Town, R.M. Environ. Sci. Technol. 2003, 37: !3945 –3952. (5) Pinheiro, J.P.; Van Leeuwen, H.P.; J. Electroanal. Chem. 2004, 570: 69–75. (6) Thurman, E.M.; Malcolm, R.L. Environ. Sci. Technol. 1981, 15:463-466. (7) Wang, Z.D.; Pant, B.C.; Langford, C.H. Anal. Chim. Acta. 1990, 232: 43-52. (8) Domingos, R.F.; Benedetti, M.F.; Croué, J.P.; Pinheiro, J.P. Anal. Chim. Acta., 2004, 521: 77-86. Acknowledgments: The authors thank the Coordenadoria de Aperfeiçoamento do Pessoal de Nivel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for financial support. WGB and JPP thank CAPES Brasil CSF scholarship 981313-6. JPP thanks Fundação para a Ciência e a Tecnologia (FCT) for funding support in project FCTANR/AAG-MAA/0065/2012) and Project Pest-OE/EQB/LA0023/2013.
310
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Humic acids as a plant growth promoter L.P. Canellas (a) *, N.O. Aguiar(b), A.C. Ramos(b), L.S. Lima (a)F.L. Olivares(b)
(a) Núcleo de Desenvolvimento de Insumos Biológicos para Agricultura, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av Alberto Lamego 2000, Campos dos Goytacazes 28013-602, Brazil * Corresponding author e-mail: [email protected] Keywords: physiological effects, humeomics, Inoculation technology, Sustainable agriculture technology Abstract Humic substances (HS) have been widely recognized as a plant growth promoter mainly by changes on root architecture and growth dynamics, which result in increased root size, branching and/or greater density of root hair with larger surface area. Stimulation of the H+-ATPase activity in cell membrane suggests that modifications brought about by HS are not only restricted to root structure, but are also extended to the major biochemical pathways since the driving force for most nutrient uptake is the electrochemical gradient across the plasma membrane. Changes on root exudation profile, as well as primary and secondary metabolism were also observed resulting in increase of maize, tomato, lettuce and sugarcane yield in tropical conditions. Humic substances concomitantly modify the structure/activity of the microbial community in the rhizosphere compartment allowing innovative technology to plant inoculation. Introduction The direct use of soluble humic substances (HS) as a Parallel to field experiments were conducted in plant growth promoter is not a novelty, but the controlled experiments to study the physiological increase of HS market as plant stimulators has effects produced by HS. For example, we use attracted the attention of new producers, businesses vibrating probe system (2) to measure ion flux on and farmers interested in knowing how such dilute lateral roots of rice seedlings. The specific H+ and concentrations can bring many benefits to sustainable Ca2+ fluxes in the root elongation zone underlying an production. The most described effect of HS is the activation of the plasma membrane H+-ATPase, Ca2+promotion of plant root system. However, primary dependent protein kinase (CDPK), coupled with an plant metabolism, diverse and complex enzymatic increased expression of the voltage-dependent machinery related with a plethora of cell processes and OsTPC1 Ca2+channels and two stress responsive more recent changes on secondary metabolism by HS CDPK isoforms, OsCPK7 and OsCPK17. These are being increasingly documented. By definition, HS results suggest that HAs act as molecular elicitors of are assemblies of heterogeneous compounds that are H+ and Ca2+ fluxes, which seem to be upstream of a insoluble in water and recalcitrant to microbial activity complex CDPK cell signaling cascade. In other and represent the stable part of earth C cycle. However, experiment, we collect root exudates (3) and observe since HS affect both plant primary and secondary significant changes in exudate profiles after fourteen metabolism including changes on exudation profile, it days. Seedlings treated with humic acids exuded fatty is pertinent to consider that HS may interfere with acids, phenols and organic acids in different amounts microorganism community in the rhizosphere. compared to other treatments while H. seropedicae Plant growth-promoting bacteria (PGPB) are a wide and H. seropedicae + humic acids exudates included a range of microorganisms that induce plant growth by large number of nitrogenous compounds mainly several processes including biological N2 fixation, heterocyclic structures. increase of nutrient availability in the rhizosphere, Results from field and laboratoy represents a step enlargement of root surface area, and enhancement of forward to understand the scientific bases to beneficial symbioses for the host. We proposed a new recommend soluble humic matter as plant growth biofertilizer concept based on the combination of HS promoter based on their beneficial driving effects on and endophytic diazotrophic bacteria (1). Such plant metabolism. The use of HS as vehicle to crop biofertilizer implies an increase of endophytic introduction of plant growth promoting bacterial open interactions associated with plant host and protection a possibility to develop technological tools for suitable of the bioinoculant in the HS hydrophobic domains agriculture. which act as carriers. A combined application of HS REFERENCES and bacteria was performed for the first time with (1) Canellas L.P. and Olivares F.L. Chemical and Biological Technologies in Agriculture 2014, 1:3 sugarcane plantlets and field experiment with maize, (2) Feijó J.A., Sainhas J., Hackett G.R., Kunkel J.G., tomato and lettuce were made. The increase on yield Hepler P.K. J. Cell Bio 1999, 144,483-496 production was significative when the plants were (3) Aulakh M.S., Wassmann R., Bueno C., submitted to some form of stress like low soil fertility kreuzwiesser J., Rennenberg H. Plant Biol 2001, 3,139-14 or water stress. Acknowledgments: CNPq Inct FOR bnfand FAPERJ for support
311
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Nature-Inspired Nanotechnology Solutions For Applications In Remediation And Agriculture A. Smith (a) *, B. White(b), C. Rice(b) (a) Address a (b) Address b. * Corresponding author e-mail:
Keywords: humic substances, organosilanes, nature-inspired technologies, remediation, agriculture Abstract Humic substances (HS) is a product of coevolution of life with its mineral surroundings. As a result, in nature, they occur as organomineral supramolecular complexes, which play multiple life-sustaining functions. The main working hypothesis here was that the chemical reactivities within these organomineral complexes could be partially reproduced by assembling interpolyelectrolyte complexes of HS with polysiloxanes, which possess high affinity for both mineral and biological surfaces. It was shown that the assembled humics-organosilane complexes were capable of immobilization onto mineral support and sequestration of waterborne contaminants (e.g., actinides in higher oxidation state, chromium (VI)). At the same time, they were capable of restoring waterproof properties of soil aggregates and caused pronounced growth stimulating effects on agriculturally important plants (wheat and potatoes). This shows good prospects for bringing nature-inspired nanotechnology solutions into the practice of remediation and agriculture. physicochemical properties of Fe in the catalytic Fenton/PCP system by EPR spectroscopy.
Introduction Pentachlorophenol (PCP) is poorly biodegradable, highly toxic therefore considerable research efforts have been devoted to the development of abiotic catalytic methods e.g. dechlorination by metal catalysts and Advanced Oxidation Processes. The Fenton system (FeII/ H2O2) offers tha basis for a ccost –effective technology for the degradation of a broad range of organic pollutants. Hydroxyl radical (HO˙) is formed during the catalytic decomposition of H2O2 by iron salts. HO˙ is a highly reactive, nonselective radical (1). Since HO˙ is nonselective, in natural aquatic samples it may react with dissolved natural organic matter (NOM) i.e. beyond organic pollutants. In this way NOM can be a significant sink of HO˙ resulting to a considerable decrease of the reaction’s efficiency. Moreover, Fe-binding by compounds, such as NOM, humic (HA) and fulvic acids (FA), can also alter the rate constant for the reaction or the redox cycle of iron and thereby change the formation rate of HO˙ . It has been demonstrated that in a catalytic Fenton system consisting of Fe, HA and H2O2, HO˙ radicals are produced (2). However, the role of HA on the HO˙ radicals appears controversial e.g. HA has been reported to either increase (3) or decrease (4) the production of HO˙. To add to this complexity, recently it has been reported that the initial catalytic conditions may influence HO˙ production (5). The effect of HA on the catalytic efficiency of the Fenton reaction still remains controversial since the degradation of organic pollutants has been reported to be either inhibited (6) or enhanced (7) in the presence of humic materials. All these cases are summarized in ref. 8. The aims of the present work were (a) to examine the role of a well characterized HA- on the catalytic decomposition of PCP by the Fenton system, (b) to frame quatitative limits regarding the Fe/HA ratio for optimal yield/rate of PCP decomposition, (c) to study the
Experimental The low-Fe HA used was a well characterised-HA sample extracted from a mining site of Greece according to the protocols of IHSS. All reactions were conducted at room temperature and in dark to exclude adverse photo-effects. The pH was adjusted to 3.5 using H2SO4 e.g. since production of HO˙ by Fenton reaction declines at higher pH, in the presence or absence of humic materials. A typical reaction mixture contained 13ppm PCP, 0.87-32.5ppm FeSO4∙7H2O, 4.35-97ppm H2O2 and 20 ppm HA. The effect of [H2O2], [Fe] and the presence of 20ppm HA was studied (Table 1). Detailed control experiments were performed using solutions containing [PCP+FeSO4∙7H2O] or [PCP+H2O2] or [PCP+FeSO4∙7H2O+HA] or [PCP+H2O2+HA]. All these systems resulted to zero conversion of PCP. Results and Discussion Fenton Catalyst in the Absence of HA:[Figure 1A] Fe-concentration appeared to have a decisive effect on PCP degradation. For example, by increasing the FeSO4∙7H2O content to 6.5ppm, the total of PCP was removed within 120 h (compare Fig. 1A, B). Increase of [H2O2] did not affect significantly the degradation of PCP (Figure S1A). For example, increase of [H2O2] by 2200% i.e. 22 times, (run 1 vs. 5) resulted to an increase of PCP removal by 18%.
312
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
A
g=4.3 EPR Signal Intensity
A
80 4
% [PCP] Remained
60
dX''/dH (au)
[FeSO 7H O] 2
0.87 ppm
40
20
run 16
(e)
run 19
0
runs 11-15
1500
3000
4500
[HA] ppm
runs 14 , 16 -20
100
B
(b) (a)
[FeSO 7H O] 4
80
(d) (c)
32.5 ppm
0
Signal Intensity
runs 4 , 6 -10
100
2
0.87 ppm
60
400 40
40
800
1200 1600 2000 2400
Magnetic Field (G)
B
runs 11-15
0
% [Fe-HA ]
20
32.5 ppm 0
time (h)
72
144
216
Figure 1. Effect of [FeSO4∙7H2O] on the removal of PCP by
Fenton reaction (A) in the absence and (B) in the presence of 20ppm HA. Catalytic conditions: 13 ppm PCP, 52 ppm H2O2, 0.87-32.5 ppm FeSO4∙7H2O. Run 4 and 14 (), run 6 and 16 (), run 7 and 17 (), run 8 and 18 (), run 9 and 19 (), run 10 and 20 ().
30 run 16 20
RFe/HA=1.17
10 0
Increase Decrease of PCP of PCP oxidation oxidation
run 19
Low R High R Fe/HA Fe/HA
3.6
HA-Modified Fenton Catalyst: [Figure 1B] FeII concentration had a prominent effect i.e. depending on the initial [FeII]:HA ratio. Noticeably, in the presence of HA, increase of H2O2 concentration had a beneficial effect which was higher than the effect of H2O2 in the absence of HA: [i] under the conditions of these reactions, HA inhibited PCP decomposition; [ii] in the presence of HA, more [H2O2] oxidant was required to achieve the same PCP decomposition as in the absence of HA [iii] further increase of [H2O2] resulted to a linear increase of the total PCP conversion at 9 days in the presence or absence of HA. Overall, under the conditions of our experiments: [i] HA appears to play a dual Janus-like role e.g. HA can act either as an inhibitor or as an enhancer of the Fenton reaction, on both PCP decomposition yield and reaction rates; [ii] the function of HA is gated by the ratio Fe/HA (RFe/HA). When Fe( mol) R Fe/HA 1.17 HA(mg) both PCP decomposition yield and reaction rates were increased otherwise they remained constant or decreased vs. the unmodified Fenton. EPR Spectroscopy [Figure 2A] shows the percent of monomeric FeIII-HA and the reduced FeII (due to the presence of HA) formed in each sample used for the Fenton catalysis of PCP, i.e. the percent of Fe (Fe III and FeII) interacting with HA (Fe-HA). It is seen that increase of the HA concentration keeping the Fecontent constant (i.e. decrease of RFe/HA ratio) resulted to an increase of the [Fe-HA] species. The downarrow in Figure 4B mark the RFe/HA=1.17, while the horizontal arrows mark the RFe/HA regimes where oxidation of PCP is increased/decreased in the presence of HA (Figure 2B).
3.0
2.4
1.8
1.2
0.6
Fe/HA (mol/mg)
Figure 2. A) EPR spectra of 0.4 mM Fe2(SO4)3∙xH2O incubated in the absence of HA, spectrum (a) and (b) (spectrum (b) has 30 % v/v glycerol) and in the presence of (c) 230, (d) 1710 and (e) 5110 ppm HA (i.e. corresponding to RFe/HA = 0.16, 0.47 and 3.5 μmol/mg respectively) for 2 h at pH 3.5. Inset plot: EPR FeIII signal intensity as a function of [HA] incubated for t 30 min () and 2 h (), the lines correspond to a linear regression of the data. B) Percent of the [Fe-HA] after incubated for 2 h at pH 3.5. Each case is labelled with the corresponding Fenton reaction with the same ratio RFe/HA
REFERENCES
(1) Rice, N.; Buxton, G.V; Nixon, A.B. J. Phys. Chem. Ref. Data 1988, 17, 513-886. (2) Paciolla, M.D. and White A.B. Environ. Sci. Technol. 1999, 33, 1814-1818. (3) Huling, S.G.; Water Res. 2001, 35, 1687-1694 (4) Lindsey, M.; Tarr, M.A.. Chemosphere 2000, 41, 409-417. (5) Ciotti, C.; Baciocchi, R.; Tuhkanen, T J. Hazard. Mater. 2009, 161, 402-408. (6) Lindsey, M.; Tarr, M.A. Environ. Sci. Technol. 2000, 34, 444-449. (7) Fukushima, M.; Tatsumi, K. Environ. Sci. Technol. 2001, 35, 1771-1778. (8) Georgi, A. Appl. Catal. B: Environ. 2007, 72, 26-36.
313
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Restoration of hyper-eutrophic Lake Pamvotis using a novel bentonite humic composite material BephosTM. M. Zamparas(a), S. Kappa(a), Y. Deligiannakis(a), I. Zacharias(a)* (a)
Department of Environmental and Natural Resources Management, School of Engineering, University of Patras, 2 Seferi Str., 30100 Agrinio, Greece * Corresponding author e-mail: [email protected] Abstract The aim of this study was to investigate the efficiency of a novel modified bentonite (BephosTM) as adsorbent for phosphate removal from natural waters with an additional estimation of sediment capping effectiveness preventing phosphorus release from sediments of eutrophic lake Pamvotis (NW Greece). BephosTM was compared with unmodified bentonite (N-Bentonite) and natural Zeolite (N-Zeolite) and commercial La-modified bentonite (PhoslockTM). The amount corresponding to 0.03 g is satisfactory for the removal of ~94.6% and ~95.4% of phosphates onto BephosTM and PhoslockTM respectively. Thus, this quantity is the optimal amount of each material to achieve high phosphate removal from natural eutrophic waters. In addition, the amount of phosphate adsorbed onto N-Bentonite, N-Zeolite, PhoslockTM and Bephos™as a function of the pH was also studied. As solution pH increases in the range of 5–7, the adsorption efficiency of Bephos™ increases gradually and reaches a maximum value (~92.3%) when the pH value is 7. When the pH increases to 9, the removal efficiency drops (~86.9%). The equilibrium adsorption capacity of BephosTM and PhoslockTM changed very little within the pH range of 5–7 and decreased in solutions with higher pH values. At pH values over 8, the clay surface becomes negatively charged thus phosphate adsorption drops. Moreover, BephosTM as a P-inactivation agent resulted in about ~91.4 % reduction of the phosphate flux from the sediments. Keywords: BephosTM, PhoslockTM, Lake Pamvotis, adsorption, phosphorus, restoration. natural materials, as specific phosphorus adsorbents from natural eutrophic water bodies. BephosTM (from the words “Bentonite” and “phosphorus”) is a novel low-cost composite material embedding Fe, Cu ions and humic acid in the interlayer space of a natural bentonite [3]. The aim of this study was to investigate the efficiency of a novel modified bentonite (BephosTM) as adsorbent for phosphate removal from natural waters with an additional estimation of sediment capping effectiveness preventing phosphorus release from sediments of eutrophic lake Pamvotis (Greece). BephosTM was compared with unmodified bentonite (N-Bentonite) and natural Zeolite (NZeolite) and commercial La-modified bentonite (PhoslockTM).
Introduction The rapid increase in human activity has substantially accelerated the eutrophication process, altering the geochemical cycles of carbon, nitrogen and phosphorous. In addition to natural sources, nutrients can enter aquatic ecosystems via point and nonpoint sources resulting from anthropogenic origins such as: (a) municipal and industrial sewage discharges, (b) runoff from fertilizers and manure applied to agricultural land, (c) from diffuse sources in catchment areas. Nonpoint sources generally are of greater relevance than point sources since they are larger and more difficult to control [1,2]. Eutrophication is posing a great threat in water quality for most of the freshwater and coastal marine ecosystems in the world. Lake restoration efforts were traditionally focused on reducing nutrient inputs from the catchment, such as sewage discharges and diffuse runoff from agricultural land. Catchment remediation works including restricting stock access to the lake, enhancing riparian buffer zones and installing constructed wetlands on the main inflow have been previously used. However, the in-lake concentration was still high enough to maintain eutrophic conditions. Thus, even in cases where this action has been quite successful, the recovery of the waterbody may still be very slow due to internal loads. Phosphorus release from the sediment into the lake water (Fig. 1) may be so intense and persistent that it prevents any improvement of water quality for a considerable period after the loading reduction [1]. Today there is growing interest in developing low-cost and effective
Study area During last decades, ecosystem of Pamvotis supported many activities such as irrigation, discharge of domestic sewages, sediment deposit, causing a serious problem in it's trophic state. Cited by a mainly rural area, human impact to the lake is expressed first of all as eutrophication. Urban pollution comes from the city of Ioannina (pop. 100.000), laying along the southwestern shoreline [3]. Moreover, an agricultural effluent of several smaller settlements and industrial wastes from the surrounding area inflow the lake. The surface water temperatures varied from 6.0°C (in January) to 27.1°C (in July) and bottom temperatures followed the same variation (i.e. from 5.89°C in January to 26.8°C in July. The pH fluctuated between 7.5 and 8.7 in surface waters appearing lower values in
314
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
different amounts (g) of each material is illustrated in Fig. 2. As shown, the amount corresponding to 0.03 g is satisfactory for the removal of ~94.6% and ~95.4% of phosphates onto BephosTM and PhoslockTM respectively. Thus, this quantity is the optimal amount of each material to achieve high phosphate removal from natural eutrophic waters. pH is considered as one of the most important parameters controlling the adsorption process at water-adsorbent interfaces. The amount of phosphate adsorbed onto N-Bentonite, NZeolite, PhoslockTM and Bephos™as a function of the pH is illustrated in Fig. 3. Figure shows that as solution pH increases in the range of 5–7, the adsorption efficiency of Bephos™ increases gradually and reaches a maximum value (~92.3%) when the pH value is 7. When the pH increases to 9, the removal efficiency drops (~86.9%). The equilibrium adsorption capacity of BephosTM and PhoslockTM changed very little within the pH range of 5–7 and decreased in solutions with higher pH values. At pH values over 8, the clay surface becomes negatively charged thus phosphate adsorption drops.
June and July. pH values above the sediment ranged between 7.5 and 8.4. Similar to temperature, stratification of pH occurred during the warm period [3]. Experimental The N-Bentonite and N-Zeolite samples used in this work were supplied by the company S&B from a mining site in Bulgaria. It is indicated that the analyzed natural zeolite contain 95% klinoptilolit (Ca Si7 Al2O18.6H2O). BephosTM was synthesized as it is reported in Zamparas et al [4]. Environmental water and sediment samples collected from the eutrophic Lake Pamvotis. Sediment was collected using a grab sampler. The samples were closed in air-sealed bags. Water sample for phosphate analysis was obtained from the water above the sediment sampling site. All the equipment used for sample collection, transportation, and preparation were free from phosphorus contamination. All the samples were brought to the laboratory in a portable fringe at 4oC. Adsorption studies of phosphate on N-Bentonite, N-Zeolite, PhoslockTM and BephosTM in contact times ranging between 15 min and 480 min were studied using natural aqueous solution. 50 mg/L of natural water from Lake Pamvotis, were added into conical flasks containing 0.03g of each material. The suspension was separated by centrifugation and the concentration in supernatant (ce) was measured by the molybdate blue spectrophotometric method using a Lambda 25 UV–Vis spectrophotometer (Perkin– Elmer,Germany). The amount adsorbed (qe) was calculated from the difference in concentration between initial (co) and the equilibrium concentration. Blank samples without any absorbent were perpetrated and monitored as a control. All experiments were carried out in duplicates. The sediment incubation experiments were carried out to estimate the efficiency of natural zeolite (N-Zeolite), natural bentonite (N-Bentonite), commercial Labentonite PhoslockTM and modified bentonite BephosTM as a sediment capping materials reducing the phosphate fluxes in Lake Pamvotis. Phosphate release measurements were conducted on well mixed wet sediments collected from Lake Pamvotis. The sediments (5 cm of thickness) were put in 5 glass cylinders (1L) with an internal diameter of 4 cm. 700 mL of deionized water was added into each column. 1 g of of natural zeolite (N-Zeolite), natural bentonite (N-Bentonite), commercial La-bentonite PhoslockTM and modified bentonite BephosTM carefully introduced into columns and one other acted as a control column simulating phosphate release from sediments. The columns were allowed to reconstitute under aerobic conditions for 5 days before starting the experiment. The experiments were run in a controlled temperature room (25 ± 10C), under anoxic conditions and absence of light. The columns were monitored for 60 days and between that time water samples were taken for determining its posphate concentration.
Figure 1. Schematic approach application in eutrophic water bodies.
of
BephosTM
Figure 2. Phosphate adsorption capacity vs amount of each material.
Figure 3. Effect of pH on phosphate uptake by NBentonite, N-Zeolite, PhoslockTM and Bephos™.
Results and Discussion The amount of phosphate adsorbed onto N-Bentonite, N-Zeolite, PhoslockTM and Bephos™as a function of
315
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
REFERENCES (1) Zamparas, M. and Zacharias, I., Science of the Total
Environment 2014 (in press). (2) Zamparas, M.; Gianni, A.; Stathi, P.; Deligiannakis, Y.; Zacharias, I. Applied Clay Science 2012, 62-63, 101-106. (3) Kagalou, I.; Tsimarakis, G.; Paschos, I. Global Nest 2001, 3, 85-94. (4) Zamparas, M.; Drosos, M.; Georgiou, Y.; Deligiannakis, Y.; Zacharias, I. Chemical Engineering Journal 2013, 225, 43-51.
316
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
A novel bentonite humic acid composite material BephosTM as an environmental-friendly tool in phosphate and ammonium management. M. Zamparas(a), Y. Deligiannakis(a), I. Zacharias(a)* (a)
Department of Environmental and Natural Resources Management, School of Engineering University of Patras, 2 Seferi Str., 30100 Agrinio, Greece * Corresponding author e-mail: [email protected] Abstract The objective of the present study was to examine the feasibility of using a novel modified bentonite as adsorbent for phosphate and ammonium removal from natural eutrophic waters. The uptake of phosphate and ammonium in adsorption kinetics A critical parameter was the initial phosphate concentration aiming to represent a eutrophic natural ecosystem. Moreover, its adsorption capacity and efficiency as a restoration tool was compared with a number of materials tested on the removal of phosphate and ammonium from aqueous solutions. Bephos™ is capable for simultaneous adsorption of both phosphorus and ammonia and this is an important advantage against other materials used to restore eutrophic water bodies. Adsorption kinetics showed that more than 90% and 70% removal of phosphate and ammonium respectively from water within 30 min. Bephos™ is potent for remediation of phosphate and ammonium at low concentrations that occur in natural water ecosystems. Keywords: BephosTM, adsorption, phosphorus, ammonium, restoration.
at pH 10 and adding 1 g of Zenith-N. Stirring addition ally for 16 h at 25 oC. The obtained material was wash ed repetitively with distilled water until Cl-free, i.e. <1 ppm Cl-, titrated with AgNO3, and then freeze-dried at -66 oC using a CHRIST-ALPHA 1-2 LD freeze dryer. Phosphate stock solution of 50 mg/L was prepared by dissolving 0.2197 g KH2PO4 in 1.0 L deionized water and dilutions of the stock solution were used in subsequent experiments. The adsorption isotherms were determined by batch equilibration of 0.02 g of each bentonite sample with 50 mL of aqueous phosphate solutions of varied initial concentrations (ranging from 0.05 to 20 mg/L). The experiments were carried out at room temperature (25 ±1 oC) and pH 7 for 24 h. After equilibration, the suspension was separated by centrifugation and the concentration in supernatant (ce) was measured by the molybdate blue spectrophotometric method using a Lambda 25 UV– Vis spectrophotometer (Perkin–Elmer,Germany) [2]. The determination limit of the analytical method was 0.01 mg PO4-3 /L. Ammonium stock solution of 1000 mg/L was prepared by dissolving 3.819 g arid NH4Cl in 1.0 L deionized water and dilutions of the stock solution were used in subsequent experiments. The adsorption isotherms were determined by batch equilibration of 0.02 g of each bentonite sample with 50 mL of aqueous ammonium solutions of varied initial concentrations (ranging from 0.5 to 500 mg/L). The experiments carried out at room temperature (25 ±1 oC) and pH 7 for 24 h. After equilibration, the suspension was separated by centrifugation and the concentration in supernatant (ce) was measured by the Phenate method using a Lambda 25 UV–Vis spectrophotometer [2]. Adsorption kinetic data of phosphate and ammonium on modified bentonite in contact times ranging
Introduction Phosphorus and ammonia in surface water originates from both point and non-point sources from anthropogenic activities such as: municipal and industrial sewage discharges, runoff from fertilizers and manure applied to agricultural land, or from diffuse sources in catchment areas [1]. Excessive amounts of nutrients entering lakes and streams trigger the growth of algal blooms and other aquatic weeds, resulting in eutrophication [1,2] Eutrophication is posing a great threat in water quality for most of the freshwater and coastal marine ecosystems in the world. Today there is growing interest in developing low-cost and effective natural materials, as specific phosphorus adsorbents from natural eutrophic water bodies. BephosTM (from the words “Bentonite” and “phosphorus”) is a novel low-cost composite material embedding Fe, Cu ions and humic acid in the interlayer space of a natural bentonite [2]. The objective of the present study was to examine the feasibility of using a novel modified bentonite as adsorbent for phosphate and ammonium removal from natural eutrophic waters. The uptake of phosphate and ammonium in adsorption kinetics A critical parameter was the initial phosphate concentration aiming to represent a eutrophic natural ecosystem. Moreover, its adsorption capacity and efficiency as a restoration tool was compared with a number of materials tested on the removal of phosphate and ammonium from aqueous solutions. Experimental The Bephos™ material was prepared by adding 6.68X 10-5 M Fe(NO3)3, 4 X 10-5 M CuNO3, 7.24 X 10-5 M AlCl3 and 8 X 10- 5 M Na2CO3 into 250 mL of lignite 1.2 mg/L solution at pH 2.3. Stirring at 60 oC for 20 h
317
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
between 15 min and 1440 min were studied using optimized conditions: pH 7, phosphate concentration 0.1 mg/L, ammonium concentration 2 mg/L, adsorbent dose 0.02 g, and 25 ±1 oC temperature . Results and Discussion Table 1 presents a comparison of adsorption capacity of with other various adsorbents for phosphate and/or ammonium adsorption. Bephos™ exhibits higher adsorption than the other modified and unmodified materials. For example, the material shows over twice the adsorptive capacity of Phoslock™ (a commercial lanthanum bentonite) while is able to adsorb ammonium in contrast to Phoslock™ which does not have corresponding capacity. From the above overview in the literature it can be concluded that the capacity of the studied materials on phosphate adsorption follow the order: Bephos™ > ferric hydroxide > alum > Al-bentonite > Fe-bentonite > Phoslock™ > modified fly ash > iron oxide tailing > geothite > red mud. Recently, there have been renewed arguments to suggest that P removal alone may not alleviate eutrophication and recognized the importance of simultaneous control of P and N. Thus, the proven capacity of Bephos™ for adsorption of both phosphorus and ammonia is an important advantage against other materials used to restore eutrophic water bodies. Moreover, in cases where one of the two factors, e.g. P or N, is the limiting nutrient of a natural water ecosystem, the use of Bephos™ is appropriate without requiring additional use of other materials.
Table 1. Comparison of phosphate adsorption capacity (qm) of tested material with some literature values. Material
qm phosphate (mg/g)
pH range
12-13.8 16.9-23.9
5.5-8.2 5.5-8.2
Phoslock Al-Bentonite
10.5 12.7
5-7 3-5
Fe-bentonite Iron oxide tailing Goethite Red mud Modified Fly Ash
11.2 8.6 6.4 0.8 9.1
5-7 3.2 5-6 2 4-10
Bephos
26.5
5-9
Alum Ferric hydroxide
Figure 2. Adsorption kinetics of phosphate and ammonium uptake by Bephos™.
.
Figure 1. XRD patterns for Zenith clay and Bephos™. As shown in Fig. 2, most of phosphate is captured during the first 15 min of the adsorption and with increase of contact time the rate of removal slowed down decreased considerably and after passage of 30 min was almost negligible. After approximately 45 min of adsorption, sorption equilibrium begins to establish. In addition, most of ammonium is adsorbed during 2 h from the start of the batch-experiment and after 5 h the isotherm reached equilibrium. REFERENCES
(1) Zamparas, M.; Gianni, A.; Stathi, P.; Deligiannakis, Y.; Zacharias, I. Applied Clay Science 2012, 62-63, 101-106. (2) Zamparas, M.; Drosos, M.; Georgiou, Y.; Deligiannakis, Y.; Zacharias, I. Chemical Engineering Journal 2013, 225, 43-51.
Adsorption kinetic data of phosphate and ammonium on the modified bentonite Bephos™ in contact times ranging between 15 min and 1440 min (24 h) are presented in Fig. 2. The plots represent the amount of phosphorus and ammonium adsorbed onto Bephos™ vs. time.
318
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Photochemical Insights into the Molecular Size of Dissolved Humic Substances Kristopher McNeill (a) *, Elena Appiani(a), Sarah E. Page(b)
(a) Department of Environmental Systems Science, ETH Zurich, Universitätstrasse 16, 8092 Zurich, Switzerland (b) Department of Earth and Environmental Sciences, University of Michigan, 1100 N. University Avenue, Ann Arbor, MI 48109, USA * Corresponding author e-mail: [email protected] Keywords: photochemistry, singlet oxygen, hydroxyl radical, kinetics Abstract Chromphoric dissolved organic matter (CDOM) is an important photochemical sensitizer in sunlit surface waters and humic substance (HS) isolates reflect well the photosensitizing properties of CDOM. Upon exposure to light, HS solutions produce a range of reactive oxygen species including singlet oxygen and hydroxyl radical. The photochemical kinetics of singlet oxygen and hydroxyl radical reacting with probe molecules offers insights into the molecular size of the HS components. The results of these kinetic investigations and their relevance to the molecular size and size distribution of HS will be discussed. Introduction The molecules that make up chromophoric dissolved organic matter (CDOM) are key lightabsorbing species in natural waters and the dominant photosensitizers in sunlit aquatic systems.1 Humic substance (HS) isolates have been used extensively to study the photochemistry of CDOM and HS solutions have been found to closely match the photochemistry of whole waters. In these systems, various reactive species are formed, including singlet oxygen (1O2 ) and hydroxyl radical (• OH). Both 1O2 and •OH are oxidizing species that are important in controlling the fate of organic pollutants in natural biogeochemical cycling of carbon and other elements. Molecular probe methods have been developed for quantifying both photochemically generated 1O2 and • OH in HS solutions.1 In the course of our previous work, we have developed lumigenic probes for these species based on chemiluminescence (1) and turn-on fluorescence (2).2,3 Application of probes 1 and 2 for the quantification of 1O2 and •OH has lead to insights regarding the molecular size of HS. In the case of 1, enhanced phototransformation, relative to well-defined synthetic sensitizer systems, led to the conclusion that 1 partitions to hydrophobic microenvironments within HS solutions and experiences elevated intra-humic concentrations of 1O2.4,5
Experimental The number average molecular weight of a DOM sample was determined from the DOM-specific HO• quenching rate constant (kDOM, L gDOM-1s-1) and the general HO• rate constant (kq, M-1 s-1 ). In this study we • selected rate constants of quenching of HO by isolated DOM material, choosing only the studies that used International Humic Substances Society (IHSS) standard isolates6 and non-standard materials isolated following Aiken et al. isolation procedures.7 The average molecular weight of DOM was obtained by dividing the general rate constant by the DOMspecific HO• quenching rate constant (kDOM) (Eq. 1). (1) Results and Discussion The quenching of •OH by dissolved organic matter is an important process in natural systems. Indeed, in many systems, it is the dominant loss process for • OH and thus controls the steady-state concentration of • OH. The rate constants for the quenching of •OH by HS have been measured by numerous investigators in a variety of systems. While there is significant variability in the quenching rate constant for a particular HS isolate using different methods, taken as a whole, the quenching rate constants fall into a narrow range, typically 1-5 x 108 MC-1 s-1. Examining a wide range of OH rate constants, it was found that a value of 5.6 x 10 9 M-1 s-1 represented the most likely rate constant between an organic molecule and •OH in aqueous solution. By dividing this typical rate constant, which is on a per-molar basis, • by the rate constants obtained for HS, which are on a per-mole-carbon basis, the average number of carbon atoms per molecule in HS was determined. Going further by using the known carbon percentage in each HS, an average molecular weight for each HS could be determined. The range is graphically represented in Figure 1.
In the present work, the quenching of •OH by HS is quantitatively examined and the implications for the number average molecular weight of HS is discussed.
319
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
is low, a component of HS can still be rather large and this minority component may account for the numerous observations that HS act like large molecules. REFERENCES
(1) Blough, N.V.; R.G. Zepp. "Reactive oxygen species in natural waters." Active oxygen in chemistry. Springer Netherlands, 1995. 280-333. (2) MacManus-Spencer, L.A.; Latch, D.E.; Kronke, K.M.; McNeill, K. Anal. Chem. 2005, 77, 1200-1205. (3) Page, S.E.; Arnold, W.A.; McNeill, K. J. Environ. Monitor. 2010, 12, 1685-1665. (4) Latch, D.E.; McNeill, K. Science 2006, 311, 17431747. (5) Grandbois, M.; Latch, D.E.; McNeill, K. Environ. Sci. Technol. 2008, 42, 9184-9190. (6) McKay, G.; Kleinman, J.; Johnston, K.; Dong, M.; Rosario-Ortiz, F.; Mezyk, S., J. Soils. Sediments. 2013, 1-7 and references therein. (7) Aiken, G. R.; McKnight, D. M.; Thorn, K. A.; Thurman, E. M. Org. Geochem. 1992, 18, 567-573.
Figure 1. Graphical representation of the relationship
between the •OH quenching rate constant for humic substance isolates and the corresponding average molecular weight.
The average molecular weight determined by this method for IHSS HS-isolates are around 500 g/mol, which is decidedly at the lower end of the estimates compared to what has been found by other methods. Assessment of molecular weight accuracy: The low molecular weights obtained by this kinetic method lead to an assessment of the factors that affect the molecular weight accuracy. As the molecular weight values are all calculated from the most-probable rate constant, kq = 5.6 x 109 M-1 s-1, which was determined by analysing the large number of previously published rate constants for organic molecules, this number is central to the estimated molecular weights. As can be seen from Equation 1, the calculated molecular weight is directly proportional to kq. As kq is essentially at the diffusion-controlled limit for a bimolecular reaction, a higher value that would lead to higher calculated molecular weight values is not possible. The other term in Equation 1 is the experimentally determined rate constants for •OH quenching. Here artificially high values would lead to artificially low calculated molecular weights. It is conceivable that artificially high values could easily be obtained. For example, the presence of co-reactants in the DOM sample, such as chloride or phosphate, would contribute to the quenching and give artificially high values. What is clear from the wide spread of rate constants obtained for the same isolate (data not shown) is that obtaining an accurate measurement of kDOM is non-trivial. Comparison to other molecular weight estimation methods: The values obtained for HS molecular weight using •OH-quenching kinetics are most in-line with methods that rely on colligative properties (i.e., vapour-phase diffusion) and diffusion (i.e., field-flow fractionation). The values obtained here are far different from those obtained by light scattering, which has a well-known bias toward large molecules. Implications: The use of quantitative reaction kinetics for the reaction of HS with •OH has provided another piece of the puzzle with regard to the molecular make-up of HS. The relatively high observed quenching rate constants on a per-molecarbon basis necessitate that the molecules that compose HS be relatively small. What is not apparent from these calculations, but equally important is that the size distribution of HS is most likely quite broad. This means that even if the average molecular weight
Acknowledgments: The authors gratefully acknowledge support of this work from a grant from the Swiss National Science Foundation (Grant No. CRSI22_127568).
320
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
In Situ Spectroscopic Study and NICA-Donnan Modeling to Characterize Functional Groups in Allochthonous and Autochthonous Natural Organic Matter and Its Interactions with Major Metal Ions Yuan Gao, Gregory V. Korshin University of Washington, Box 352700, Seattle, WA,USA, 98195-2700 [email protected] Keywords:natural organic matter, spectroscopy, in situ, characterization, metals, NICA-Donnan model AbstractThein situstudy of NOM interactions with sodium and hardness cationsprovides insights into both the mechanisms of these interactions and the extent of metal-NOM binding. This in-depth understanding can be achieved via comparison of the data of model calculations carried out using NICA-Donna model of NOM interactions with cations and, on the other hand, examination of the features of the absorbance spectra of NOM at varying system conditions. DAS results demonstrate the nature of the interactions between metals andNOM functional groups and reveal the presence of specific features with maxima at characteristic wavelengths. Several alternative parameters, such asthe differential logarithms of NOM absorbance spectra measured at a selected characteristic wavelength were found to strongly correlate with the amount of NOM-bound calcium and magnesium cations. These parameters appear to correlate with the Donnan volume as well. Therefore, numerically processed absorbance spectra of NOM can be used to quantify the amount of metal specifically or non-specifically bound to NOM. Introduction Natural Organic Matter (NOM) is one of the most important factors controlling water quality and properties in many biogeochemical processes and engineered systems. NOM originates from materials of biological origin within the water body (e.g., algae), or from debris of terrestrial plants and soil sources adjacent to the water body 1is referred to as autochthonous and allochthonous NOM, respectively. Multiple processes, such as the transport and toxicity of heavy metals are strongly impacted by the interactions between metal cations and NOM, in particular, carboxylic and phenolic groups in NOM. Many techniques have been used to characterize interactions between NOM and metal ions, such as ion-selective electrodes, ISE 2,3, X-ray absorption fine structure spectroscopy (EXAFS) 4, and nuclear magnetic resonance (NMR), and many details such as the quantification of metals bound to functional groups and structural geometry of NOM-metal complexes can be elucidated. However, these techniques virtually always require pre-concentration or pre-treatment of the NOM and NOM-metal complexes, which, beside constituting a difficult to overcome complication in practical terms, could alter the characteristics of NOM and its complexes 5, and the high concentrations of NOM and metals required are often above concentration of environmental interest. Therefore, a need to develop an effective method for characterization of NOM and NOM-metal binding properties on a structural level that can be applied to unaltered NOM at the low concentrations found in environmental systems is highly demanding. Differential absorbance spectroscopy (DAS) represents an powerful in situ approach to describe the deprotonation of NOM chromophores, which are moieties in NOM tend to absorb ultraviolet (UV) and visible light and are closely related to the functional groups. The merits of this methods are that a) no pretreatment or preconcentration is required for water
sample with any NOM levels; b) extremely subtle changes in NOM chromophores can be precisely measured; c) differential absorbance spectra are rich in interpretable features, d) interactions of NOM with different species can be probed and e) DAS data can be interpreted based on existing NOM protonation and metal complexation theories, particularly, Non-Ideal Competitive Adsorption model that accounts for ion binding by discrete functionalities separated into the carboxylic and phenolic moieties, and non-specific Donnan electrostatic interactions (NICA-Donnan). Experimental NOM samples were obtained from International Humic Substance Society (IHSS), including standard Suwannee River fulvic acid (SRFA), standard Suwannee River humic acid (SRHA) to represent allochthonous NOM and Pony Lake NOM as autochthonous NOM. To characterize the change of chromophores by common water treatment approaches, chlorination was applied to alter these NOM. After the addition of appropriate amount of metal stock solution (e.g., Na+, Ca2+) and a 30-min equilibrium time, aliquots were taken from solutions with varied metal concentrations and then the corresponding absorbance spectra were recorded by a Perkin-Elmer Lambda 18 or Shimadzu 8700 spectrophotometers. High-performance size-exclusion chromatography (HPSEC) was carried out to investigate changes of apparent molecular weight (AMW) and quantify size changes indicated by DAS data features. Results and Discussion Metal effects on differential spectra:DAS profiles reflect the contributions of dissimilar NOM functional group feature the evolution of several characteristic peaks or band that develop as the metal increases from its reference level. Differential absorbance spectra (DAS) for Na+, Ca2+ and Mg2+ are illustrated in Figure
321
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
the differential absorbance at 320 or 390 nm might be indicative of the specific aspects of NOM-hardness ions interactions. A strong linearity between spectroscopic parameter DAS390 and concentrations of hardness cations bound to NOM calculated by NICADonnan model can be obtained. That is, the DAS at the characteristic wavelength 390 nm is well correlated with the calculated total bound metal comprising metals bound to carboxylic groups (denoted in the model used in this study as FA1) and phenolic groups (FA2) and electrostatically bound metals in the Donnan phase. Therefore, the results shown above confirm the applicability of the concept that measurements of the intensity of DAS at these selected wavelengths could yield results indicative of the extent of divalent cation-NOM interactions 0.002
DOC-normalized differential absortbance (Lmg- 1 cm -1)
(a)
2.50E-04 5.00E-04 1.00E-03 2.50E-03 0.0008
5.00E-03 increase of Ca (M )
1.00E-02
0.0004
275
325
375
425
475
525
Wavelength (nm)
0.0020
0.005
pH=5
1.25E-04 0.0012
225
0.010
0.01
0.008
0.0016
0
DOC-normazlied differential absortbance (Lmg- 1 cm -1)
Differential absortbance (Lmg-1 cm -1)
1 and Figure 2. For varying concentrations of Na+: 1) the intensity of the DAS spectra at lower pH is lower than those at higher pHs. Prior research 6 has shown that pH could affect the degree of deprotonation in NOM and consequently the electrostatic conditions of NOM molecules. As NOM comprises molecules with functional groups that have varying pKa values including the two most important moieties of carboxylic and phenolic groups with pK ranges 2~3.8, and 7.2~10.9, respectively. Therefore, due to the partial deprotonation of the functional groups at lower pH range, NOM molecules are less negatively charged compared to those in higher pH range;2) at higher pHs (e.g., pH=9), a peak at ~ 250 nm and a broad band from 300 to 400 nm evolves as Na+ increases. Figure 2 shows the evolution of DAS spectra as divalent cation (calcium) concentrations increase. The DAS spectra for these divalent cations are distinct from those generated in the presence of varying sodium concentrations as the characteristic peaks for calcium and magnesium appear at wavelength 320 and 390 nm at higher pHs (e.g., pH=9). As Na+ is not able to specifically bind to functional groups of NOM, the formation of the characteristic peaks at 320 and 390 nm may be associated with specific interactions between Ca2+ and Mg2+ with functional groups in NOM, especially the phenolic groups whose activity is expected to increase when pH is close or above the range of their pKa. pHalso strongly affected the interactions between NOM and these hardness cationsas the interactions between NOMs and divalent cations are affected by the charging behavior on NOM and the deprotonation of the functional groups.
0.055
(b)
1.25E-04
increase of Ca (M )
2.50E-04
0.0016
5.00E-04 1.00E-03 2.50E-03
0.0012
5.00E-03 1.00E-02
0.0008
0.0004
0.105 0.006
0.155 0.0000
0.205
225
275
375
425
475
525
Figure 2.Differential absorbance spectra NOM of SRFA as a function of calcium concentrations (M): (a) pH=5, and (b) calcium, pH=9.
increase of Na (M )
0.002
0.000
325
Wavelength (nm)
0.004
225
275
325
375
425
475
525
Wavelength (nm) Differential absortbance (Lmg-1 cm-1 )
0.010
pH=9
REFERENCES
0.005
increase of Na (M )
0.008
0.01
(1)
0.055 0.105 0.006
(2)
0.155 0.205
0.004
0.002
(3)
0.000
225
275
325
375
425
Wavelength (nm)
475
525
Figure 1.Differential absorbance spectra (DAS) of SRFA
(4)
Spectroscopic parameters indicative of metal-NOM interactions:We observe that the two characteristic
(5)
for varying NaClO4 concentration (in mol/L) at pH=5 and pH=9.
peaks located at 320 and 390 nm appear in the differential spectra for Ca2+ and Mg2+ but are much weaker or absent for the monovalent cation Na+ whose interactions with NOM are dominated by non-specific electrostatic interaction. We therefore hypothesize that
(6)
322
Thurman, E. M. Organic geochemistry of natural waters; Springer, 1985. Christl, I.; Metzger, A.; Heidmann, I.; Kretzschmar, R. Effect of Humic and Fulvic Acid Concentrations and Ionic Strength on Copper and Lead Binding. Environ. Sci. Technol.2005, 39, 5319–5326. Christl, I. Ionic strength-and pH-dependence of calcium binding by terrestrial humic acids. Environ. Chem.2012, 9, 89–96. Korshin, G. V.; Frenkel, A. I.; Stern, E. A. EXAFS Study of the Inner Shell Structure in Copper(II) Complexes with Humic Substances. Environ. Sci. Technol.1998, 32, 2699–2705. Dryer, D. J.; Korshin, G. V.; Fabbricino, M. In Situ Examination of the Protonation Behavior of Fulvic Acids Using Differential Absorbance Spectroscopy. Environ. Sci. Technol.2008, 42, 6644–6649. Benedetti, M. F.; Van Riemsdijk, W. H.; Koopal, L. K. Humic Substances Considered as a Heterogeneous Donnan Gel Phase. Environ. Sci. Technol.1996, 30, 1805–1813.
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Ecotoxicity assessment of functionalized activated charcoal on the activity of the enzyme acetylcholinesterase and its effect in the presence of pesticides a)
a)
(b)
(b)
b)
M.S. Vianna( *, E.H. Novotny( , T.S. Silva , B.R. Ribeiro , L.A. Dittz ( (a)
(b)
Embrapa Soils, R. Jardim Botânico, 1024, CEP 22460-000, Rio de Janeiro, Brazil. Integrated Laboratory of Agro-environmental Analysis (LIAAM)/IBELGA – C.E.F.F.A. Rei Alberto I
* Corresponding author e-mail: [email protected] Keywords: Acetylcholinesterase; biochar; ecotoxicity; pesticide. Abstract The study of soil organic matter of the “Terras Pretas de Índios” showed an efficient compost model for improving the physical chemistry properties of the soil. The charred materials have the condensed aromatic structures but it does not provide carboxylic groups, that are important for its reactivity and its contribution to soil CEC. The objective of this study is to evaluate the ecotoxicity of functionalized charcoal compounds (humic acids like material – HALM) through its sub-lethal kinetic behaviour of the enzyme AChE even in the presence of pesticides. Activated charcoal was chemically oxidized using sodium hypochlorite. Brains of rats were used for ecotoxicity assay. The IC 50 (half maximal inhibitory concentration) for AChE was 65 mg L-1 of HALM. The presence of HALM (10 mg L-1) reduced the AChE inhibition by the tested pesticides (methomyl; methyl parathion; and atrazine) probably due a decrease of pesticide availability to form stable complexes with AChE. Introduction The study of soil organic matter (SOM) of the “Terras Pretas de Índios” (TPI) (archaeological extremely fertile soil found in the Amazon) showed an efficient compost model for improving the physical chemistry properties of the soil and provides carbon sequestration. The charred materials have the condensed aromatic structures that ensure their recalcitrance in the soil, and hence is an efficient material for carbon sequestration (half-life ranging from decades to millennia), but it does not provide carboxylic groups, that are important for its reactivity and its contribution to soil CEC, as found in the SOM of TPI. Studies indicate that it is possible to functionalize these materials chemically (oxidation) (1) However, it is important to evaluate the risks that the use of biochar, and its products, as a soil amendment can represent to the environment. Ecotoxicological tests reveal whether and in what quantity chemical substances, isolated or in mixtures, are harmful to the environment, and how and where their effects are expected to be shown in living matter. The effects can be acute or chronic and result in death or changes in morphology, physiology and histology, manifesting altered growth, reproduction, metabolism, and behavior of test organisms (bioindicators). These tests provide information about and identify possible risks and negative physical and chemical alterations that the environment can suffer through the introduction of a specific substance. They provide the concentration at which the tested chemical substance has a toxic potential, serving as a preventive system of protection and warning. Besides it is necessary to access its possible interactions with other agricultural inputs, such as pesticides. Therefore the toxicity tests
based on a selective and sensitive biomarker for possible sub-lethal effects are needed. The choice of the enzyme acetylcholinesterase (AChE) as an indicator of ecotoxicity was based on the following criteria: (1) high sensitivity to the presence of different classes of pesticides (including being used as a sensor for quantification); (2) use of a protein fraction capable to metabolize organophosphate pesticides in their anticholinesterase way; (3) molecule widely used as bioindicator in ecotoxicity tests with the soil fauna. The objective of this study is to evaluate the ecotoxicity of functionalized charcoal compounds (humic acids like material – HALM) through its sublethal kinetic behaviour of the enzyme AChE. Additionally, has been described kinetics of AChE in the presence of HA with different concentrations of commercial formulations of known pesticides with anticholinesterase activity and acute toxicity in humans (methomyl; methyl parathion; and atrazine). Despite the common mechanism of anticholinesterase action for methomyl and methyl parathion, the latter acts as an indirect inhibitor. However, the metabolite of parathion (paraoxon) is a potent anticholinesterase agent. Atrazine, although a herbicide (AChE is not the target molecule), presents a significant effect on cholinesterase metabolism and it has described ecotoxicological tests based on in AChE (2). Experimental Activated charcoal was chemically oxidized using sodium hypochlorite. Afterward, the HALM was obtained according the method suggested by IHSS (alkali solubilization; precipitation at acid pH; dialysis and freeze drying of the acid insoluble fraction).
323
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Results and Discussion Kinetic characterization of AChE: The protein fraction used in ecotoxicity tests shows Michaelis constant (Km) of 0.4 mmol L-1 and maximum reaction rate (Vm) of 1.0 mmol min 1 mg-1 of protein. These values show the affinity of AChE for the substrate used in the tests, expressed by Km and efficiency in its hydrolysis, represented by Vm. The protein material obtained from the rat brain, although a simple fractionation procedure, reached a satisfactory kinetic profile. It can be considered that the substrate used in the kinetic assays has affinity for the AChE molecule enough to compose essays with rapid and sensitive kinetic response fraction. AChE inhibition curve in the presence of different concentrations of HALM: [Figure 1] The catalytic activity of the enzyme AChE decreased by 50% in the presence of 65 mg L-1 of HALM (IC50). Activity of R. norvegicus AChE (%)
100
Y= [104,8 - 39,9/(1 + e
(x -42,5)/ 14,4
of pesticide availability to form stable complexes with AChE. This indicates a potential for environmental protection (inhibition of pesticides toxic effect), however could also to require a dose adjustment when HALM is used as soil amendment. On the other hand, the HALM and pesticides showed no synergism on their effects on AChE. 100
Activity of R. norvegicus AChE (%)
A unpurified brains extract of rats Wistar (Rattus norvegicus) was used for ecotoxicity assay. The brains were removed and washed with distilled water (4 °C); processed using a tissue homogenizer; subjected to saturation with ammonium sulphate; and centrifuged at 300 g at 4 °C. The catalytic activity of AChE of the was measured under experimental conditions obtained by experimental design (23 center composite). Measurements of enzyme activity of AChE was performed by spectrophotometric method, measuring the absorbance (412 nm) of the formed product from acetylthiocholine (ATCh) metabolism (3). This measuring was performed by adding the substrate ATCh (3.2 10-4 mol L-1) and DTNB (3.2 10-4 mol L-1). The incubation time of the assays was 2 h at 37 °C.
)] + 39,9
50
30
40
50
60
70
80
70 60 50 40 30 20
20
40
60
80
100
120
140
Ecotoxicological tests assess the effects on AChE in experiments "in vivo" and "in vitro" generally consider their lethal effect on test organisms or the in vitro effect on AChE extracted these organisms (4).In this work the study of sub-lethal toxicity of functionalized charcoal and its effect in the presence of pesticides using enzymatic assays was employed. The effect on the toxicity of atrazine AChE has already been described tests Earthworms species (2) showing the sensitivity of the ecotoxicity biondicador to agricultural environments. The HALM had an inhibitory effect on AChE (IC50 of 65 mg L-1), on the other hand, at low concentration (10 mg L-1) it reduced the inhibitory effect of the tested (methomyl; methyl parathion; and atrazine) pesticides, indicating a need of dose adjustment, on the other hand this indicates a potential for environmental protection by HALM.
60
20
80
Figure 2. Effect of the HALM presence in the inhibition kinetics of AChE by pesticides: All assays were performed in quadruplicate and the standard deviation ≤ 0.02./ Y= [A1 – A2/(1 + e (x – X0)/dx] + A2 .
70
10
)] + 39,9
Pesticides Concentration ( g L )
80
0
(x -42,5)/ 14,4
-1
90
40
Y= [104,8 - 39,9/(1 + e
90
REFERENCES
90
(1) Linhares, C. R. ; Lemke, J. ; Auccaise, R. ; Duó, D. A. ; Ziolli, R. L. ; Kwapinski, W. ; Novotny, E. H. Pesquisa Agropecuária Brasileira, 2012, 47, 693-698. (2) Stenersen, J; Brekke, E.;Engelstad, F. Soil Biol. Biochem. 1992, 21, 1761-1764. (3) Ellman, G.L. et al. Biochemical Pharmacology. 1961. 7, 88-95. (4) Caselli F., Gastaldi L., Gambi N., Fabbri E. 2006. Comparative Biochemistry and Physiology, Part C 143, 416– 421
-1
Concentration of HALM (mg L )
Figure 1. Acetylcholinesterase (AChE) inhibition curve in the presence of different concentrations of humic acids like materials obtained from functionalized charcoal (HALM). Catalytic conditions: sodium phosphate buffer 0.04 mol L-1 (pH 7.4). All assays were performed in quadruplicate and the standard deviation ≤ 0.006.
Acknowledgments: Financial support: CNPq (590059/2011-4); Post-Doctoral fellowship M.S. Vianna (CAPES: PNPD 2480/2009).
Effect of the presence of HALM in the kinetics of inhibition of AChE by pesticides: [Figure 2] For the initial tests the concentration of 10 mL-1 was chosen for HALM to avoid the its inhibitory effect on AChE activity. So the inhibitory action on AChE would be exclusively due the presence of pesticides. The presence of HALM (10 mg L 1) reduced the AChE inhibition by the tested pesticides (methomyl; methyl parathion; and atrazine), probably due a decrease
324
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Chloride interference during analysis of total dissolved organic carbon and individual humic and hydrophilic substances using wet chemical oxidation methods A. van Zomeren (a) *, R. Koper(a), J.J. Dijkstra(a), R.N.J. Comans(b)
(a) Energy Research Centre of The Netherlands (ECN), Post Office Box 1, 1755 ZG Petten, The Netherlands (b) Wageningen University, Department of Soil Quality, P.O. Box 47, 6700 AA Wageningen, The Netherlands. * Corresponding author e-mail: [email protected] Keywords: up to 6 keywords Abstract Wet chemical oxidation methods (WCO) for the analysis of dissolved organic carbon (DOC) usually have a relatively low detection limit (±1 µgC/L) and are, therefore, interesting for DOC analyses in environmental samples. However, these matrices might contain relatively high concentrations of other ions (e.g. chloride) that can affect the DOC oxidation efficiency. The aim of this work was to study the influence of chloride ions on the oxidation efficiency of humic substances (HS) and weak organic acids. Results show that the oxidation efficiency correlates well with the proton dissociation constant of the organic acids. Distinct differences in the DOC recovery between several organic acids, aquatic HS and the soil HS were found, which are probably related to the origin of the HS. The results of this study clearly indicate that the type of organic carbon is important for the oxidation behavior under conditions of high chloride background concentrations. substances (HS) and weak organic acids in aqueous samples with different background concentrations of chloride. In addition, we have tested nine weak organic acids of different nature to investigate effects of the chemical structure of these substances on the level of chloride interference in these measurements. Knowing these effects will help to improve analytical methods to determine analysis of HA and FA.
Introduction Analysis of dissolved organic carbon (DOC) is widely used as a characterization method for the sum of all organic carbon species in water. Over the past decades, several commercially available analysers have been developed. These analysers can be mainly divided into two categories; analysers that are based on catalytic combustion of DOC and analysers that are based on wet chemical oxidation (WCO). The latter category of DOC analysers usually has a relatively low detection limit (<<1 mg C/L) and is, therefore, an interesting technique for DOC analyses in environmental samples (e.g. groundwater, porewater in soils, surface water or seawater). In addition, fractionation procedures to distinguish humic and fulvic acids from other DOC fractions have been developed (1) and standardized (ISO 12782-4 and ISO 12782-5). These procedures use HCl to acidify the samples for precipitation of humic acid and to establish an acidic environment for adsorption of fulvic acid to the DAX-8 resin. However, these matrices might contain relatively high concentrations of chloride and it is known that chloride affects the DOC oxidation efficiency (2-5). The process of chloride interference in the analyses of DOC using WCO has been systematically studied by Aiken (2). He concluded that chloride is oxidized by the persulfate ion (S2O82-), thereby reducing the persulfate concentration and, hence, a loss of oxidation power. Although Aiken (2) found chloride interference in the analyses of several organic molecules, the reaction mechanisms were not elucidated. In addition, the oxidation method using persulfate in combination with UV irradiation was found to eliminate the chloride interferences (2). The aim of this work was to study the influence of chloride ions on the oxidation efficiency of humic
Experimental Samples Prepared solutions of IHSS standard HA and FA (Elliot soil and Suwannee river) were used to study the effect of chloride on these typical natural organic compounds. Stock solutions of the HS were prepared at concentrations of about 5 mg C/L. In addition, solutions of about 5 mg C/L were prepared using nine different organic acids (acetic acid, propionic acid, butyric acid, oxalic acid, valeric acid, malonic acid, benzoic acid, glutaric acid and potassium hydrogen phthalate). Chloride was added to obtain a range in chloride concentrations. A selection of the data is presented in this paper, the complete overview is presented in (6). DOC analyses A Sievers 900 Portable TOC analyzer was used for DOC analyses. Total carbon is oxidized to CO2 using a UV-persulfate oxidation technique (15% (NH4)2S2O8), while DIC is evolved as CO2 by the addition of H3PO4 (6 M). The evolved CO2 from both steps is separated from the solution with a semi permeable membrane and subsequent conductometric detection at the other side of the membrane. The detection limit is about 0.03 µg C/L.
325
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The performance of the Sievers DOC analyser was checked independently with a Shimadzu TOC 5000a analyser. DOC was determined as the difference between total dissolved carbon (catalytic combustion at 680 oC) and inorganic carbon (acidification with H3PO4). The evolved gasses from the liquid samples (total dissolved carbon and dissolved inorganic carbon, DIC) were detected for CO2 by a nondispersive infrared detector. The detection limit of the Shimadzu TOC 5000a analyser is about 1 mg C/L. .
quenching of the chain reactions of hydrogen peroxide photolysis and the subsequent radical formation. Apparently, the structure and/or molecular properties of the organic acids play an important role in the DOC recovery under these more difficult oxidation conditions. Oxidation efficiency of humic substances as a function of the chloride concentration Figure 2 shows the DOC recovery of the organic acids (in 1 M chloride) as a function of the proton dissociation constants (pKa1) of the individual acids (6). The correlation between the DOC recovery and the pKa1 of the organic acids is R2=0.87. Apparently, acids that more strongly dissociate in water (i.e. lower pKa) are more easily oxidized. The pKa1 of the organic acids is a parameter reflecting part of the chemical structure of the molecule. However, the precise mechanisms by which the oxidation efficiency of DOC during WCO is influenced in the presence of chloride remains unknown. Our new findings imply that the analyses of DOC by chemical oxidation methods should include an assessment of the chloride concentrations in the samples. The results of this study clearly indicate that the type of organic carbon is important for the oxidation behavior under conditions of high chloride background concentrations.
Results and Discussion Oxidation efficiency of organic acids and humic substances as a function of the chloride concentration The results of the DOC analyses (represented as the calculated recovery) as a function of the log chloride concentration are presented in Figure 1. The shape of the DOC recovery curves as a function of the chloride concentration varies substantially for the different organic acids and HS. Distinct differences in the DOC recovery between the aquatic HS (Suwannee river) and the soil HS (Elliot soil) were found and these differences are probably related to the origin of the HS as was identified by Pomes et al. (7). At a log chloride concentration of 2.55 mg/L, most substances are not affected by chloride. Only acetic acid and valeric acid (not shown) show a substantial loss in their recovery to about 50% at a log chloride concentration of 2.55 ppm. At higher chloride concentrations, a decrease in the DOC recovery becomes more important for almost all substances. The DOC recovery of malonic acid remains relatively high over the entire range of chloride additions while the recovery of oxalic acid seems to increase to over 100% at increasing chloride concentrations. We currently have no explanation for the high recovery of oxalic acid. The results are not influenced by inorganic carbonate which was only 2% of DOC.
Figure 2. Recovery of DOC as a function of the pKA1 for different organic acids (6). REFERENCES
(1) van Zomeren, A. and Comans, R.N.J. Environ. Sci. Technol. 2007 41, 6755-6761. (2) Aiken, G.R. Environ. Sci. Technol. 1992, 26, 24352439. (3) Peyton, G.R. Mar. Chem. 1993, 41, 91-103. (4) McKenna, J.H. and Doering, P.H. Mar. Chem. 1995, 48, 109-114. (5) Liao, C.H., Kang, S.F., and Wu, F.A. Chemosphere 2001, 44, 1193-1200. (6) van Zomeren, A., Koper, R., Dijkstra, J.J. and Comans, R.N.J. Submitted 2014. (7) Pomes, M.L., Larive, C.K., Thurman, E.M., Green, W.R., Orem, W.H., Rostad, C.E., Coplen, T.B., Cutak, B.J., and Dixon, A.M. Environ. Sci. Technol. 2000 34, 42784286.Rice, N.; Buxton, G.V; Nixon, A.B. J. Phys. Chem. Ref. Data 1988, 17, 513-886.
Figure 1. Effect Recovery of DOC for different organic
acids and HS (initial concentration was 5 ppm) using wet chemical oxidation (Sievers) as a function of the log chloride concentration (6).
These results shown in Figure 1 clearly indicate the difference in sensitivity of organic acids towards oxidation under increasing chloride background concentrations. The remarkably low DOC recovery of acetic acid might be related to the radical scavenging properties of acetic acid that was noted by Liao et al. (5). These authors conclude that acetic acid causes
Acknowledgments: This work was partly funded by the Ministry of Infrastructure and the Environment as part of ECN’s environmental research program.
326
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Germination Efficiency Assessment of Corn Husk Biochar C. C. Bueno (a) *, C. H. Watanabe (a), M. T. Domingues (a), L. F. Fraceto (a), A. H. Rosa (a) (a) Department of Environmental Engineering, Sao Paulo State University, Sorocaba, SP, Brazil * Corresponding author e-mail: [email protected] Keywords: germination, corn husk, biochar, soil-less bioassay Abstract Biochar is the solid by-product of pyrolysis which is mainly carbon based. For being touted as a promising soil conditioner, the certification of biochar quality is essential to its agronomical usage. In addition, since the maize crop originates several types of cellulosic residues, such as husk, it is important to find out a valuable usage for them. In this context, corn seeds were sown in a soil-less Petri dish with aqueous solution and nine different types of biochar corn husk. The seeds germination and its vitality were evaluated by the first germination count (FGC) and the germination speed index (GSI). The results show that biochar produced at 300ºC was the best growing medium for corn seeds germination by the means of FGC and GSI, exhibiting 70% of corn seedlings in the fourth day, as well as the highest germination speed index: 10.5 against 40% and 4.3 for the control, respectively. Introduction The quality of an agricultural product is evaluated not only for its intrinsic value, but as well as the sustainable technology that sustains them. In the current agricultural production system, the inputs, especially fertilizers and conditioners, may occupy more than a quarter of the total budget to obtain a good crop. This statement endorses the importance of this segment in agriculture. Biochar is a solid residue rich in carbon produced by the thermal decomposition of organic matter/biomass (1), also being a stable form of organic waste. In recent years, the biochar has attracted attention for its environmental and agricultural applications as an efficient and versatile material (2). The char from biomass is a multifunctional material that is used primarily as a soil conditioner to immobilize metals, to increase pedological drought resistance, and to recover and to fertilize the soil. Since the temperature of production (pyrolysis) can change the physical composition, chemical and porosity structure, and pH (3-4). However, the quality of biochar needs to be evaluated to guarantee its agronomical security. Since the maize crop is an agricultural commodity harvesting high volumes, the amount of organic waste produced in biomass form is also high. The aims of the present work were (a) to examine the role of the raw corn husk biomass (BMPM) and its biochar on the germination index, (b) to propose a value-added product, and (c) to study the biochar agronomical quality.
muffle furnace. A fixed amount of dry sample (15.0 g) was placed in a stainless steel horizontal tube-shaped reactor with an entrance for nitrogen gas. Slow pyrolysis (5°C/min) of the samples was analysed for nine different regimes of temperature: 250ºC (BC250), 300ºC (BC300), 400ºC (BC400), 450ºC (BC450), 500ºC (BC500), 550ºC (BC550), 600ºC (BC600), 700ºC (BC700), and 800ºC (BC800). The retention time of the samples produced by slow pyrolysis on targeted temperature was 120 minutes. Soil-less Petri dish bioassay: Ten corn seeds (Zea mays) were sown in Petri dishes on a layer of filter paper moistened with ultrapure water. Eleven treatments were conducted, where control was the seed sown in ultrapure water only and, BMPM was the treatment which the corn husk milled was added and 0.5 g. The other treatments were setup for 0.5 g of biochar produced in different temperatures. After that, 20 mL of ultrapure water were added to each treatment. All Petri dishes were covered with lids and incubated in the dark at 35ºC for 72h. First germination count (FGC) was conducted with the germination test, by computing the percentage of normal seedlings on the fourth day after installation of the test. Finally, the rate of germination was checked every 24h, in order to calculated the germination speed index (GSI). The GSI was calculated by the Equation 1 (6):
Experimental Pyrolysis of corn husk: Samples of corn husk were used to manufacture biochar. All the samples were previously washed with water to remove dust and microorganisms and then, dried in an oven for 48 hours at 80°C (2). Subsequently, the dried samples were ground in a mill with fixed rotation of 1730 rpm, since the size of the biomass particles is a significant parameter in the production of biochar, directly affecting the yield of pyrolysis. The pyrolysis process was performed in laboratory scale by means of a
where G1, G2 and G3 were the number of plants computed in the first, second and final count and N1, N2 and N3 were the number of days ofsowing associated to the first, second, and third counts. Results and Discussion Direct seeding is widely used in agricultural crops, such as maize, for example. Thus, two controls were made: one with ultrapure water and one made with
327
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
raw corn husk biomass to evaluate the germination behaviour under these conditions. As can be seen in Figure 1, the two controls showed no significant differences between them and, therefore, they were taken as a base for the investigation of biochar treatments. All biochars produced have suffered complete pyrolysis, with the exception of biochar produced at 250°C. This material showed brown colour and similar characteristics to the controls. The control treatments, BMPM control, and BC250 had a pH between 5.5 and 5.9 after 72h of germination. On the other hand, on the other controls, the pH ranged from 6.5 to 7.4. The water entered into the system germination had an initial pH of 6.5. Thus, it can be seen that the materials did not undergo pyrolysis left the slightly acidic environment, when compared to systems with biochar.
BMPM
80
C o n tro l
F G C (% )
100
60 40 20 0
T re a tm e n ts
Figure 2. First germination count (FGC) conducted with the germination test, by computing the percentage of normal seedlings on the fourth day after installation of the test.
15
5
BM PM
C o n tr o l
IG S
10
0
T re a tm e n ts
Figure 3. Index of germination speed (IGS) due the different temperatures made biochar presence.
This germination test proved to be a reliable procedure to differentiate between the effects on seedling growth, when applying different types of biochar directly in contact with them.
Figure 1. Effects of biochar presence on the corn
germination. A, C and E: Beginning of germination test (0h) and; B, D and F: The end of the test (72h). Treatments on horizontal: Control, BMPM and, BPM300.
REFERENCES
(1) Dai, Z.; Meng, J.; Muhammad, N.; Liu, X.; Wang, X.; Liu, H.; Wang, H.; He, Y. J Soil Sediment. 2013, 13, 9891000. (2) Zhao, X.; Zhao, W.; Ouyang, F.; Hao, C.; Lin, F.; Wang, S. et al. Bioresour. Technol. 2013, 147, 338-344. (3) Uchimiya, M.; Wartelle, L.H.; Klasson, K.T.; Fortier, C.A.; Lima, I. M. J. Agric. Food Chem. 2011, 59, 25012510. (4) Cantrell, K. B.; Hunt, P. G. .; Uchimiya, M.; Novak, J. M.; RO, K. S. Bioresour. Technol. 2012, 107, 419-428. (5) Oliveira, A. B.; Gomes-Filho, E. Rev. Bras. Sementes. 2009, 31, 48-56.
The first germination count was taken was as baseline for determining the inhibition of germination possibly caused by the biochar treatment. The treatments with BC250, BC450, BC500, BC550, BC700, and BC800 demonstrated lower rates than those presented controls. Since phytotoxic compounds are mostly water soluble, it is reasonable for these treatments to contain substances which act as allelochemicals. The IGS for BC300 was the highest suggesting that soluble compounds present in this biochar have provided, with the water, the activation of the metabolic processes of the seeds, culminating in the increased growth of embryonic axes observed in the test. In addition, the BC300 treatment showed the highest length of developing roots. The roots of this treatment were strongly linked with the biochar, even after washing.
Acknowledgments: The authors would like to thank FAPESP and CNPq for the support.
328
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Probing the photolytic-photocatalytic degradation mechanism of DEET in the presence of different humic and fulvic acid models by scavenging and EPR spectroscopy M. Antonopouloua, Konstantinoua*
C.G.
Skoutelisa,
C.
Daikopoulosb,
Y.
Deligiannakisa,
I.K.
a
Department of Environmental and Natural Resources Management, University of Patras, Seferi 2, Agrinio 30100, Greece b Department of Materials Science and Engineering, University of Ioannina, Ioannina 45100, Greece Keywords: DEET; EPR; Humic; Fulvic acid; Scavengers Abstract The present work provides a detailed investigation of the photocatalytic degradation mechanisms of DEET in the presence of different humic and fulvic acid models based on scavenging and EPR measurements. Scavenging experiments indicated the HO• as the main species in the DEET oxidation in the presence of humic acids (LHA and HALP). In the presence of FA, 3DOM* accounted for DEET degradation. Spin-trapping measurements by electronic paramagnetic resonance spectroscopy, using the spin-trap DMPO, followed by computer simulation analysis of the spectra was used to monitor the evolution of photogenarated HO• in the photocatalytic systems and the generation of organic radical intermediates. acid Like Polycondensate (HALP) with aromatic-ring rich properties and c) a soil FA (4). Irradiation procedure: All photocatalytic experiments were carried out in a Suntest XLS+ apparatus from Heraeus (Germany) equipped with a xenon lamp (2.2 kWatt) and special glass filters (λ< 290 nm). Α tap water cooling circuit was used for keeping the temperature up to 25°C. Irradiation experiments were performed using 600W/m2 irradiation intensity. Analytical procedures: Determination of DEET was performed by a Dionex P680 HPLC equipped with a Dionex PDA-100 Photodiode Array Detector and a Discovery C18 column (from Supelco (Bellefonte, PA, USA). The mobile phase was a mixture of LC-grade water H2O (50%) at pH 3 (adjusted with formic acid) and acetonitrile (50%) with a flow rate of 1 ml min-1. DEET was measured at 210 nm. EPR experiments: Electron paramagnetic resonance (EPR) spectra were recorded with a Bruker ER200D spectrometer at room temperature, equipped with an Agilent 5310A frequency counter. The spectrometer was running under homemade software based on Lab View (5). Simulation of the EPR spectra of holes and electrons was done by using the software SIMFONIA by Brucker.
Introduction Over the past several years, TiO2 mediated photocatalysis is well-established as a promising advanced oxidation process (AOP) that has demonstrated ability to degrade a broad range of organic pollutants (1). Key advantages of TiO2 photocatalytic treatment processes include the lack of mass transfer limitations, operation at ambient conditions, the material’s low cost, resistance to photocorrosion and chemical corrosion as well as the potential use of solar irradiation (1). In practical applications of photocatalysis for organic pollutants treatment, the presence of natural organic matter (NOM) and other photoactive dissolved and particulate constituents can influence the degradation kinetics and even shift the initial catalytic mechanism (2). Considering that humic and fulvic acids account for the significant fraction of natural organic carbons in surface waters acting as an inner filter, a radical scavenger and/or a precursor of reactive species, a better understanding of the mechanisms and the reactive species involved is a great demand (3). Therefore, the purpose of this study is the investigation of the photocatalytic mechanism of DEET a representative aquatic contaminant resistant to conventional water treatment processes and fully eliminated by TiO2 photocatalysis and the determination of the predominant reactive species involved in the presence of different humic and fulvic acid models by scavenging experiments and EPR spectroscopy techniques.
Results and Discussion Preliminary experiments: Preliminary photolysis experiments in the absence and presence of humic and fulvic acids were carried out at the initial DEET concentration of 10 mg L-1 to assess the contribution of NOM in the photodegradation of DEET (data not shown). Direct photolysis was able to achieve only around 10% degradation of DEET. The addition of NOM was found to increase the photodegradation of DEET leading to almost 30-40% after 240 min of simulated solar illumination due to the additional contribution of indirect photolysis. Scavenging studies indicated that excited state dissolved organic matter (3DOM*) are responsible for the major photolytic
Experimental Materials: DEET was residue analysis grade (SigmaAldrich, USA). TiO2 Ρ25-Degussa (Germany) was used for all degradation experiments. All solvents used were pesticide residue analysis grade (Merck, Darmstadt, Germany). Sorbic acid was purchased from Sigma–Aldrich. The humic and fulvic acids used were: a) a well characterised Lignite HA (LHA) mainly of aliphatic character; b) a synthetic Humic
329
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
degradation of DEET whereas HO• play minor role during the photolytic process in all cases.
> isopropanol > furfuryl alcohol. According to the scavenging experiments, 3DOM* and e-aq participated in DEET degradation, in consistence with the proposed sensitization mechanism (electron transfer to TiO2) in the literature (6). A lower contribution was associated to HO•.
Contribution of 3DOM* and reactive species to the photocatalytic degradation of DEET: The contribution of 3DOM*, 1O2, HO• and e-aq in the degradation of DEET in the presence of different humic and fulvic acids was evaluated with the addition of specific scavengers (Fig. 1). The extent of decrease in the DEET degradation efficiency that induced by the scavenging reagents, indicates the importance of the corresponding active species and 3DOM*. 1 .0
EPR Spectroscopy. Electron Paramagnetic Resonance data reveal that [i] DMPO-OH radical production was not influenced severely by the presence of DOM. [ii] in all cases DEET degradation resulted in formation of a DEET-radical adduct that is characteristic of a methy-based radical formed near the surface of the TiO2 particle.
(a ) L H A
0 .8
0,006
0,004
2 -i-P rO H F u rfu ry l a lc o h o l C h lo ro -e th a n o l S o rb ic a c id W ith o u t s c a v e n g e r
0 .2
0 .0
0
10
20
30
40
50
60
Irra d ia tio n tim e (m in ) (b ) H A L P
1 .0
0 .8
C /C 0
3300
DEET-radical
0
10
20
30
40
50
3340
3360
3380
3400
3420
3440
3460
3480
Figure 2. Room temperature EPR spectra of DEET in the presence of (a) P25 TiO2 and LHA. The time indications refer to in-situ irradiation of the sample in the EPR cavity.
60
Irra d ia tio n tim e (m in ) (c) F A
1 .0
3320
Magnetic Field (Gauss)
2 -i-P rO H F u rfu ryl a lc o h o l C h lo ro -e th a n o l S o rb ic a c id W ith o u t s c a v e n g e r
0 .2
0 .8
Overall the present data reveal that the effect of DOM on the photocatalysis of DEET is not interfering with the OH radical pathway but rather with the e-pathway.
0 .6
C/C 0
0,000
-0,004
0 .4
0 .4 2-i-P rO H Furfuryl alcohol C hloro-ethanol S orbic acid W ithout scavenger
0 .2
0 .0
HA-radical 0,002
-0,002
0 .6
0 .0
DMPO-OH
Dark 3 min 7 min 12 min
0 .4
dx''/dH (A.u.)
C /C 0
0 .6
0
10
20
REFERENCES
30
40
50
(1) Konstantinou, I.K.; Albanis T.A. Appl.Catal.B: Environ. 2003, 42, 319-335 (2) Doll T.E.; Frimmel F. Water Res. 2005 39, 403–411. (3) Santoke, H.; Song H.; Cooper W.J.; Peake B.M. J. Hazard. Mater. 2012, 217-218, 382-390 (4) Giannakopoulos, E.; Drosos. M.; Deligiannakis, Y. J. Colloid Inter. Sci. 2009, 336, 59-66. (5) Grigoropoulou, G.; Christoforidis, K.C.; Louloudi, M.; Deligiannakis, Y. Langmuir 2007, 23, 10407-10418 (6) Vinodgopal, K.; Kamat, V.P. Environ. Sci. Technol.1992, 26, 1963-1966
60
Irra d ia tio n tim e (m in )
Figure 1. Degradation kinetics of DEET in the presence of (a) LHA, (b) HALP, (c) FA with the addition of various scavengers An extensive inhibition, around 80-85% in DEET degradation was occurred with the addition of isopropanol indicating the essential participation of HO• in the reaction mechanism in the presence of humic acids (LHA and HALP). When sorbic acid was added to the both photocatalytic systems in the presence a significant inhibitory effect was also observed. In contrast a partial inhibitory effect was observed with the addition of furfuryl alcohol and chloro-ethanol, indicating that 1O2 and e-aq did not participate significantly in the photocatalytic mechanism. In the presence of FA, the inhibitory effect of the scavengers on the degradation of DEET was shown to be in the following order: sorbic acid > chloroethanol
Acknowledgments: This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALIS. Investing in knowledge society through the European Social Fund.
330
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Water-soluble organic matter from different composts: Characterization and effects on maize seeds germination H. Monda*, V. Cozzolino, R. Spaccini, A. Piccolo
Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l'Ambiente, l'Agroalimentare ed i Nuovi Materiali (CERMANU), Università di Napoli “Federico II”, via Università 100, 80055 Portici, Italy. * Corresponding author e-mail: [email protected] Keywords: Compost water extracts; seed germination; 13C-CPMAS NMR; DRIFT; TGA Abstract: Water-soluble organic matter extracts (C.WOM) were obtained from an artichoke compost (C.Cyn,), a tomato/woodchips compost (C.C1) and a maize compost (C.Mais) and then tested on maize seeds germination bioassay. C.WOM was further characterized by spectroscopic and thermal analysis such as CPMAS−13C NMR, DRIFT-IR, thermal gravimetric analysis and differential scanning calorimetry. The germination percentage resulted generally decreased, while root and shoot length resulted increased in presence of Mais C.WOM, which showed the highest presence of hydrophilic moieties, whereas C.WOM from C1 and Cyn resulted more aromatic. These results confirm the role of organic matter in stimulating plant growth, in particular when the content of potentially bioavailable hydrophilic components is predominant as in the case of C.WOM. experiments were replicated 5 times and data obtained were statistically analyzed by one-way analysis of variance (ANOVA). CPMAS-NMR Spectroscopy of composts Fine-powdered samples were analyzed by solid-state NMR spectroscopy (13C CPMAS NMR) on a Bruker AV300 Spectrometer equipped with a 4 mm wide-bore MAS probe. NMR spectra were obtained by applying the following parameters: 13,000 Hz of rotor spin rate; 2 s of recycle time; 1 ms of contact time; 30 ms of acquisition time; 4000 scans. Samples were packed in 4 mm zirconium rotors with Kel-F caps. DRIFT IR Spectroscopy of C.WOM Diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) spectra of C.WOM samples were recorded with a PE Spectrum-One spectrometer, equipped with a diffuse reflectance accessory. Thermogravimetric analysis and differential scanning calorimetry of C.WE Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) curves were obtained by air combustion of each sample in a simultaneous thermal analyzer (STA 6000-Perkin Elmer). The initial and final temperatures were 30°C and 700°C, respectively, with an increasing temperature rate of 10°C min−1.
Introduction Naturally organic matter (NOM) is known to exert significant and direct influences on plant growth and metabolism, affecting morphological, physiological and biochemical processes (1). The application of natural organic products on agricultural crops is hence considered a promising innovative and valuable technology as sustainable management of agroecosystems. However, no unique or simple relationship has been found among the chemical structure of organic molecules and biological functionalities of agricultural crops. Therefore, a detailed molecular characterization is an essential requirement to elucidate the interaction of the intrinsic structural complexity of NOM with plant biochemical activity. Here we present the preliminary results of a research study focused on the effects of water-soluble fractions from three on farm composts, of different sources and composition, on the germination of maize seeds. Materials and methods Compost and water-soluble fractions The composts were selected on the basis of their mixture composition and identified as C.Mais (composted maize residues), C1 (composted tomato residues) and C.Cyn (composted artichoke residues). The water soluble organic matter extract (C.WOM) was obtained through the following extraction procedure: 100 g of each compost were suspended in 1000 ml of distilled water and mechanically shaken for 24 h. The suspension was centrifuged at 2500 rpm for 15 min and filtered through a 0.45 μm Whatman filter. Plant material and germination experiment Maize seeds (Zea mays L.) were placed in Petri dishes, and added with either 5 ml of distilled water (control) or different C.WOM fractions (1:10 v/v dilution). Seed germination was achieved in a growth chamber at 25°C in the dark setting humidity at 85%. All
Results and Discussion Effect of C.WOMs on maize seeds: The application of C.WOMs produced a decrease in the relative amounts of germinated seeds in respect to control, thereby suggesting a time lag effect on the germination rate (Fig. 1), which may be related to an adaptation to the more complex composition of nutrient solutions. Conversely, the primary root length of treated seeds increased significantly with the addition of water extracts of Mais and Cyn composts, meanwhile a decreasing effect was observed with C1 C.WOM treatment. A positive effects of Mais C.WOM was also revealed by the analysis of lateral
331
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
C Y N C .W O M
roots elongation, whereas a significant inhibition of lateral root emergency was measured on germinated plants following the application of C1 and Cyn C.WOMs. Finally, no major differences, between control and water compost extracts, were found for the shoot length development, except for Mais C.WOM which showed the best general performance (Fig. 1.a). st
1 Root nd
2 Root Shoot
length (cm)
6
(a)
%T
(b)
2
20
1
CNTR
CYN C1 Sample
MAIS
0
CNTR CYN C1 MAIS Sample
Figure 1. (a) Effect of water-soluble organic matter (C.WOM) on maize roots and shoots length. (b) Effect of
water-soluble organic matter (C.WOM) on maize seeds germination.
13C-CPMAS-NMR Spectra of compost: The 13C CPMAS NMR spectra of bulk composts (Fig.2) were characterized by strong signals in the O-alkyl-C (60110 ppm) region, revealing a final composition dominated by carbohydrates. The presence of different alkyl chains (0-45 ppm) derived mainly from various lipid compounds, plant waxes, and polyesters. The signal at 56 ppm and the broad bands around 116 and 130 ppm may be associated with lignin monomers as well as with condensed aromatic moieties. Finally, the broad signal at 174 ppm indicates the content of carbonyl groups in either aliphatic acids or amino acid moieties. The steady largest amount of polysaccharides shown by the NMR spectra of both C.C1 and C.Cyn samples, suggested a lower humification degree of these mature composts. Infrared Spectroscopy of C.WOM: A similar composition of structural functional groups was revealed by the DRIFT IR spectra of the three C.WOM samples (Fig. 3). The differences in the IR spectra of C.WOMs were mainly related to the stronger intensity of the peaks in the 1035-1104 cm-1 interval of water extract from C-Mais sample, thereby suggesting a larger release of hydrophylic bioavailable compounds in the C.WOM of Mais as compared with C.WOM from C1 and Cyn.
NMR Intensity (arb. un.)
25 00
2 00 0 c m -1
1 50 0
10 00
Thermal analysis of C.WOMs: The thermogravimetric diagrams of C.WOM samples showed a lower weight loss (-32%) for the water extract from Mais C.WOM. Conversely, a significant larger weight loss, around 45% and 60%, were found for C.WOM of C1 and Cyn respectively, thereby suggesting a more complex structural composition. In DSC curve of Mais-C.WOM, the presence of lowtemperature exothermic peak and the absence of the high-temperature signal indicated the predominant bio-labile structures in the water extract from maize compost. In fact, the first exothermic peak is currently assigned to the thermal degradation of carbohydrates and decarboxylation of carboxylic groups. On the contrary, the progressive larger intensity shown by the second exothermic signal for the DSC the C1-C.WOM and Cyn-C.WOM samples was mainly related to the thermal breakdown of complex aliphatic and aromatic moieties. Conclusion The bioactivity assays were positively influenced by the treatment with Maize C.WOM, while both C1 and Cyn C.WOM samples caused a general decrease in the development of maize seedling, when compared to control. A significant stimulation effect on shoot and roots elongation was observed after the application of C.WOM from maize compost, characterized by a large content of hydrophilic bio-labile components. These results are consistent with previous findings which highlighted the role of bioactive hydrophilic components of humic substances on biological activities of maize crops (1-3). In the present experiment the main hydrophobic character of maize compost may have promoted the preservation of stable bio-labile components (4), thereby allowing a larger release of active hydrophilic molecules in the water soluble extracts.
40
3
C .W O M
Figure 3. DRIFT IR spectra of C.WOMs
60
4
M A IS
3 00 0
80
5
0
100
% Percentage
8
7
C 1 C .W O M
REFERENCES (1)
Nardi S.; Muscolo A.; Vaccaro S.; Baiano S.; Spaccini R.; Piccolo A.; Soil. Biol. Biochem. 2007, 39, 31383146. (2) Dobbss, L.B.; Canellas, L.P.; Olivares, F.L.; Aguiar, N.O.; Peres, L.E.P.; Azevedo, M.; Spaccini, R.; Piccolo, A.; Façanha, A.R.J. Agric. Food Chem. 2010, 58, 3681-3688. (3) Vaccaro S.; Muscolo A.; Pizzeghello D.; Spaccini R.; Piccolo A.; Nardi S. J. Agric. Food Chem. 2009, 57, 10267-11276. (4) Spaccini, R.; Piccolo, A.; Conte, P.; Haberhauer, G.;Gerzabek, M.H. Soil. Biol. Biochem. 2002, 34, 1839-1851.
CYN C1 M A IS 200
150
100 [p p m ]
50
0
Figure 2. CPMAS−13C NMR spectra of compost
332
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Iron oxide-vermiculite associations affect the selective retention of organic matter M. Sodano, D. Said-Pullicino, A.F. Fiori, M. Catoni, M. Martin, L. Celi*
Soil Biogeochemistry, Dept. of Agricultural Forest and Food Sciences, University of Torino, Italy * Corresponding author e-mail: [email protected] Keywords: DOM, fulvic acids, ligand exchange, cation exchange, conformational rearrangement Abstract. Sorption of organic matter (OM) onto soil minerals affects OM dynamics as well as the identity, crystal growth rate, and surface properties of mineral phases. We aimed to understand mineral surface modifications brought about by different Fe (hydr)oxides-phyllosilicate associations and the implications on OM sorption. Paddy soilderived OM was used in sorption isotherms on mixed mineral phases obtained by precipitating different amounts of Fe (hydr)oxides on vermiculite. Results evidenced that the surface properties of vermiculite strongly drive Fe (hydr)oxide precipitation and consequently OM adsorption mechanisms. The change in surface charge with increasing Fe content resulted in a higher retention of OM. Adsorption isotherms of N-containing compounds and FT-IR carboxyl vibrational shifts revealed the occurrence of two adsorption mechanisms, one driven by electrostatic attraction of N-containing compounds by the negatively charged vermiculite surface, and another involving ligand exchange of aromatic compounds with the positive oxides precipitated in localized nucleation sites. Introduction The sorption of organic matter (OM) onto soil mineral phases is known to affect OM turnover, stabilization and loss on one hand, and the identity, crystal growth rate, and surface properties of mineral phases on the other. In particular poorly crystalline (hydr)oxides can show a high retention capacity due to their large surface reactive area and positive charge at pH<8-9. However, in soil these oxides are often present not as separated pools but associated or precipitated, coating other mineral particles, such as phyllosilicates. Different properties, such as electrical charge, specific surface area and porosity of phyllosilicates can therefore be modified by the association with (hydr)oxides (1; 2). The result is a complex system with a different reactivity towards organic matter, which depends on the kind of phyllosilicate involved (3; 4), the percentage of Fe coverage (2), the degree of interaction between the two minerals (association or precipitation) (5), and the chemical composition of the soil solution (6). The type and degree of (hydr)oxidephyllosilicate association are particularly dynamic in soils subjected to alternating redox conditions, and may thus play an important but still not well understood role in soil OM turnover (7). Our aim was to understand mineral surface modifications brought about by different Fe (hydr)oxides-phyllosilicate associations and the implications on OM sorption.
Experimental Four vermiculite-hydrous Fe oxides systems (FeVM) at different percentage of Fe (1.3, 2.8, 4.7, 5.6 mol Fe kg-1) were prepared by adding different volumes of 0.025 M Fe(NO3)3 at pH 2.0 to a suspension of VM. All mineral phases were characterized by X-ray diffraction, specific surface area (SSA) and electrical charge. In order to mimic all organic compounds that can be sorbed on Fe (hydr)oxides surfaces and released into the solution under anoxic conditions, two different OM pools, i.e. the water extractable organic matter (WEOM) and fulvic acids (FA), were extracted from the Ap horizon of a paddy soil (Haplic Gleysol), purified and pooled together (thereafter called DOM). Sorption isotherms were carried out at pH 5.5 and in 0.005M KCl. The different mineral phases were dispersed in KCl and then 5.0 mL of the suspensions were added to 25 ml of 0 to 180 mg C l-1 DOMcontaining solutions. The DOM-mineral systems were shaken for 16 hours at 20°C in the dark. The suspensions were then centrifuged and the amount of OM remained in the supernatant at the equilibrium was determined by elemental analysis. The pH, absorptivity coefficient, and Fe content were determined on the filtered supernatants before and after sorption. Electrophoretic mobility and infrared spectra were run on the suspensions before and after sorption. Results and Discussion Mineral phases: Vermiculite is a 2:1 phyllosilicate consisting of ideal tetrahedral and octahedral layers. The XRD spectra of Fe-VM systems showed an increase of the 6-line ferrihydrite peaks following Fe increase, which caused a variation of SSA from 20 to 38 m2 g-1 and electrical charge from -39 to + 8.0 mV.
333
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
and the positively charged oxides precipitated in localized nucleation sites. Moreover, the absorbance data at 254 nm indicated that selective adsorption of aromatic compounds was an important process for all (hydr)oxide-covered substrates, particularly at high C loadings, as a consequence of OM conformational rearrangements.
Figure 1. Sorption isotherms of DOM on vermiculite (VM) and Fe oxide-vermiculite (xFe-VM) systems at pH 5.5 in 0.005 M KCl. Linear lines represent Langmuir fits, while dash lines Sigmoidal fits.
Sorption isotherms and mechanisms: The amount of DOM adsorbed on VM was negligible up to Ce 52 mg C l-1 and then increased to reach a plateau at 10 mg C g-1 (Fig 1a). DOM adsorption was adequately described by a sigmoidal model. Conversely, in the presence of Fe (hydr)oxide, DOM showed a higher affinity for the surfaces since the lowest adsorbate concentrations even in the 1Fe-VM substrate. In the latter, DOM sorption reached a plateau at 25 mg g-1, although the data were not well described by both the sigmoidal and Langmuir models. On the following FeVM system, DOM sorption increased to a Qmax of 67 mg C g-1 while on the 3Fe-VM system (Fig 1b), Qmax was halved but both Langmuir models presented a higher fit compared to 1Fe-VM. The highest Fe containing system showed a lower adsorption and a minor affinity constant than 3Fe-VM. If adsorption isotherms were drawn considering the dissolved nitrogen fraction (TDN) only VM and 1FeVM showed a sigmoidal trend of the TDN adsorbed amount as a function of the TDN remaining into the solution at the equilibrium, suggesting electrostatic attraction of N containing compounds by the negatively charged vermiculite surface. The FT-IR spectra of DOM at pH 5.5 and the difference spectra [(xFe-VM)-DOM – (xFe-VM)] are reported in Fig. 2. Comparing the difference spectra with that of DOM we observed a shift of the vibrational bands relative to –C=O asymmetric (1629 cm-1) to lower frequencies (1588 cm-1), increasing with Fe content, while symmetric stretch (1396 cm-1) moved to 1410 cm-1. FT-IR carboxyl vibrational shifts revealed that ligand exchange became the dominant mechanism between the negative DOM compounds
Figure 2. Transmission Fourier transform infrared (FT-IR spectra of OM before adsorption (a) and the differential FTIR spectra of OM adsorbed on Fe oxide-vermiculite systems 1Fe-VM (b), 2Fe -VM (c), 3Fe-VM and 4Fe-VM (d).
REFERENCES
(1) R.M. Cornell, U. Schwertmann 1996. The Iron Oxides, VCH, Wheinheim. (2) A. Dimirkou, A. Ioannou & C. Kalliannou. 1996. Synthesis-identification of hematite and kaolinite hematite (k-h) system. Communications in Soil Science and Plant Analysis, 27, 1091-1106. (3) L. Celi, G. De Luca & E. Barberis. 2003. Effects of interaction of organic and inorganic P with ferrihydrite and kaolinite-iron oxide systems on iron release. Soil Science, 168, 479-488. (4) R. Celis, J. Cornejo & M.C. Hermosin. 1998. Textural properties of synthetic clay-ferrihydrite associations. Clay Minerals, 33, 395-407. (5) A.R. Saidy, R.J. Smernik, J.A. Baldock, K. Kaiser, J. Sanderman & L.M. Macdonald. 2012. Effects of clay mineralogy and hydrous iron oxides on labile organic carbon stabilisation. Geoderma, 173, 104-110. (6) A. Hanke, M. Sauerwein, K. Kaiser & K. Kalbitz. 2014. Does anoxic processing of dissolved organic matter affect organic-mineral interactions in paddy soils? Geoderma, 228229, 62-66. (7) Z. Karim. 1984. Characteristics of ferrihydrites formed by oxidation of FeCl2 solutions containing different amounts of silica. Clays & Clay Minerals, 32, 181-184.
334
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Molecular characteristics and bioactivity of humic acids isolated from vermicomposts D. Martinez-Balmoria, R. Spaccinib, N. O. Aguiarc, E. H. Novotnyd, F. L. Olivaresc, Luciano P. Canellas a* (a) Departamento de Química, Universidad Agraria de La Habana, San José de las Lajas, Cuba (b) CERMANU, Università di Napoli Federico II via Università 100, 80055 Portici (c) NUDIBA – Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av.Alberto Lamego, 2000, Campos dos Goytacazes, 28013-602 Rio de Janeiro, Brazil (d) Embrapa Solos, Rua Jardim Botânico, 1024, Rio de Janeiro 22460-000 Rio de Janeiro, Brazil * Corresponding author e-mail: [email protected] Keywords: vermicompost, humic acids, thermochemolysis, plant bioactivity Abstract Vermitechnology is an effective composting method which transforms different residual biomasses into nutrient rich organic fertilizer. In fact, the mature vermicompost is a renewed organic source suitable to provide humic substances with high biological activity. The chemical characteristics of humic acids isolated from different vermicomposts, produced with cattle manure, sugarcane bagasse, sunflower cake from seed oil extraction and filter cake from sugarcane factory, were accessed by thermochemolysis and 13C solid state NMR spectroscopy. More than 200 different molecules were found and were possible to identify chemical markers on humic acids according the nature of organic source. The large hydrophobic character of humic extracts and the preservation of altered lignins derivatives confer to HAs the ability to induce lateral root emergence in maize seedlings. thermochemolysis allow a useful qualitative and quantitative measurement of structural components and their relationship with biological activity (7). The aim of this study was to evaluate, by 13C- NMR and thermochemolysis, the molecular characteristics of humic acids isolated from five mature vermicomposts of different composition, as related to the bioactivity response in the lateral root emergence of maize seedlings.
Introduction Vermicomposting is the post-thermophilic biodegradation of agricultural biomasses through the interaction between earthworms and microorganisms. The positive effects produced by the application of mature vermicompost on plant development, it is related to, both, the large content and availability of biologically-active plant promoters represented by the hormone-like humic acids (HA) produced during vermicomposting (1; 2). In fact, the humic like organic matter isolated from vermicomposts showed an high biological activity (3) thereby acting as an effective plant growth promoter (4). HAs isolated from vermicompost effectively induced the synthesis of plasma membrane (PM) H+-ATPase in a typical auxinlike response, thereby significantly enhancing lateral root emergence (1; 2). However, the effectiveness of HA from vermicompost as bio-factors, strongly depend on the quality of raw organic biomasses and on the final molecular composition of humified organic compounds. Non-destructive spectroscopic methods such as the 13C Cross-Polarization Magic-Angle-Spinning Nuclear Magnetic Resonance (13C-NMR) is extensively used to identify content, distribution and biochemical modification of molecular components in natural organic substrates and compost materials. The plant physiological response, following the application of HS from different vermicomposts, has been previously related to the hydrophobic molecular characteristic of humic components, as measured by solid state NMR analysis (5; 6). Additional complementary molecular characterization of complex matrices may be obtained by the combination of NMR spectra with the structural information provided by thermally assisted hydrolysis and methylation reaction (thermochemolysis) followed by gas chromatography-mass spectrometry. The
Experimental The HS were isolated from five different vermicomposts prepared from the following substrates: cattle manure (A), cattle manure+sugar cane bagasse (1:1 w/w as dry mass) (B), cattle manure+sunflower cake (1:1 w/w as dry mass) (C), cattle manure+sugarcane bagasse+sunflower cake (1:1:1 w/w/w/ as dry mass) (D) and filter cake from sugarcane factory (E). The 13C CPMAS NMR spectra, thermochemolysis and bioactivity assay of HS were conducted as reported elsewhere (5; 7) Results and Discussion. Thermochemolysis of HS released more than 200 different molecules, which were identified as methyl ethers and esters of natural compounds. Most of humic components were from plant and microbial origin and were mainly represented by lignin derivatives, followed by alkyl biopolymers, nitrogenous compounds, terpenes and sterols products (Table 1). The 13C-NMR spectra of HS showed an overall composition dominated by lignin and lipid components and minor amount of polysaccharides and carbohydrates (Fig. 1).
335
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Table 1 Total yield (g g-1 d.w.) of main thermochemolysis products released from HAs
Lignin G6/G4 S6/S4 G Alkyl Sterol N-deriv
A 950 2,6 4,7 2,8 291 40 148
B 4271 1,6 2,7 0,6 1274 130 1618
C 2976 1,9 2,2 2,3 942 305 818
D 65 nd 4,7 0,8 228 212 49
sugarcane bagasse (Tab 1). However the most abundant aromatic monomers found in the pyrograms of HS-B were the 3-(4-methoxyphenyl)-2-propenoic and 3-(4,5dimethoxyphenyl)-2-propenoic derivatives, whose accounted for about the 40% of total lignin content. These molecules originate from either the side chain oxidation of lignin units but also from the aromatic domains of plant biopolymers, whose important relative contribution in herbaceous plant may had overrated the total content of lignin in the humic extracts from sugarcane. Moreover the higher values of lignin structural index found in the pyrograms of HS-A and E (Tab. 1), suggested the occurrence of wider an intense decomposition process of lignin biopolymers in humic fraction from cattle manure and filter cake. The most advanced humification may hence have promoted a preferential accumulation of small active aromatic fragments which should allow a more prompt physiological response as compared to partial undecomposed lignin residues (8). In addition to the content of specific molecules, also the ratio of hydrophobic to hydrophilic moieties is considered an important characteristic for the bioactivity of humic extracts. The role of humic hydrophobicity may be explained by the selective preservation, of active biofragments, which may be successively released by conformational changes of humic associations in solution (2; 5). In fact, plants treated with humic substances enhance exudation of organic acids, thereby leading to a modification of structural arrangement and to a release of active molecules in rhizosphere. Although the role of hydrophobic humic components needs further evaluation in order to gain a deeper insight of the structure-activity relationship, the association of detailed molecular characterization and bioactivity essays are unavoidable requirements for a more accurate and valuable utilization of humic material as plant-growth promoters and for a comprehensive understanding of the plant-SOM interaction.
E 870 2,1 2,7 0,4 239 118 409
HA-A HA B HA C HA D
HA E
Figure 1 13C CPMAS NMR spectra of vermicompost HAs
All the HS from mature vermicomposts showed an improvement from 36 to 135%, in the number of lateral roots, in respect to control plants (Fig. 2). The most hydrophobic humic acids isolated from cattle manure and filter cake biomasses, provided the best stimulation on lateral roots emergence, while the effect decreased in HA from sugarcane bagasse, sunflower cake and mixed materials, characterized by the progressive lowering of hydrophobic and aromatic molecular components (Table 2). HA-E HA-D HA-C
REFERENCES
(1) Canellas, L.P., Okorokova-Façanha, A., Olivares, F.L., Façanha, A.R., 2002. Plant Physiol. 130, 1951-1957 (2) Dobbss, L.B., Canellas, L.P., Olivares, F.L., Aguiar, N.O., Peres, L.E.P., Azevedo, M., Spaccini, R., Piccolo, A., Facanha, A.R., 2010 J. Agric. Food Chem. 58, 3681-3688 (3) Canellas, L.P., Dobbss, L.B., Oliveira, A.L., Chagas, J.G., Aguiar, N.O., Rumjanek, V.M., Novotny E.H., Olivares F.L., Spaccini R., Piccolo A., 2012. Eur. J. Soil Sci. 63, 315-324 (4) Canellas, L.P., Olivares, F.L. 2014.Chemical and Biological Technologies in Agriculture (5) Aguiar, N.O., Olivares, F.L., Novotny, E.H., Dobbss, L.B., Balmori, D.M., Santos-Júnior, L.G., Chagas, J.G., Façanha, A.R., Canellas, L.P., 2013. Plant Soil 362, 161-174 (6) Aguiar, N.O., Novotny, E.H., Oliveira, A.,L., Rumjanek, V.M., Olivares, F.L., Canellas, L.P., 2013b. J. Geochem. Expl. 129, 95–102. (7) Martinez-Balmori, D., Olivares, F.L., Spaccini, R., Aguiar K.P., Araújo, M.F., Aguiar, N.O., Guridi, F., Canellas, L.P., 2013. J. Anal. Appl. Pyrol. 104, 540-550 (8) Quaggiotti, S., Ruperti, B., Pizzeghello, D., Francioso, O., Tugnoli, V., Nardi, S., 2004. J. Exp. Bot. 55, 803-813.
HA-B HA-A
Figure 2. Increase of lateral root emergence in maize seedling treated with vermicompost HAs compared to control
These results are consistent with previous findings (3; 5; 7) where it was observed the humic fractions with larger hydrophobicity were able to provide a steady high bioactivity, the presence of aromatic lignins compounds being closely related with the ability of HA to induce lateral root emergence. Table 2 Hidrophobicity and aromaticity of vermicompost HAs as calculated from NMR chemical shift regions (2; 5) A B C D E HB 1.79 0.79 0.82 0.76 0.99 Ar 27.4 23.5 22.4 24.0 27.9
An apparent inconsistency with this hypothesis is represented by the results of thermochemolysis, which showed the largest lignin content in the HS from
336
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Organic Carbon sequestration in cultivated soils by in Situ Catalyzed Polymerization of Soil Organic Matter R. Spaccini (a) *, A. Piccolo(a)
(a) Centro Interdipartimentale per la Risonanza Magnetica Nucleare, Università di Napoli Federico II via Università 100, 80055 Portici * Corresponding author e-mail: [email protected] Keywords: Organic carbon sequestration, agricultural soils, humic molecules, biomimetic catalyst, in situ soil organic matter polymerization Abstract An innovative technology based on in situ Soil Organic Matter (SOM) photo-polymerization with biomimetic catalysis, was successfully employed for the attainment of Organic Carbon (OC) sequestration in three Italian agricultural sites. The analyses of, both, bulk soils and water stable aggregates revealed that the catalyst assisted oxidative coupling reaction of humic molecules, promoted an effective SOM stabilization and a stable OC incorporation in soil particle-sizes of each experimental field. After three year the OC fixation of SOM polymerization treatment, produced, as compared to control samples, an increase that ranged from 3 to 6 ton OC ha-1 in different soils, without exogenous OM addition. These results indicate that the agricultural application of current available innovative methodologies appear as a suitable SOM management practice for SOC sequestration, and may then become a reliable techniques to turn agricultural soils into OC sinks. photo-oxidative natural conditions, thereby significantly reducing C mineralization and CO2 emissions. Here we reports the outcomes of an Italian national project in which the innovative methods for SOC sequestration, based on in situ SOM photopolymerization, was extended at field level within common agricultural practices of crop production. We aimed to verify on three cultivated soils whether the new approach based on a catalyst-assisted photopolymerization reaction may promote the SOC sequestration through in situ SOM stabilization.
Introduction The acknowledged progressive decrease of SOM in European agricultural lands, accelerate the decline of soil fertility, thus also contributing to global CO2 emissions. These effects are even more emphasized in marginal areas of Mediterranean regions, which are characterized by low OM inputs and increased GHG emissions. The appraisal of forecasting analyses indicate that European agricultural soils could potentially store up to 19 Mt C year-1. An important role in SOC sequestration is assigned to humified organic matter which is the most abundant and persistent pool of SOM, and represents the principal potential sink of OC in the biosphere. A novel understanding of humus chemistry has shown that the stable soil humified pools are build up by natural heterogeneous biomolecules, self-assembled by weak interactions in supramolecular associations (1, 2). The supramolecular nature of SOM implies that the humic components may undergo to chemical stabilization by coupling them into larger molecularweight materials, thereby strengthening the intermolecular interactions and reduce the SOM mineralization process. Specific technologies based on biomimetic catalysts, such as biocompatible metal-porphyrins, that mimic the activity of oxidative enzymes, can be successfully employed in the catalysis of photo-oxidative coupling of humic phenolic derivatives which is expected to stabilize the SOM. An effective oligo-/poly-merization of humic aromatic components have been already achieved with the catalytic action of water-soluble ironporphyrin activated by either a chemical oxidant or solar radiations (3, 4). Preliminary laboratory experiments were carried out in order to test the effectiveness of biomimetic technology to improve the in situ SOM stabilization in complex multiphase systems such as rhizo-microcosms, soils and model systems (5, 6). The results laboratory tests revealed that soil addition with iron-porphyrin could stabilize the SOC, under
Experimental The field experiments were performed on three different agricultural soils, located in the University farms of Torino, Piacenza and Napoli (Tab. 1). The soil treatments were performed on 1 x 1 m field plots (n=4) cultivated with wheat (Triticum Durum) for three years, with the following managements: - FeP: ploughing at 35 cm depth, followed by surface harrowing with addition of mineral fertilizer and 10 kg ha-1 of a biomimetic catalyst; - Control: ploughing at 35 cm depth followed by surface harrowing with addition of mineral fertilizers. The synthesis of water-soluble iron–porphyrin (FeP) and soil fractionation into water stable aggregates were described elsewhere (7) Table 1. Textural composition (%) and TOC (g kg -1) content of soils from field experiments Field site Sand Silt Clay TOC Torino 36.9 56.2 6.9 12.5 Piacenza 17.9 47.1 35.0 17.2 Napoli 43.0 24.1 32.9 10.5 Results and Discussion After three year of soil addition with iron-porphyrin solution, the in situ polymerization treatment produced noticeable effects on the SOM stabilization in FeP plots, revealing an effective OC sequestration in all
337
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
experimental fields. A significative preservation of TOC was found in the catalyst-assisted OM photopolymerization treatment for the coarse textured soil of Torino (Fig .1). Even though no statistical difference was shown by Fe-P plots as compared to control samples, the occurrence of effective OC fixation was indicated by the stacked data of global OC of soil aggregates (Fig. 1). In fact, a strengthen association between organic components and soil particle sizes was revealed by the raised OC recovery found in Fe-P samples, which retained more than the 97 % of bulk TOC (Fig.1), with a net gain of 1.2 g kg-1 in OC amount respect to Control soil. t0
I year
II year
III year
gOC kg-1
14
biomimetic catalyst, revealed a final improvement of TOC preservation of 1.1g kg-1 with respect to conventional management practices. A peculiar differences among the experimental sites was represented by the variable time response to soil amendment with biomimetic catalyst. While a prompt effect on the in situ OM fixation, was obtained by the initial application of FeP treatments in the silt-sandy soil of Torino, an apparent time lag were found in the first soil amendment of biomimetic catalyst of, both, the silty-clay loam and clay loam soil of Piacenza and Napoli. The combination of low applied amounts and water solubility of metallo-porphyrin, with the larger clay content of heavier textured soils, may have favoured a strong interaction with the wide specific inner- and out-side surfaces of clay minerals and a consequent initial quenching inhibition of the catalytic activity (8). The progressive annual addition of amending iron-porphyrin solutions could have then counterbalanced the attenuation allowing an effective chemically assisted oxidative coupling reaction of organic components, also in clay rich soils. The data of, both, bulk samples and cumulative yields of soil aggregates for the FeP plots of Torino, revealed an overall steady SOM maintenance respect to Control samples. Conversely the results of Piacenza and Napoli, indicated that soil amendment with biomimetic catalyst was capable to significantly enhance the TOC content, as compared with starting initial conditions, thereby suggesting an effective incorporation of recent OC inputs from either plant roots, microbial activities or crop residues. The extension at farm scale of the in situ SOM stabilization treatment corresponded to an effective incorporation, of about 3, 5 and 6 ton OC ha-1, for the experimental sites of Torino, Piacenza and Napoli in the order, without exogenous organic matter addition. The feasible agricultural application of current available alternative methodologies appear as suitable SOM management practices, which may be also combined with the most promising conventional techniques (e.g. organic farming) and then liable to become a viable practice to turn agricultural soils into OC sinks
10 6 C
FeP
C
FeP
C
FeP
Figure 1. Time variation of TOC content in bulk soils (whole bars) and water stable aggregates (stacked bars) of soil treatments in Torino experimental field
A large OC preservation was shown by the soil samples added with iron porphyrin (18.4 g kg-1) in comparison with control (17.0 g kg-1), for the experimental field of Piacenza (Fig. 2). This trend was confirmed by the cumulative results of aggregate-size separates; in fact, the persistent larger global OC yield of particle sizes from FeP amended soil (+1.5 g kg-1), further indicated that the in situ OM polymerization promoted a stable incorporation of SOC in water stable aggregates.
gOC kg-1
20 t0
I year
II year
III year
14 8 C FeP C FeP C FeP Figure 2. Time variation of TOC content in bulk soils (whole bars) and water stable aggregates (stacked bars) of soil treatments in Piacenza experimental field
gOC kg-1
12
t0
I year
II year
REFERENCES
(1) Piccolo A (2002). Adv Agron 75:57–134 (2) Nebbioso A, Piccolo A (2011) Biomacromolecules 12: 1187–1199 (3) Šmejkalova, D., Piccolo, A. (2005). BioMacromolecules 6: 2120-2125 (4) Fontaine B, Piccolo A (2012) Environ Sci Pollut R 19(5): 1485-1493 (5) Piccolo A, Spaccini R, Nebbioso A, Mazzei P (2011) Environ Sci Technol 45: 6697–6702 (6) Nuzzo A, Piccolo A. (2013) BioMacromolecules 14: 1645-1652 (7) Spaccini R, Piccolo A (2012) In: Carbon Sequestration in Agricultural Soils; Piccolo, A., Ed.; Springer-Verlag, Heidelberg pp 61−105 (8) Nuzzo A, Piccolo A (2013). Mol Catal A: Chem 371: 8−14
III year
8 4
C FeP C FeP C FeP Figure 3. Time variation of TOC content in bulk soils
(whole bars) and water stable aggregates (stacked bars) of soil treatments in Napoli experimental field
The attainment of stable OM fixation with in situ polymerization treatment, was confirmed by the results of Napoli experimental field (Fig. 3). The TOC content found in bulk soil (11.6 g kg-1) and in the sum of fractions (11. 4 g kg-1) for soil amendments with
Acknowledgments: This work was supported by the national FISR-MIUR project: “MESCOSAGRSustainable Methods for Organic Carbon Sequestration in Agricultural soils”.
338
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Peat Structure Of The Northern Region Of Russia L. Parfenova(a), S. Selyanina(a, b), K. Bogolitsyn(a, b), M. Trufanova(a)*, A. Orlov(a), A. Tomson(c), V. Strigutskiy(c) E. Maltseva(d) (a) Institute of ecological problems of the North, UB of RAS, Arkhangelsk, Russia (b) Northern (Arctic) Federal University, Arkhangelsk, Russia (c) Institute for Nature Management NAS of Belarus, Minsk, Belarus (d) Institute of petroleum chemistry SB RAS, Tomsk, Russia * Corresponding author e-mail: [email protected] Keywords: peat, structure, nanoparticles Abstract: The structural organization of polymer peat matrix was studied by the means of the methods of light, atomic force microscopy and dynamic light scattering at different levels of dimensional hierarchy (macro-, micro-and nanolevels). It was established that individual macromolecules of humus biopolymers had globular structure and size about 3-10 nm. It seems that larger particles (5-100 nm) with elements of asymmetry have associative nature. Elemental composition of peat is C-43.9 %, O2-46.4 %, H-5.9 %, N-0.3 %, others elements - 3.5 %. Introduction According to modern concepts all natural objects are an ordered, structured, hierarchical system. Particularly the study of the structure of natural objects is necessary at different hierarchical levels, revealing of relationship between structure and properties of fundamental and applied nature. Peat is a unique natural renewable organic material. Its physic-mechanical properties, chemical properties are connected with the structure characteristics. So the purpose of the research is to study the structural organization of the polymer matrix of peat and its components at different levels of dimensional hierarchy.
peat has loose structure, where little decomposed moss fragments are in contact with a small amount of humus (Figure 2). The cells of micro-structure of polymer peat matrix are filled with particles aggregate nature, formed on the base of humus substances and carbohydrate complex of peat. Microstructure describes the internal structure of aggregates (associates), which is usually referred to coagulation, i.e. movable highly elastic structures. The interaction between the elements of the units is carried out by means of molecules and water layers, mainly due to the hydrogen bonds (2).
Experimental Samples of peat were taken in the summer lowwater period in 2009-2013 to the North-West of Russia (Ilas marsh array at weather station Brusovitca) by the method of drilling. By their native state peat is the polydisperse, hetero-porous system having macro - and microstructure. Structural organization of peat was examined by light microscopy using laboratory microscope Axio Scope A1 Zeiss complete with digital camera Canon G10. Editing of images was made by using the licensed software AxioVison Rel.4.8. The study of particle sizes of humus nature biopolymers at the nanoscale was done using the atomic force microscope Multimod 8 Bruker and device of particle size Horiba LB 550. Background status of microelements peat was evaluated by x-ray fluorescence spectroscopy (fundamental parameter method).
-10 10 30 50 70 Figure 1 - Cut peat deposits of Ilas bog By the means of atomic force microscopy the images were taken that allowed to fix the size of nanoparticles of individual macromolecules of aromatic biopolymers and supramolecular aggregates (Figure 3). Globular nature of individual macromolecules is stated. Their dimensions do not exceed 10 nm, which are comparable with the sizes of nanoparticles of other biopolymers of lignohumic nature. Larger particles (5-100 nm) with elements of asymmetry, evidently, have associative nature.
Results and Discussion Macrostructure of a polymeric peat matrix is a flexible framework formed by weaves of fibrous residues of peat-producing plants (1). Peat macrostructure (visible to the naked eye) depends on the dynamics of peat accumulation. Sphagnum type of peat with a small and medium degree of decomposition is the most widespread in the Northern wetlands. This peat macrostructure is spongy with the transition to the folded with the increase of peat deposit depth (Figure 1). Next pictures show that the
339
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
peat soils of the North have less nitrogen and carbon (C-43.9 %, O2-46.4 %, H-5.9 %, N-0.3 %, others elements - 3.5 %).
а)
Conclusions The structural organization of polymer peat matrix are studied by the means of the methods of light, atomic force microscopy and dynamic light scattering at different levels of dimensional hierarchy (macro-, micro-and nanolevels). Macrostructure of a polymeric peat matrix is loose and depend on peatproducing plants. Microstructure has aggregative nature. Supramolecular particles are characterized by asymmetry elements. Supramolecular particles in solutions are in dynamic equilibrium with separate globular macromolecules of 3-10 nm size. This is comparable with the size of nanoparticles of other biopolymers of lignohumic nature. Elemental composition of northern peat has less nitrogen and carbon, then Siberia peat.
b)
c) d) Figure 2 - Micrographic images: a) sphagnum; peat depth: b) 0-20 sm; c) 20-50 sm; d) 50-70 sm
REFERENCES (1) Lishtvan, I.I., Basin, E.T., Gamayunov, I.I., Terentiyev, A.A., 1989, 304 p. (2) Lishtvan, I.I., Basin E.T., Kosov V.I., 1985, 240 p. (3) Inisheva T.I., Ecology, 2002. № 4. P. 261266. Acknowledgments: The authors are grateful to M. Surso (IEPS UB RAS) for pictures made by method of light microscopy and D. Chukhchin (NArFU) for pictures made by method of atomic force microscopy.
Figure 3 - AFM image of the nanoparticles of biopolymers of humus nature of peat.
Research carried out with the support of the Program of interregional and interdepartmental basic research of RAS (project no 12-With-5-1017), the Russian Foundation for basic research (RFBR project no 1203-90018-Bel_a), Program of oriented basic research of RAS 12-5-3-008-ARCTIC, Programs of the Presidium RAS № 4 (project no 12-P-5-1021) using the equipment of the CCU SE "Arctic" (NArFU) and CCU CT RF Arctic (IEPN, IFNA UB RAS)
As a component of wetland ecosystems peat is an active participant in the carbon cycle and its significant reservoir. Studying the elements of carbon balance in wetland ecosystems due to possible climate changes is especially topical problem in the Euro-Arctic region, where the amount of wetland is very high (80 %). However, the carbon balance of forest and peat-bog ecosystems of the North of Russia was a little studied. The study of the elemental composition of peat soils showed that in comparison with elemental composition of Siberia peat (3) the
340
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
About The Feature Of Peat Composition Formed In The Northern Environment S. Selyanina(a, b)*, L. Parfenova(a), K. Bogolitsyn (a, b), M. Trufanova(a), A. Tsyganov(c), T. Sokolova(c), V. Pekhtereva(c), M. Bogdanov(b). (a) Institute of ecological problems of the North, UB of RAS, Arkhangelsk, Russia (b) Northern (Arctic) Federal University, Arkhangelsk, Russia (c) Institute for Nature Management NAS of Belarus, Minsk, Belarus * Corresponding author e-mail: [email protected] Keywords: peat, extractives, “green” solvents, bitumen. Abstract: Peat composition of Northern territories of Russia was studied. The report presents comparative characteristics of bitumen extraction by organic solvents from peat of North of Russia, Western Siberia and Byelorussia. Extractives were studied by the methods of gas-liquid chromatography and chromato-mass spectrometry. The hypothesis about the influence of climatic conditions on the process of peat accumulation was experimentally confirmed. It was shown that the difference of Arctic and Subarctic peat from Western Siberia and Byelorussia peat is in a low content of bitumen and a smaller variety of individual low-molecular compounds. The peat formed in conditions of the European North of Russia, is the most universal material for extraction of “green” solvents and implementation of the calorific value of peat, due to its low ash content. Introduction Peat deposits are the main ecosystems in the Arctic and Subarctic region. More than 20 % of Russian reserves of peat are concentrated in this region. Formation process of peat, its structure, physical, physical-chemical and chemical properties of its components are studied insufficiently. It is important, because the peat chemistry researches are aimed for the creation and verification of new directions of peat technologies and equipment for production of different types of the peat products. Resource potential of the peat directly depends on its composition, influencing on the direction of peat processing. For example, on the basis of peat humus substances various drugs has been developed; they are used as fertilizers and plant growth regulators, fodder, biologically active additives (BAA), production bioelectronic materials. Peat extractives are multicomponent mixture of alcohols, esters, fatty acids, triterpenoids, carbohydrates and other biologically active components. They are used like extracts of plants in cosmetics and medicine. In addition, the well known way of peat processing is its usage as an energy source.
Results and Discussion Figure 1 shows the dependence of the maintenance of various groups of components in peat on the depth of deposits. There is a zone of fluctuations in groundwater levels - depth up to 50 cm. In this horizon the decrease of ash and hydroscopic properties, and increasing of the Klason lignin content (neutralized by acids) is seen. But the content of bitumen is smoothly increases almost in 2 times at the studied depth. This is due to, apparently, the process of biological degradation of high-molecular components of the peat polymer matrix, proceeding with the formation of low-molecular bitumen.
Experimental Natural material was collected during expedition, described by standard facilities, degree of decomposition, the botanical composition of peat forming plants. Peat samples were of the same type according to the degree of decomposition, the depth and the absence of direct anthropogenic and technogenic effects. Samples of peat were taken of various depths of deposit. Extractive substances were isolated from peat samples by various organic solvents using the method of reflux distillation with infusion. Extractives were studied by means of the methods of gas-liquid chromatography and chromatomass spectrometry. Composition of peat samples presented by the content of the arches, extractives, Klason lignin, humus biopolymers, humin, and compound of peat bitumen was studied.
Figure 1 - Dependence of the peat composition from depths of deposit 1 – ash content; 2 – bitumen content; 3 - moisture content; 4 – lignin content; 5 – humus content. Comparative characteristics of bitumen extraction by various organic solvents from peat of the North of Russia, Western Siberia and Byelorussia are presented. The study of extraction process by organic solvents (Figure 2) shows that the fullest possible extraction of bitumen from northern peat is performed by method of extraction by ethoxyethanol (method of reflux distillation with infusion). There
341
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
were samples of the same type according to the degree of decomposition, the depth and the absence of direct anthropogenic and technogenic effects. The hypothesis about the influence of climatic conditions on the process of peat accumulation was experimentally confirmed. It was shown that the difference of Northern peat from Western Siberia and Byelorussia peat is in a low content of bitumen and a smaller variety of individual low-molecular compounds. a)
b) Figure 3 - The abundance of compounds with different number of carbon atoms in the hydrocarbon chain in the fractions: a) unsaturated fatty acids, b) saturated fatty acids, isolated from peat extracts: 1 - Nothern, 2 Belarus, 3 - Western Siberia
Рисунок 2 - – Influence of organic solvent on the extractives output а) at temperature 25 С; b) at temperature of boiling solvents: 1 - ethanol; 2 hexane; 3 - etoxite; 4 - ethyl acetate; 5 – tetrachlorethilene The research of composition of free and bounded acids (Figure 3) shows that they are represented mainly by monobasic saturated aliphatic acids (C10C26); their contents is 83-92 %. Unsaturated acids: quantified oleic, tetracosenic and dibasic azelaic have been identified. Saturated aliphatic acids are prevalent in the peat extract of Western Siberian and Byelorussian peats (1, 2). When comparing the composition of the peat extracts the high content of saturated compounds in combined acids in the peat from Subarctic zone with prevalence of hexadecanoic acid (C16) among them has been observed. Alcohols with normal structure (C14-C27), tocopherol, stigmasterol and sitosterol, and aliphatic hydrocarbons (C15-C29) were identified in the neutral fraction. It should be noted that 36 compounds in the neutral fraction of northern Russia and 47-49 compounds in the neutral faction of Siberian and Belorussian peat were identified. New data about peat resource potential of the northern territories of Russia have been obtained from the point of view of extraction by the promising “green” solvents: high-molecular humus, biologically active low-molecular compounds (waxes, resins). It was established that the structure of peat polymer matrix is stable macro-component formation with humus compounds and lignin in its composition.
Conclusions The peat formed in conditions in the Northern Russia environment is the most universal material for extraction of “green” solvents and implementation of the calorific value of peat, due to its low ash content. REFERENCES (1) Bel'kevich P.I., Golovanov N.G., Dolidovich E.F. Nauka i tehnika, 1989. 127 p. (2) Shinkeeva N.A., Maslov S.G., Arhipov V.S. Vestnik TGPU, 2009, V.3 (81). P.116-119. Acknowledgments: Research was carried out with the support of the Program of interregional and interdepartmental basic research of RAS (project № 12-With-5-1017), the Russian Foundation for basic research (RFBR project № 12-05-90011-Bel_a), Program of oriented basic research of RAS 12-5-3-008-ARCTIC, Programs of the Presidium of RAS № 4 (project № 12-P-5-1021) by means of the equipment of the CCU SE "Arctic" (NArFU) and CCU CT RF Arctic (IEPN, IFNA UB RAS).
342
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Study of humic substances from some Amazon regions assessed by 3D fluorescence spectroscopy and PARAFAC Cleber Hilário dos Santos,(a,b) Gustavo Nicolodelli(a), Renan Arnon Romano(a,c), Amanda Maria Tadini(a,b), Célia Regina Montes (d), Stephane Mounier(e), Débora Marcondes Bastos Pereira Milori(a)* (a)
* Embrapa Instrumentação, P.O. Box 741, 13560-970, São Carlos, SP, Brazil Instituto de Química de São Carlos, Universidade de São Paulo, P.O. Box 780, 13560-970, São Carlos, SP, Brazil (c) Instituto de Física de São Carlos, Universidade de São Paulo, P.O. Box 369, 13560-970, São Carlos, SP, Brazil (d) NUPEGEL, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, P.O. Box 09, 13418-900, Piracicaba, SP, Brazil (e) Université de Toulon, PROTEE, EA 3819, 83957 La Garde, France * Corresponding author e-mail: [email protected] (b)
Keywords: Humic acid; three-dimensional fluorescence; fluorophores; PARAFAC. Abstract The Amazon rainforest has the most biodiversity in the world and represents a huge carbon reservoir, both in the vegetation and inside the soil. The understanding of the carbon cycle in this biome is very important, especially on issues addressing climate change. In the present paper, humic acid from a toposequence of an Oxisol-Spodosol system associated with kaolin was studied using fluorescence emission-excitation matrix (EEM) combined with parallel factor analysis (PARAFAC). The method allowed us to identify two fluorophores with CORCONDIA 84.2%. The results for the Humiluvic Spodosol seem to corroborate the model of the supramolecular structure of humic acid, because the ratio between the fluorophores changes along the profile. Introduction Soil organic matter (OM) is a key component in the quality and sustainability of soil. It is formed by organic fractions with different lability. The study of the vulnerability of soil organic carbon due to anthropogenic activities and/or climate change is important for building simulation models to predict future scenarios and to assist in taking remedial environmental decisions. Humic substances (HS) are OM that is highly decomposed and the most stable carbon type in the soil. Spectroscopic techniques are usually used to study the chemical structure of humic substances. The Amazon rainforest has a large and dynamic carbon pool, but this carbon can be released to the atmosphere due to deforestation, non-conservative land use, and climate change. In the Amazon, the relationships between soil carbon stocks and carbon in natural vegetation are poorly understood. Furthermore, the permanence of carbon in the forest soil is greatly influenced by the soil type and associated vegetation. The Amazon has a wide area of Spodosols characterized by thick sandy horizons overlying clayey horizons often associated with Oxisols in Oxisols-Spodosols systems (1). In the literature, there is a consensus that in Spodosols the organic matter on the soil surface is degraded and translocated along the profile, accumulating itself in deeper soil layers. However, the OM stored at depth in this kind of soil is still little studied. This study aimed to characterize humic acids obtained from the Oxisol-Spodosol system employing fluorescence spectroscopy. Due to the complexity of the chemical structure of humic acids, it is usually
very difficult to extract quantitative information from fluorescence emission spectra. For this reason, excitation-emission matrix (EEM) fluorescence spectroscopy, combined with parallel factor analysis (PARAFAC) (2), was chosen for this study. Experimental The samples were collected in São Gabriel da Cachoeira, Amazonas state, Brazil. The soil profile analysed is a Humiluvic Spodosol. The soil sampling was done at depths according to the soil horizons until reaching rock. The profile shows the following sequence of horizons: A1 and A2 (surface organomineral); E (sand horizon), Bh and Bhs (intermediate organo-mineral horizons); T (transition between soil and rock); K1 and K2 (lower set of layers of kaolin with infiltrations of OM). Altogether 8 samples were collected. Soil samples were dried at room temperature, sieved to remove roots (2.0 mm), ground and sieved again (106 µm) in order to obtain homogeneous samples. HA was extracted from these soil samples using the method recommended by the International Humic Substances Society (IHSS). Humic acid solutions were prepared using NaHCO3 0.05 mol L-1. The ideal concentration for each HA sample was determined by UV-vis spectroscopy. For pH around 8.0, absorption at 254 nm was kept lower than 0.1. This procedure is necessary to mitigate molecular interactions (inner filter effect) (2). A UV-vis spectrometer SHIMADZU (model UV-1601PC) was used. Fluorescence spectra were acquired at excitationemission mode (EEM) using a Perkin Elmer Luminescence Spectrometer model LS-50B. The scan
343
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
ranges were 240 to 700 nm for emission and 220–510 nm for excitation. The spectra were obtained with a 290 nm filter and an excitation increment of 10 nm, totalling 30 scans. PARAFAC was used to extract information from the data obtained by classical fluorescence spectroscopy EEM, allowing us to identify the contribution of the intensities of the most representative fluorophores.
The contributions of the fluorophores in each horizon are presented in Figure 3. The results show that the chemical structure of HA changed along the profile. For superficial and spodic horizon (Bhs), the HA structure has a greater contribution of Fluorophore 2. From the transition horizon (Tr), the contribution of Fluorophore 1 increases and becomes greater than Fluorophore 2. Various models proposed in the literature that try to explain the structure of humic substances, highlight the macromolecular model (5) in which the molecular components of HS are produced by secondary synthesis reactions of degradation products and the fragments formed by macromolecular aggregates are connected by strong covalent bonds. A new vision has resulted in the development of an HS model, which is composed of supramolecular aggregates of degradation products coming together by entropic interactions and non-covalent bonds (6). If we consider the macromolecular model for humic acid, the ratio between the different fluorophores of a molecule should be kept constant along the soil profile. However, in our results, this was not observed. Indeed, a sharp reversal occurs after the transition layer. Thus, it seems that structures identified as Fluorophore 1 more readily cross all the soil profile and tend to accumulate on the transition horizon and in the kaolin pores.
Results and Discussion Figure 1 shows two examples of the EEM fluorescence spectra obtained for HA.
Excitation (nm)
500
(a) A1
0
450
4,6
400
9,2
350
14
300
18
250 300 350 400 450 500 550 600 650 700
Emission (nm)
(b) K2
0
450
25,0
400
50,0
350
75,0
300
100
7
250 300 350 400 450 500 550 600 650 700
Intensity of each fluorophore (a.u.)
Excitation (nm)
500
23
120
Emission (nm)
Figure 1. Total fluorescence spectra in EEM mode obtained
for the HA samples (concentration 10 mg L-1, pH 8.0) from the horizons (a) A1, (b) K2 of Humiluvic Spodosol.
(a)
Humiluvic Spodosol Fluorophore 1 Fluorophore 2
6 5 4 3 2 1 0 A1
A2
Bh
Bhs
Tr
K1
K2
Horizon/Layer
The core consistency diagnostic (CORCONDIA) is an effective tool for determining the appropriate number of components in PARAFAC models. For the proposed model using two components, the CORCONDIA was around 84.2%. Figure 2 shows the two fluorophores identified using PARAFAC (2).
Figure 3. Contributions to the fluorescence of Fluorophores 1 and 2 of the HA extracted from Humiluvic Spodosol in sampling profiles.
REFERENCES
(1) Lucas, Y.; Montes, C.R.; Mounier, S.; Cazalet, M.L.; Ishida, D.; Achard, R.; Garnier, C.; Melfi, A.J. Biogeochemistry of an Amazonian podzol-ferralsol soil system with white kaolin. Biogeosciences 2012, 9, 37053720, 2012. (2) Luciani, X.; Mounier, S.; Paraquetti, H.H.M.; Redon, R.; Lucas, Y.; Bois, A.; Lacerda, L.D.; Raynaud, A.; Ripert, A. Mar. Environ. Res. 2008, 2, 148-157. (3) Milori, D.M.B.P.; Martin-Neto, L.; Bayer, C.; Mielniczuk, J.; Bagnato, V.S. Humification degree of soil humic acids determined by fluorescence spectroscopy. Soil Science 2002, 167, 739-749. (4) Stevenson, F.J.J. Humus Chemistry. New York: Wiley Interscience 1994, 443. (5) Schulten, H.R.; Schnitzer, M. Soil Sci. 1997, 162, 115-130. (6) Piccolo, A. Soil Sci. 2001, 166, 810-832.
Figure 2. Fluorophores 1 and 2 obtained by PARAFAC
method and descriptive of the three-dimensional fluorescence (EEM) of the HA extracted from Humiluvic Spodosol and Yellow Oxisol.
Acknowledgments The authors thank FAPESP, CNPq, CAPES and EMBRAPA for their financial support of this study.
Based on the spectral profile shown in Figure 2, Fluorophore 1 seems to be associated with more complex and more humified HA (3,4).
344
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The effect of humic acid on the removal of pharmaceuticals from aqueous solutions George Z. Kyzas 1, Dimitrios N. Bikiaris 1, Dimitra A. Lambropoulou2,* Laboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki GR 54124, Greece 2 Laboratory of Environmental Pollution Control, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki GR 54124, Greece * E-mail address of presenting author: [email protected]
1
ABSTRACT SUMMARY: A modified chitosan (grafted with sulfonate groups and cross-linked with glutaraldehyde) was synthesized in order to examine the effect of humic acid (HA) on the adsorption equilibrium of a pharmaceutical compound. The results show that increasing the concentration of HA, the maximum adsorption capacity decreases.
was then mixed with a complex of chlorosulfonic aciddimethylformamide and stirred for 1 h in a water bath at 50 °C. The reaction mixture was then diluted by a small quantity of deionized water, filtered, and precipitated by pouring into ethanol 95% (400 mL). The precipitate was dissolved in deionized water, neutralized by a saturated Na2CO3 solution, and dialyzed against deionized water. After dialysis, the product was dried and stored in a desiccator. Then, a cross-linking procedure was realized with GLA as reagent (0.5 wt%) at 60 °C for 1.5 h. The final grafting degree (GD) was determined on the basis of the percentage weight increase of the final product relative to the initial weight of chitosan GD = (W 2-W1)/W1 (where W1 and W2 denote the weight of chitosan before and after grafting reaction, respectively). So, the grafting degree was found 2.2. Adsorption/desorption experiments were conducted in 20-mL amber vials using a batch approach. All experiments were run in duplicate. The residual concentration of PRM was measured spectrophotometrically by monitoring its UV absorbance at 263 nm (model U-2000, Hitachi). A detailed description of experimental procedure is given below, where [PRM]0 (mg/L) is the initial PRM concentration, pH is the pH of the aqueous solutions (fixed with micro-additions of HCl or NaOH), T (°C) is the temperature, m (g) is the mass of the adsorbent used, V (mL) is the volume of adsorbate, N (revolutions or full rotations per minute, abbreviated as rpm ) is the agitation rate of the shaking machine and t (h) is the contact time. For all experiments three different values of HA concentrations (2.5, 5.0, 20 mg/L) were selected to investigate their influence to adsorption of PRM onto CsSLF. Effect of initial PRM concentration: [PRM]0=0500 mg/L; pH=10 (optimum value found from (i)); m=0.02 g; V=20 mL; T=25 °C; N=160 rpm; t=24 h. The equilibrium data resulted were fitted to the Langmuir-Freundlich (L-F) isotherm model [3]:
INTRODUCTION: Pharmaceuticals are of scientific and public concern as newly recognized classes of environmental pollutants and are receiving considerable attention with respect to their environmental fate and toxicological properties over the last 15 years. This problem is directly linked with the existence in waters/wastewaters of humic compounds and especially humic acids (HA). Humic acid is a principal component of humic substances, which are the major organic constituents of soil (humus), peat, coal, many upland streams, dystrophic lakes, and ocean water. It is produced by biodegradation of dead organic matter. It is not a single acid; rather, it is a complex mixture of many different acids containing carboxyl and phenolate groups so that the mixture behaves functionally as a dibasic acid or, occasionally, as a tribasic acid. The presence of humic acid in water intended for potable or industrial use can have a significant impact on the treatability of that water and the success of chemical disinfection processes. Therefore, industrial effluents are even mistreatable. In this study, a chitosan derivative (poly-β-(14)-2amino-2-deoxy-D-glucose) grafted with sulfonate groups was synthesized and tested as adsorbent for the removal of a particular pharmaceutical compound; Pramipexole dihydrochloride ((6S)-N6-propyl-4,5,6,7tetrahydro-1,3-benzothiazole-2,6-diamine, PRM) is used widely all over the world for its unique pharmaceutical activity and on the basis of recent drug usage trends [1]; therefore, treatment of wastewater by high polluted levels of PRM is required and urgent needed. The novelty of this study is based on the coexistence of humic acids on the adsorbate (PRM) in various concentrations. Which is the effect of humic acids on the adsorbent use of chitosan derivatives? How did humic acids influence the crucial parameters of adsorption (isotherm etc)? The latter are some of crucial questions replied with this study.
Qe=
Q m bC e1/n 1+ bCe 1/n
where Qe (adsorbed PRM weight/adsorbent weight) is the equilibrium concentration in the solid phase; Qm is the maximum amount of adsorption (adsorbed PRM weight/adsorbent weight); b is the L-F constant; n is the L-F heterogeneity constant. The adsorption capacity in equilibrium (Qe) was calculated using the mass balance equation: Q = V
EXPERIMENTAL METHODS: For the synthesis of sulfonate-grafted chitosan adsorbent (CsSLF) [2], a mixture of dichloroacetic acid (5 mL) and formamide (50 mL) was added into chitosan (4.0 g) and stirred to be an homogenized solution. This
e
C0
Ce
m
where C0 and Ce (PRM weight/liquid volume) are the initial and equilibrium PRM concentrations in the liquid phase, respectively.
345
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
RESULTS AND DISCUSSION: The effect of grafting reactions to the physical structure and the appearance of chitosan can be observed from SEM images. All derivatives of chitosan (Cs, CsSLF) had an irregular shape owing to the grinding. The nearly total smooth surface of Cs (Fig. 1a) was changed to CsSLF. It is readily observed that the drying method caused the collapse of any porous microstructure of the particles. This is possibly due to hydrophilic interactions between the water molecules and the carboxyl, hydroxyl and amino groups on the macromolecular chains of the prepared materials. According to BET analysis, the surface area of CsSLF was 2.9±0.3 m2/g, while the non-grafted derivative (Cs) had only 0.9±0.2 m2/g. The above values belong to the typical ones of non-porous materials, as bibliographically chitosan is characterized. In the field of swelling, CsSLF showed swelling ~300% at pH=10. The above percentages are commonly observed in chitosan adsorbents, given the powdered– nature of the adsorbents and the single cross-linking method with GLA followed in the current study (and not dual cross-linking with GLA and some ionic reagent as sodium tripolyphosphate).
150
L-F fitting
Cs
Qe (mg/g)
120
90
60 HA: 0 mg/L HA: 2.5 mg/L HA: 5.0 mg/L HA: 20.0 mg/L
30
0
0
100
200
300
400
500
Ce (mg/L)
Fig.2: Isotherms of Cs under various concentrations of HA. 240 L-F fitting
CsSLF
210
Q e (mg/g)
180 150 120 90 60
HA: 0 mg/L HA: 2.5 mg/L HA: 5.0 mg/L HA: 20.0 mg/L
30 0
0
100
200
300
400
500
Ce (mg/L)
Fig.3:Isotherms of CsSLF under various concentrations of HA
It is obvious that the phenomenon is antagonistic, but the Qm of the chitosan derivative remains high. COO- H2 N
S
+
CH3
+ COO- H3 N
N
GLA
(a) (b) Fig. 1: SEM images of (a) Cs, and (b) CsSLF.
-
O3SO
O
Based on previous study, the optimum pH for the system Cs (or CsSLF) – PRM was alkaline (10). Therefore, all isotherm tests were carried out at pH=10. The most crucial parameter of each adsorbent-adsorbate system is the adsorption isotherms. The maximum theoretical adsorption capacity shows how suitable is a material for the pollutant studied. Fig. 2 shows the effect of the presence of humic acid on the adsorption isotherm of Cs. The experimental were fitted to L-F fitting. Based on L-F theoretical calculation, Qm was 181 mg/g without existence of HA in the adsorbate solution (CHA = 0 mg/L). However, increasing the HA concentration to 2.5 mg/L, a decrease was observed for the maximum adsorption capacity of Cs (Q m = 151 mg/g), which corresponds to 17% reduction. Increasing the HA concentration to 5.0 and 20.0 mg/L, a sharp decrease was observed for the maximum adsorption capacity of Cs (CHA = 5.0 mg/L: Q m = 74 mg/g, ΔQm = 51%; CHA = 20.0 mg/L: Q m = 50 mg/g, ΔQm = 33%). As it was shown, the after a gradual decrease in the cased of 2.5 mg/L HA, the next reduction was very intense, implying complex antagonistic interactions between HA and PRM for the adsorption onto Cs. Similar observations were taken for the case of CsSLF. (CHA = 0.0 mg/L: Q m = 339 mg/g; CHA = 2.5 mg/L: Qm = 252 mg/g, ΔQm = 25%; CHA = 5.0 mg/L: Q m = 146 mg/g, ΔQm = 43%; CHA = 20.0 mg/L: Q m = 134 mg/g, ΔQm = 8%).
OSO3 H2C O -
O
H2C OSO3-
S
COO- +H3 N
O3SO
-
O
GLA H2+-OOC N CH3
n
N
Fig.4: Antagonistic adsorption of PRM versus HA for adsorption onto CsSLF.
CONCLUSIONS: Chitosan grafted with sulfonate groups and cross-linked with glutaraldehyde was synthesized in order to examine the effect of humic acid (HA) on the adsorption equilibrium of a pharmaceutical compound. The results show that increasing the concentration of HA, Qm decreases. The higher reduction was for CsSLF: (CHA = 2.5 mg/L: ΔQm = 25%; CHA = 5.0 mg/L: ΔQm = 43%; CHA = 20.0 mg/L: ΔQm = 8%). REFERENCES:
[1] S.A. Hollingworth, et al., Pharmacoepidemiology and Drug Safety, 20 (2011) 450-456. [2] J. Miao, et al., Desalination, 181 (2005) 173-183. [3] C. Tien, Adsorption Calculations and Modeling, ButterworthHeinemann, Boston, U.S.A., 1994.
Acknowledgments
The support for this study was received from the Greek Ministry of Education (GGET) and through the research program “Excellence II (ESPA2007-2013/EPAN II/Action “ARISTEIA II)” under the title “Advanced microextraction approaches based on novel nanopolymers to measure pharmaceuticals, personal care products and their transformation products in the aquatic environment” (project acronym: PoL-PPCPs-TPs), which is gratefully appreciated.
346
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Effect of humic substances on the quality of essential oils of medicinal plants Alireza. Sardashti 1*, Amin. Ganjali 2 and Ali. Kordi 3 1,2,3 Department of Chemistry, faculty of Science, University of Sistan and Baluchestan, P.O. box 98135-167, Zahedan,Iran *Correspodence author: [email protected] Keywords: medicinal plant; humic substances; root; quality of essential oil Abstract Environmental factors cause changes in growth of medicinal plants as well as the quantity and quality of their essential oil composition. About the plant A .sieberi as its root was exposed to humic acid, the percentage of oxygenated terpeniod from the whole essential oil became 81.39%that in comparison with blank sample, an increase of 24.29% was observed. When the root got Sapropel rather than humic acid, we observed only 21.99% increase in the percentage of composition. As the root of plant Semenovia suffruticosa received extracted humic acid, the percentage of oxygenated terpeniod increased to 5.98%. While by adding the Sapropel, the increase was only 0.48%. Comparison of the percentage of essential oil composition, after addition of humic substances to their roots, in primary stage of growth depends on Factors such as physiological structure, the kind of the growing place soil, climate and the amount of sun-shine. Introduction Soil is one of the important components of basic resources, so that it is considered as main bed of plant cultivation and also, as a unique environment for all kinds of lives(1).Ecological movement of present time and fear of water and soil contamination by chemicals from usage in soil more than ever. Because their productive effects on physical, chemical and biological properties of soil organic materials are considered as important elements in soil fertility. They not only increase fertilizer efficiency and promote plant growth, but can reduce the potential of groundwater contamination. Plant growth research involving humic substances at the university of Minnesota in the Departments of horticulture and soil, water and climate has centered on owth chamber,greenhouse,and golf green experiments, Both basic and practical research components are underway to investigate the ‘how and why’ of humic substances interactions with plant and soil ecosystems(2). Experimental The plant was collected, from Taftan Mountain located 30 km away from khash city of Baluchestan region in June 2009. Plant identification was carried out by Dr. Mozaffarian Botanist in the Research Institute of Forests and Rangelands in Tehran-Iran (Mozaffarian, 2007). Essential oil decomposition was den by GC/MS technique (Bilia et al., 2002) .For preparing a Laboratory sample, we dried it under the shadow of sun Light and made it power by a grinder (20 days). The oil yield was for sample collected in the Following in Figures 1 and 2 for 4condition. Essential oil decomposition was den by
GC/MS technique (Bilia et al., 2002) .We extract humic acid from the soil of Nahakhoran forest of Gorgan in north of Iran, according to IHSS (International Humic substances society) protocol (Davies et al., 2001; Stevenson, 1994) and then purify it. Since, Sapropel has not more than 30% humic acid therefore our extracted humic acid has stronger effect.We solve the Sapropel of a 20g package in 400 ml water then sprinkle them along with 3 litter water on the base of root a little on plant leaves in an area about half acre, in the month of mars when the plants wake up from winter sleep and start to grow. In this area it does not , receive, Sapropel by some of the plant and they are considered as blank samples. Results and Discussion A .sieberi plant was collected at flowering stage. The percent of terpeniod in the essential oil whether oxygenated or hydrocarbon have increased from 66.05 %( when Sapropel solution was sprayed to leaf) 83.17 %( when humic acid and solution of Sapropel on the root of plant), 88.43 %(when humic acid on the root of plant), and 78.64% %( blank sample). The percentage of oxygenated terpeniod compounds increased in the essential oil of two samples (when humic acid and solution of Sapropel) the percentage of terpeniod hydrocarbon compounds decreased in three states, comparing to blank sample.The percentage of oxygenated terpeniod compounds in the essential oil of blank sample with a percentage of 57.10, but it increased in the essential oil of the sample whose root had received extracted humic acid. Semenovia Suffruticos plant was collected at flowering stage. The percentage of terpeniod whether oxygenated or hydrocarbon have increased from 70.12 %( blank
1 347
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
sample), 55.21 %,( solution of Sapropel at root) 58.30 % (addition of humic substances to root )and 65.46 % (solution of Sapropel on the leaves).Figure 1. Show that after addition of humic substances to root, the percentage of oxygenated terpeniod in essential oil compositions in this plant was more than that of blank plants has increased
considerably. So, their anti-microbial property increases. The percentage of some compositions increased and of some other, decreased in comparison with blank samples some compositions were totally removed and were displaced by new compositions (3,4).
Figure 2.The variation of total terpeniod in the essential oils from A .sieberi and Semenovia -Suffruticosa
Figure1.The variation of oxygenated terpeniod in the essential oils from A .sieberi and Semenovia Suffruticosa
Conclusion Addition of Humic substances to the root of stadied plants has resulted in growth of plant organs such as leaves. Addition of Humic substances (extracted humic acid and Sapropel) causes biologic activity and decrease of flowering stage in plants. The reasons behind this fact, is a better " complex – making" property of Humic acid than Sapropel that results in assimilation of heavy metals such as Pb and Cd ions, so that the quality of essential oil composition improves(5).
(2)Clapp C E,Liu R, Cline V W,Chen Y and Hayes M H B (1998).Humic substances for enhancing turfgrass growth. The Royal Society of Chemistry, Cambridge, 227-233. (3)Liu K, Rossi P. G, Ferrari B(2007). Composition irregular terpenoids chemical variability and Antimicrobactertial Corsica Jordan et Fourr, Photochemistry. 68:1698-1705. (4)Masoudi S, Monfared A,Rustaiyan A, Chalabian F(2005).composition and Antibacterial Activity of the Essential oils of Semenovia dichotoma (Boiss), Journal of Essential oil Research,1-6. (5)Davies G, Ghabbour E, Steelink. C(2001).Humic Acids: Marvelous products of soil chemistry. J chem.Edu. 78, 12:1609-1613.
REFERENCES (1) Mogimi J(2007). Introducing some of pasture species in Iran, Arvan publishing, Iran.,94-102.
348
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Characteristics of Humic Acids from Kazakhstan Coals E.Yermoldina(a)*, J.Kairbekov(a), I.Dzheldybaeva(a), A.Zharmagambetova(b) (a)
Al-Farabi Kazakh National University,Kazakhstan D.Sokolskii Institute of Organic Catalysis & Electrochemistry, Kazakhstan * E-mail: [email protected] Keywords: humic acids, physico-chemical properties of humic substances, (b)
Abstract Physicochemical characteristics of humic acids (HA) of coals of two deposits of Kazakhstan has been studied. Experimental data showed that the studied humic acids consisted of the multi-component mixture of high molecular, polyfunctional aromatic and heterocyclic compounds substituted with various functional groups. The obtained HA were used as catalyst modifiers for hydrogenation of coal disstilates Introduction Recently many countries including Kazakhstan intensively study chemical processing of coals. One of the priority areas of coal chemistry is development methods of humic substance extraction from lignite charcoal and their various application. Their ability to various chemical transformations opens the new possibilities for obtaining various materials from them for different purposes [1-2]. In this paper we studied the physicochemical characteristics of HA of the two Kazakhstan coal deposits (Oy-Karagaj and Mamyt). Experimental Characteristics of coals of both deposits are presented in the Table 1. Characteristics Moisture total, (WP, %) Moisture analytical sample. (Wa, %) Ash dryness (AC, %) Ash working (A0, %) Volatiles ashless substances (Vdaf , %) C total dryness (Cdaf, %)
Mamyt 9.0 3.0
Coal samples were treated with 2.0 % KOH for 30 minutes at 333 K and constant stirring. The resulting mixture was centrifuged. The extract was poured with equivalent amount of 5 % HCl (to the pH = 2-3). Precipitated humic acid is separated from the fulvic acids by filtration. Humic acids and the coals have been studied by IR spectroscopy (VERTEX 70V, German). Scanning electron microscopy (H-600, Japan) was used for comparison of HA treated and untreated with HCl. Results and Discussion The IR-spectra confirms the presence of methyl, carboxyl, nitrogen and sulfur-containing groups. Absorption bands corresponding to -C = O in the COOH and quinones were observed in the spectra of both humic acids. We tried to count the number of oxygen-containing groups in the HA using Ca-acetate and Ba(OH)2 procedures [3]. The results of quantified analysis of carboxyl, phenolic and carbonyl groups are shown in the Table 2.
OyKaragai 7.8
11.3 10.3 34.8
12.0 10.8 35.8
73.1
74.1
H total dryness (Hdaf, %) S total dryness (Sdaf, %)
4.7 0.3
Calorific value of a combustible, highest analytical (Q Aв, кJ/mol) Calorific value of a combustible,. highest working (QРв, кJ/кg) Calorific value of a combustible, lower working (QРн, кJ/кg) С/Н 100 Н/С
Functional composition, mg-eq/g СООН СООН ОН С=О ОН 7.0 2.7 4.3 1.9 6.8 3.2 3.6 1.9
Sample
рКα
4.7 0.1
HAОК HAМт
6.9 7.0
29.2
15.6
Table 2. Functional analysis of humic acids of Oy-Karagai (HAOK) and Mamyt (HAM) coals.
28.0
28.5
26.8
26.8
15.5 6.5
15.5 6.5
The total content of oxygen-containing groups in HAOK and HAM is in the range of 6.8- 7.0 mg-eq/g, including 2.7- 3.2 of carboxyl , 4.3-3.6 mg-eq/g of phenol and 1.9 mg-eq/g of carbonyl groups. Analysis of the spectra leads to the conclusion that they are multi-component mixture of macromolecule polyfunctional compounds of heterocyclic and aromatic units substituted with various functional groups. HA extracted from the of the coals of both deposits were studied by electron microscopy. SEM images of the HAOK and HAM are similar. Humic acids are
Table 1 . Physical and chemical characteristics and elemental composition of "Mamyt" and "Oy-Karagai" brown coal deposits
349
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
well-structured uniform particles with the sizes of 710 microns (Figure 1).
Figure 1. SEM image of the humic acid extracted from Mamyt coal
HA were hydrogenated on 1%Pd/aluminosilicate catalyst under mild conditions (400C, atmospheric hydrogen pressure). According to the IR spectroscopy data, the absorption bands characteristic for quinones are disappeared in the spectra of hydrogenated HA. Thus, the study of physicochemical properties of humic acids show that they are multi-component mixture of high molecular polyfunctional compounds of aromatic and heterocyclic nature. The hydrogenated humic acids were used as modifier of the supported catalysts. It has been found that the introduction of humic acids in the catalyst composition leads to an increase of their activity in the hydrogenation of coal distillates REFERENCES
(1) Steinberg, E.W.; Meinelt, T; Timofeyev, M; Bittner, M; Menzel, R. Env. Sci .Pollut. Res . 2008, 15 (2), 128- 135. (2) M. De Nobili; Contin , M. ; Leita, L.Can.J. Soil. Sci. 1990, 70,531-536. (3) Yerrmoldina, E; Aubakirov, E; Myltykbaeva, J. В. КаzNU. Chem. 2011, 1(61), 488-492 .
Acknowledgments: Work is executed on MON RK grant on a priority 5.1. Basic researches in the field of natural sciences according to the program «To develop scientific bases of processing of combustible minerals and receiving new materials».
350
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Application of humic acids for the removal of reactive dyes from model wastewater T. Weidlich*
Address: Institute of Environmental and Chemical Engineering, Faculty of Chemical Technology, University of Pardubice, Studentska 95, CZ-53210 Pardubice, Czech Republic * Corresponding author e-mail: [email protected] Keywords: reactive dye; acid dye; cationic dye; ion pair; chlorotriazine; Abstract By the production of reactive dyes in aqueous solution and subsequent isolation by filtration the aqueous filtrate saturated with these reactive dyes are obtained. The dissolved reactive dyes are simply removed by addition of sodium or potassium humate and heating of the mixture at elevated temperature for the short time period. After the completion of nucleophilic substitution reaction, cooling and acidification of the cooled reaction mixture the coagulation of humic acids with chemisorbed reactive dyes occurs. Most of dissolved reactive dye is removed from the aqueous solution by the above mentioned process. The isolated modified humic acids are effective sorbents for cationic dyes or heavy metal cations. Introduction Reactive dyes are the most common dyes used due to their advantages, such as bright colors, excellent colorfastness and ease of application (1,2). In a reactive dye a chromophore contains a substituent (reactive group) that reacts with the substrate. Most of these dyes are based on formation of azo group introduced by diazotization of appropriatte aromatic amine and subsequent coupling process. Diazotisation and coupling processes are important for the manufacture of APIs and represent the essence of azo dye manufacture. Azo dyes are the predominant colourant family, accounting for over 50 % of all commercial organic dyes. Among the typical raw materials for diazotization and coupling are halogenated aromatic anilines, phenols and aromatic sulfonic acids, which can contribute to the AOX load of waste water streams (3). Sodium nitrite is added in excess to a solution or suspension of the arylamine in aqueous mineral acid solution in a diazotisation tank. The reaction is cooled to 0 °C by adding ice or by cooling with brine. In a separate tank, the coupling component is dissolved in water and alkali. Both solutions are clarified by filtering and added to the coupling vessel. Clarifying may be necessary on completion of the reaction (by filtration over SiO2, Al2O3 or charcoal) to remove unreacted amine and salty, resin-like or oily by-products, followed by precipitation of the product (usually by salting out or pH change), filtration, washing, dissolving and, e.g. spray drying to yield the standardised dyestuff. Due to their good solubility, reactive dyes are common water pollutants occured in the aqueous mother liquors produced during separation of dyes by salting-out and filtration. Unfortunately, reactive dyes are resistant to bacterial activity and biological treatment alone would take a long time to be effective (4-6). Mother liquor, often containing high loads of refractory COD and possibly AOX (if halogenated starting materials are used), high salt loads from
salting out. Wash-water containing lower loads of refractory COD and possibly AOX (if halogenated starting materials are used). The common treatment techniques for aqueous waste streams from diazotation and azo coupling are adsorption on activated carbon, chemical oxidation or wet oxidation (3). However, occurrence of reactive group bonded to the dye stucture enables effective removal of reactive dyes by nucleophilic substitution reaction with suitable nucleophile. Humic acids obtained simply from young brown coal (leonardite, oxyhumolite) serve as the cheap and simply applicable reagent for removal of reactive dyes from the aqueous solution by the heating in alkaline conditions. Humic acids with chemisorbed reactive dyes are removed from the alkaline aqueous solution by simple acidification (7). Experimental The sodium and potassium salts of HA (NaHA or KHA) used were industrial samples obtained from Humatex company (Czech Republic) by extraction of oxyhumolite in aqueous hydroxide, sedimentation of undissolved matter and evaporation of alkaline solution of humic acids. All reactions were conducted on air. 50 grams of NaHK were added to the 1 L of aqueous filtrate from the isolation of Reactive Red dye (concentration of dye was 5mM according to the measurement of absorbance at Amax, COD = 80,500 mg O2/L) and the mixture was heated at 90-100 o C for 2 hours. After cooling to the room temperature the mixture was acidified with 16 wt.% H2SO4 (pH = 2.5) and stirred for 30 minutes. The precipitated humic acid was isolated by filtration. The chemical oxygen demand of obtained filtrate was COD = 4950 mg O2/L. Results and Discussion The removal efficiency based on sodium or potassium salts of HA was tested on 11 reactive dyes:[Figure 1]
351
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
COONa NaOOC
N
CH3 CH2 N Ph
N N
Cl
N
N
NaO3 S
O ONa O
+
O
COOH CH R
N
SO3 Na
O
NaO
CH3
OH
H
COONa
NaO
N NH
O
NaO
COONa
N
SO3 Na
H
O
O
O
O
N
O
O NH
Reactive Red 45:1
O
cukr
R CH CONHR
COOH HOOC
o
95 C/30 min. 2. + H
N
+
OH HN
N N N
HO 3S
O
HO
COOH
H 2 CH3 C N
H
COOH
O
O O OH
HO N Ph
SO 3H
N
O O
COOH CH R H
O
O
O
O
O
N
O
O
cukr
NH R CH CONHR
Figure 1. Scheme of the nucleophilic substitution of
chlorine in chlorotriazine reactive dye Reactive Red 45:1 by phenolic group of HA (structure HA according Stevenson (8).
The obtained removal efficiency was mostly above 80 %. 100
removal efficienc y (%)
90 80 70 60 50 40 30 20 10 0
Abbreviation of removed dye
Figure 2. Effect of chemisorption of reactive textile dyes on humic acid in alkaline conditions at elevated temperature.
The obtained humic acids modified by reactive dyes were tested as possible sorbents for cationic dyes. REFERENCES
(1) Yang X., Al-Duri B. Chem. Eng. J. 83, 15 (2001). (2) Mahony T.O.; Guibal, E., Tobin J. Enzyme Microbiol. Technol., 31, 456 (2002). (3) Integrated Pollution Prevention and Control. Reference Document on Best Available Techniques for the Manufacture of Organic Fine Chemicals, August 2006, available on-line: http://eippcb.jrc.ec.europa.eu/reference/BREF/ofc_bref_0806 .pdf (4) Elwakeel, K.Z. and Rekaby, M. J. Hazard. Mater. 188, 10 (2011). (5) Robinson, T.; McMullan, G.; Marchant, R. and Nigam, P.: Bioresour. Technol. 77, 247 (2001). (6) Pearce, C.I.; Lloyd, J.R. and Guthrie, J.T.: Dyes and Pigments 58, 179 (2003). (7) Weidlich T., Socha F.: Czech Patent CZ20120669 (2013). (8) Stevenson F.J. in the book: Humus Chemistry, John Wiley, New York, 1982.
352
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Measurement of ion exchange properties and complexation of extracted humic acid from forest soil
A.R. Sardashtia ,M.M.Maorofdoostb a,b Department of Chemistry,Faculty of Science, University of Sistan and Baluchistan, P.O.Box 98135-674, IRAN *Corresponding author e-mail : [email protected] Keywords: Forest soil, extracted humic acid, ion exchange, Zinc ion Abstract Presence of weak acidic functional groups in humic acid, enable them to act as a cation exchanger between soil and plant. The exchange process is slow and depends on the pH of the medium. The presence of nitrogen in the structure of humic acid also causes to be a source of nitrogen in the soil which helps the growth of plants. Thus, the soil containing more humic acid can act as a better fertilizer. Zinc ion has been selected because its role is important in the physiological system. In this studies batch method has been used and the amount of exchanged zinc ion with respect to dried matter at pH 6.75 is 1.24mmol/g per dry matter. This amount has been measured by flame atomic absorption spectrophotometery and the calculated ionic exchange constant for an initial concentration, 4.00×10-3 M, is 2.28×10-2. Introduction Humic substances (Hs) are heterogeneous mixtures of acidic, randomly polymerized, polydiserse, high-molecular-weight organic macromolecule have widely different chemical function groups in ortho position arise humic acid have high capacity ion exchange and more stable chelates for heavy metals(1,2).Capacity of ion exchange isn't equal in soils with different carbon percentages, because cation exchange capacity decreases by methalation. (Only for humic acid containing carboxylic and phenol functions). Moreover Omat (R-) is more stable in high pH, therefore, reacts with more Zn+2, cation of this complex is insoluble in high pH, it can be solve using pyrophosphate salt. So humic acid with complex structure is important in agriculture, industry, and environment (3). Experimental Materials All chemical were of analytical reagent grade (Merck, Germany). ,(Titrisol, Merck, Germany) ml /g stock solution using adjustable micropipettes (Gilson, France). Standards were acidified to 1% with nitric acid. Apparatus (ELM 1400 rpm, Germany)., IR-470 Shimadzu (Japan) ,digital pH meter CD620 with glacial calomel electrode (Zag shimi, Iran), Flame Atomic Absorption Spectrometer (Philips, Pu 9100 X, UK), Milli-Q Plus filter apparatus (Millipore, USA) Sample Samples were derived from soil with high organic and low iron content, taken from Naharkhoran forests of Gorgan in the North of Iran. The separation of organic fractions is, after
an initial physical (sieve) removal of stones and leaf litter. Then washed with 0.5 M HCl and several times with DDW. According to the protocol of the IHSS (2,3). Fixation of Zn+2 ions We used discontinue or in pot ion exchange method for reaction between humic acid and Zn+2 solution. cationic exchange kinetic (due 48 h study) was slow, Fig. 2.First make a acetate buffer solution (HOAc/OAc-) contain 1g purified humic acid with various volume of 7.70×10-3 M Zn+2 at 0.02 M NaNO3 in 200 ml polyethylene Erlenmeyer and agitate slowly to complete ion exchange between Zn+2 and humic acid's sites under N2 for two days. Then separate the solid phase of mixture by centrifugation at 6000 rpm. Measure [Zn+2] in liquid phase (CL) with FAAS. Also the obtained result by used formula’s=(C0CL)V/1 g humic acid Measurement with FAAS shows that relation between absorbance and concentration in 7.70×10-6 – 4.62×10-5 M concentration of zinc is linear. Results and Discussions The potentiometer titration method with 0.5 M NaOH on the humic acid extract was studied. the result obtained shows that carboxyl benzoic is pka= 4.70 and phenol function is pka= 8.70.capacity exchange for Zinc ion The results of the kinetic studies on Zinc ion by potentiometer titration is 1.25 mmol/g shows in table1 that fixation mechanism has a low kinetic fixation mechanism and it is the same for other bivalence’s. Finally, it will be fixed according to Kinetic studies The best time period for ion exchange reaction[ 3,4,5] is 48 h.
353
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
1 0.9 0.8 0.7
Y
0.6 0.5 0.4 0.3 0.2 0.1 0 0
0.2
0.4
0.6
0.8
1
X
Figure. 1. The variation logarithmic of limit distribution coefficient of zinc on humic acid as a function of pH for 4.00×10-3 M of Zinc. The carboxyl function is ionized 99.50% in pH= 7 and phenol function 95.24% ionization in pH= 10, completely in pH > 10, and less than 1% at pH=7. Zinc hydroxide precipitate in pH > 7 therefore phenol function can't react with Zn+2. In the other words, if this group could react slope in fig 3 would be 2(6,7). But in ammonia buffer solution (NH3/NH4+) with at pH= 9.25 amount of fixing of zinc is 1.81mmol/g per dry matter. This amount is more than the complexing capacity of carboxyl function of Zinc. This could be due to the formation of a complex between the carboxylic and phenol groups of humic acid with Zinc ion. Maximum coefficient distribution at pH= 6.75 in table 2 is 103 and limit coefficient distribution when CL0 is 105.in table 1 and amount of fixing of zinc in this pH is 1.24 mmol/g. per dry matter. The detection limit of humic acid with respect to Zinc ion is 4.00×10-3 M which equals three times of equilibrium point of CL at pH= 6.75.then is calculated Kex =2.28×10-2, at intercept -1.562 and at slope 0.867in Fig 1 . From the Kex value found it is possible construct partition diagram of the zinc between the solution and the carboxyl benzoic and phenol groups in the humic acid. We observe by plotting the curve Y=f(x) that for cefully react limited to carboxyl benzoic groups We have: (8). We observed that this cation essentially fix to the carboxyl-benzoic groups that each Zn+2 ion exchanges only a single H+ ion in fig. 2 and relative error is 0.71% and CV=1.25 %.
Figure. 2. The variation ionic fraction of Zn+2 in the humic acid as a function of ionic fraction of Zn+2 in solution. the preconcentration of diluent solutions before a quantitative measurement (2). REFERENCES (1)Davies,G ;Ghabbour,E.A ; Cornelius ,S.J. chem. Ed. Chem. Wisc. Edu 2001. Vol 78 No 12, 1609-1613. (2)Stevenson, F. J. Humus chemistry, 2nd ed. : John wiley & Sons Newyork 1994. (3)Robertson, A. P ; Oleckie, J. environ Sci. Technol 1999,33, 786-795 . (4)Gardea-Torresdey ,J.L; et al .Journal of Hazardous materials 1996,48,191-206 (5) Sardashti, A and. Rumeau,M.J. Analusis 1999 , 5, 27 ,432-435. (6)Matsuda, K.; Ito, S. soil Sci. Plant Notr (Tokyo) 1970, 1-10. (7)H ussain, K.; Rumeau, M.; Bulletin de la societe chimique de France 1979,3-4, 73-81. (8).Rumeau, M.; Sardashti , A.; 3e Collogue du groupe Franscais de l'IHss nature et fonctions des matieres organiques dans l'environment centre de recherches de l' INra, verseillesFrance 2-3 jun 1999.
Conclusions The extracted humic acid from Naharkhoran forest soil of Gorgan with an average of cationic exchange capacity (CEC) is about 3.1mmol/.per dry matter for carboxyl-benzoic groups .stability constant of complex is calculated 186. Reqarding suitable selectivity of humic acid which depends on pH,it can be used for the extraction and separation of metal ions and for
354
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Electrooxidation Extraction of Humic Acids from Coal Tailing and its Characterization by 13C NMR CP/MAS. Renzon Cosme Pechoa*; Eduardo Albuquerque Brocchia; Deborah Pinheiro Dickb (a)
Department of Science of the Materials and Metallurgy – PUC-RIO Brazil Institute of Chemistry, Department of Physical Chemistry, UFRS, Porto Alegre, RS - Brazil * Corresponding author e-mail: [email protected] (b)
Keywords: Acid Humic, coal, electrooxidation
Abstract Humic acids (AH's) are generated naturally by decomposition of organic matter and the activity of microorganisms. This work begins condensate elemental carbon without the presence of micro-organisms, but using the method of electrooxidation achieves the functionalization of coal tailings (RJ), followed by physical and chemical procedures that allow the extraction of humic acids (AH's). The characterization of these AH's was carried out by elemental chemical analysis and 13C NMR spectroscopic method by CP/MAS, determining a high aromaticity index (IA), H/C <1, as well as functional groups (carboxylic carbonílicas and phenol) of the order of 20%, contained in its structure.
Introduction Coal is a natural fossil fuel found at great depths. Although the extraction and beneficiation of coal being responsible for environmental impacts in the generation of DAM, it is an important source of raw materials for power generation as well as in ironmaking and steelmaking. Brazilian coals are considered to have a low quality grade for presenting high levels of ash and sulfur. At the time of coal milling generates a large amount (30 - 40%) of coal tailings (RJ) with high sulfur content, toxic metals (Fe, Pb, Zn, Cu, Al and Si) and 17% Alternatively representing an organic carbon source for the supply of humic acid (HA). Experimental The sample of coal tailings are previously dried at 60 ° C for one day then milled to a size of <250mesh according to figure 1. The electrolysis was carried out in the electrochemical cell consisting of three compartments (anode, cathode and reference (SCE)) at a potential of 1.2V (V vs SCE), the carbon concentration was maintained at 0.015 g / ml. It has been reported that iron can help in the oxidation of carbon (Dhooge et al. 1992). According to Patil et al. 2006 reports that the current densities increase during the addition of Fe2 + at 10 to 12 times the current density so Fe ions. Thus were added Fe2 + (100 mM) in anode solution consisting of 40% H2SO4.
355
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
charging it positively, providing the conditions for connection with oxygen ions that are also released. The addition of ions Fe+2 acts as a catalyst in the process of electro-oxidation, shown in Figure 2.
Fig. 2 Efficiency by obtaining AH electrooxidation methods and IHSS .
Characterization by elemental analysis, HA obtained by electro-oxidation had an atomic ratio (H / C) of 0.9 (Table 1) thus indicating the predominance of very condensed aromatic structures. Table 1 –Results of the elemental composition of AH and RJ
Fig. 1 Flowchart of the functionalization process, extraction and purification.
Figure 2 shows the 13C CP/MAS RMN spectra of AH obtained by electro-oxidation dominated by of signal aromatic, aliphatic, and carboxylic regions.
Elemental analysis It was determined elemental composition (C, H, N and O) and H/C ratio (degree of aromaticity) of humic acid (HA). 100 mg of the purified HA was used for analyzing in the Thermo Electron equipment, Flash model, using as the standard acetanelida. 13 C RMN CP/MAS: RMN spectra were obtained applying the following parameters: 14mHz of rotor spin rate; 1s of recycle time; 1ms of contact time; 4000 scans. Sample were packed in 3,2 mm, The overall chemical shift range is usually divided into the following main resonance regions: alkyl-C (0-110 ppm); aromatic-C (110-160 ppm); carboxil- and carbonyl-C (160-200 ppm). The relative areas obtained from the integration of the above-cited regions were used for the semi-quantitative evaluation of different organic components Results and Discussion There is a greater efficiency in obtaining the AH was the electrooxidation reaching 13.4% compared to 3.4% of IHSS. During the electrolysis of H2SO4 ions H+ were produced which is consequently responsible for the polarization on the particle surface coal,
Figure 2 –The 13C-CP/MAS-MNR spectra of AH and RJ
356
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The values found for the integration of the relative peak areas or 20% confirming the attribution of signals from carbons containing oxygen. The degree of aromaticity was around 61.1% indicating an aromatic structure. This fact corroborates the H/C<1 Table 1 –Relative values of integrated peak areas of NMR spectra of AH
Sistema Brasileiro de Classificação de Solos, Tese Doutorado, UFRJ, Instituto de Agronomia, 2009. (6) DHOOGE P. M., Electrochemical society, 1983, v. 130, p. 1539-1542. Paciolla, M.D. and White A.B. Environ. Sci. Technol. 1999, 33, 1814-1818. (7) PATIL P., DE ABREU Y., MARQUEZ A., BOTTE G., Characterization of electrooxidized Pittsburgh, 2007, No. 8 Coal. Fuel 4, v. 86 p. 573–584. (8) SPACCINI, R e. PICCOLO A., Molecular characterization of compost at increasing stages of maturity. Chemical fractionation and infrared spectroscopy, J. Agric. Food Chemical, 2007, v. 55, p. 2293 – 2302.
Acknowledgements The authors thank CNPq and the Brazilian Network of Coal for the research support as well as the PUC-Rio and UFRGS for the study opportunity
Conclusions
The electrochemical method showed an efficiency of 13.4% AH, while the method of IHSS generated 3.4% AH. Demonstrating that it was possible to generate so AH from waste coal.
The 13C NMR spectroscopic techniques and elemental chemical analysis used in this study showed the functionalization of HA obtained by the electrochemical method, indicating a structure more aromatic than aliphatic, content of 20% of structures containing oxygen (carboxylic, phenolic and carbonyl).
REFERENCES (1) BOTTE G., SATHE N., Assessment of coal and graphite electrolysis on carbon fiber electrodes, 2006, v. 161, p. 513-523 (2) JINGDONG M, CHEN N, XIAOYAN C., Characterization of humic substances by advanced solid state NMR spectroscopy: Demonstration of a systematic approach, Organic geochemistry, 2011, v. 42, p. 891–902. (3) COSME R., Extraction of humic substances from tailings of mineral coal beneficiation: Alternative methods and characterization for possible applications. Doctoral thesis, DEMA, PUC-Rio, 2013. (4) DICK, D.P., BARROS DA SILVA L., VASCONCELLOS A., KNICHER H., Estudo Comparativo da Matéria Orgânica de Diferentes Classes de Solos de Altitude do Sul do Brasil por Técnicas Convencionais e Espectroscópicas. Revista Brasileira de Ciência do solo, 2008, v. 32, n. 6, p. 2289-2296.. (5) FONTANA A., Fracionamento da Matéria Orgânica e Caracterização dos Ácidos Húmicos e sua Utilização no
357
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
The effect of humus rich sludge on lettuce growth G. Ntzala1, P.H. Koukoulakis2, P. Kokkinos3, I.K. Kalavrouziotis3* 1. University of Patras, Department of Environmental and Natural Resources Management,G. Seferi 1, Agrinio, Greece 2. Soil Science Institute, of Thessaloniki, GR-570 01, Thermi, Thessaloniki 3. School of Science and Technology, Hellenic Open University, GR 26222, Patras, Greece *Corresponding author: Associate Professor Dr. Ioannis K. Kalavrouziotis, Hellenic Open University, School of Science and Technology, Tsamadou 13-15 & Saint Andrea, 262 22 Patras, Greece, e-mail: [email protected]. ABSTRACT The purpose of this work was to study the effects of high levels of sludge application on the growth of lettuce (Lactuca sativa L. var. longifolia), and to assess the extent of soil pollution with heavy metals. A greenhouse pot experiment using a randomized block design in four replications and six sludge treatments was conducted. It was shown that the plant height was statistically and significantly affected by the applied sludge levels. Similarly, the sludge had a statistically significant effect on fresh and dry matter yied of lettuce leaves. These results showed that the addition of sludge had positive effect on lettuce plant growth and development. The application of sludge decreased the soil pH and increased the electrical conductivity. Potentially, this rise of pH may in the long run increase the solubility of some heavy metals such as Zn, Co, Cr in addition to the available heavy metals added to soil by the direct application of sludge. Also, an increase of the electrical conductivity may constitute a constraint for the normal plant development. A significant increase of Mg of dry matter of lettuce plant by the applied sludge was also attained, which supplanted the available soil Mg, potentially affecting plant growth. The Pollution Load Index (PLI) value was found 1.09 under the effect of sludge. This, value suggested that the experimental soil was only slightly polluted be the applied sludge, probably because the decomposition of the organic complexes of the heavy metal was not completed as a result of the low ambient temperature. In conclusion, the applied sludge increased lettuce plant height, fresh and leaf dry matter, and leaf dry matter Mg content, while it did not caused substantial soil heavy metal pollution, due to the low calculated value of the PLI. A greenhouse pot experiment using a randomized block design in four replications and six sludge INTRODUCTION Sludge, is a byproduct of wastewater treatment. It treatments (Table 1) was conducted. Sludge was contains organic matter (humus) approximately 3035%, which favors the physical properties of soil derived from the wastewater processing plant (addition of organic matter to soil, aggregation of (WPP) of Messolonghi, W. Greece. Lettuce soil particles, improvement of soil structure, water (Lactuca sativa L. var longifolia) was used as test holding capacity). It also contains essential plant crop. The soil was a light sandy loam (SL), with nutrients (N, P, K, Ca, Mg, Fe, Zn, Mn, Cu, and B) slightly acid pH, low salinity, moderate organic contributing to soil fertility, and relatively large matter content, and adequacy of Olsen P, Zn, Mg, amounts of various heavy metals such as Cd, Cr, Mn and Cu. The experiment was conducted in pots Co, Pb, and Ni, which may render its application to of total volume of 6 L, containing 10 kg of air dried soils problematic. Therefore, it requires special soil, of known moisture content (12,5% determined management. These metals may become toxic at at 100oC). Soil samples were taken from each pot high concentrations, and therefore they may cause twelve (12) and (18) weeks, respectively, after serious problems to the environment, to plants, and transplanting. At the same time, whole plant to human health. Exposure to these heavy metals samples were also taken. Both soil and plant may cause kidney damage and other health samples (roots, leaves) were analyzed according to abnormalities (WHO, 1992). In spite of the above standard methods for the determination of macro unfavorable effects, sewage sludge has long been and micro nutrients (Jackson, 1957, Lindsay and used as fertilizer, especially as supplementary Norvell, 1978, Olsen et al. 1954; Lanyon and Heald, source of N and other macro and micronutrients, as 1982, Richards, 1954), and heavy metals (Ure, well as an amendment for the improvement of soil 1996, Soltanpour et al., 1996, APHA, 1996, AOAC, physical properties (Pereira et al., 2011; NRC 1996; 1996), respectively. Metcalf and Eddy, 1991). However, the sludgeborne metals, have been reported as having lower RESULTS AND DISCUSSION phytotoxicity than the added metals, that is why phytotoxicity symptoms are very rarely observed Table 1: Experimental Sludge treatments even after application of large amounts of sludge Treatment Dry sludge Wet sludge s (Adriano, 2001). The purpose of this work is to g/pot Kg/ha g/pot Kg/ha study the effects of high levels of sludge application S0 0 0 0 0 on the growth of lettuce (Lactuca sativa L. var, longifolia), and to assess of the extent of soil S1 22.6 600 41.8 1110 pollution with heavy metals. S2 45.2 1200 83.6 2220 S3
67.7
1800
125.2
3330
S4
90.3
2400
167.1
4440
S5
112.9
3000
199.1
5550
Effect of sludge and TMWW in plant height MATERIALS AND METHODS
358
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
It was shown that the plant height was statistically and significantly affected by the applied sludge levels. It was also shown that the increase of plant height started much later than the planting date of lettuce at the time at which the conditions of greenhouse had been improved. It should be noted that greenhouse was not heated. The gradual increase of air temperature contributed to decomposition and mineralization of sludge organic matter and as a result to the release of nutrients (N, P and other micro-nutrients) which favored the plants growth.
Figure 2:Effect of the applied sludge levels (t/ha) on pH and electrical conductivity (EC) of soil, during both samplings
Effect of sludge on the nutrient and heavy metal content of dry of heads of lettuce heads and roots A significant increase of Mg of dry matter of lettuce plant by the applied sludge (sig. P 0,042) was also attained, which supplanted the available soil Mg, potentially affecting plant growth (Figure 3).
The impact of sludge on soil pollution Among the main objectives of this work was to study and evaluate the level of soil pollution. This was made possible by calculating the Pollution lLoad Index ((PLI) which is given by the following equation (Tomlinson et al., 1980). The PLI value was 1,09 under the effect of sludge. This, value suggested that the experimental soil was only slightly polluted be the applied sludge (Cabrera et al 1999), probably because the decomposition of the organic complexes of the heavy metal was not completed as a result of the low ambient temperature.
CONCLUSIONS In conclusion, the applied sludge: • increased lettuce plant height. • increased fresh and leaf dry matter, and leaf dry matter Mg content. • it did not caused substantial soil heavy metal pollution, as shown by the low calculated value of the pollution load index (PLI = 1.09).
Figure 1: Effect of the applied sludge levels (t/ha) on the fresh (A) and dry (B) matter yield of lettuce head. (mean effect of both plant samplings)
REFERENCES Adriano,D.C.,2001 Trace elements in terrestrial environment Biochemistry, Bioavailabiolity and Risks of Metals 2nd Edition Springer 2001 New York
Effects of sludge on fresh and dry matter yields Similarly, the sludge had a statistically significant effect on fresh and dry matter yield of lettuce leaves (Figures 1). These results showed that the addition of sludge had positive effect on lettuce plant growth and development. In the context of the present study, the effects of sludge on some physical and chemical characteristics i.e. on pH, and electrical conductivity (EC), organic matter, and on macroand micro-nutrients and heavy metals were also studied. As shown in Figure 2, the application of sludge decreased the soil pH and increased the electrical conductivity. Potentially, this rise of pH may in the long run increase the solubility of some heavy metals such as Zn, Co, Cr in addition to the available heavy metals added to soil by the direct application of sludge. Also, an increase of the electrical conductivity (EC), may constitute a constrain for the normal plant development (Figure 2)
AOAC, 1996. Official Methods of Analysis of AOAC International, 16th Edition Publication International Suite AOAC, 481 Frederic Ave. Gaithersburg, Maryland 2027-2417, USA. APHA 1995. Standard Methods for Examination of water and wastewater. Method 3110, 19th Edition, American Public Health Association Washington, D.C. USA. Cabrera, F., Clemente, L., Diaz Barrientos, E., Lopez, R., Murillo J.M., 1999. Heavy metal pollution of soils affected by the Guadiamar toxic flood. The Science of the Total Environment 242: 117-129 Lanyon, L.E., and Heald, W.R., 1982. Magnesium, Calcium, Strontium and Barium. In: Page, A.L., Miller, R.H., and Keeney, D.R., (eds) Methods of soil analysis, Part 2, Agronomy 9, American Society of Aeronomy Madison, Wisconsin, pp 247-262. Lindsay, W.L., and Norvell, W.A., 1978.
359
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Development of DTPA test for zinc, iron manganese and copper, Soil Sci. Soc. Amer. J. 42: 421-428. Metcakf and Eddy Inc. 1981.Wasrewater engineering, treatment, disposal and reuse, New York mcgwaw and Hill NRC,1996 Use of reclaimed water and sludge in Food Crop Production. National research Council, USA p34 Olsen, S.R., Cole, J.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of available phosphorus in soils by extraction with sodium bicarbonate, Circular 939 USDA, Washington USA. Pereira,B.F.F., Z.L.He, P.J.Stoffela and A.J.Melfi, 2011. Reclaimed wastewater effects on citrus nutrition, Agricultural Water Management 98:1828-1833 Richards, L.A., 1954. Diagnosis and improvement of saline and alkaline soils, Agricultural Handbook No 60 USDA, Washington, D.C., USA. P 94.
Soltanpour, P.N., C.W. Johnson, S.M. Workman, J.B. Jones, Jr. and R.O. Miller, 1998. Advances in ICP emission and ICP mass spectroscopy Adv. Agron. 64: 28-113. Tomlison L., Wilson G., Haris R., Jeffery D.W., 1980. Problems in the assessments of heavy metal levels in estuaries and formation of a pollution index. Helgol Meeresunters 33: 566-575. Ure, A.M., 1995. Methods of analysis for heavy metals in soils. In: Alloway, B.J. Heavy Metals in Soil, 2nd Edition, Blackie Academic and Professional An Imprint of Chapman and Hall, London, p. 58. WHO 1992, Cadmium, environmental Health Criteria vol. 134, Geneva World Health Organization, ppl-280
360
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Seasonal changes of fluorescent dissolved organic matter in coastal waters off Visakhapatnam, Bay of Bengal, India N.V.H.K. Chari, Nittala S. Sarma*., P.Sudarsana Raro, R. Kiran, G. Chiranjeevulu, K. N. Murthy, P.Venkatesh Marine chemistry laboratory, School of Chemistry, Andhra University, Visakhapatnam 530003, India *Corresponding author: Email: [email protected] Abstract Visakhapatnam is at the centre of the east Indian coast. Although far away from any major estuary. its coastal region receives inputs from rivers like Ganga in the north and Godavari in the south. It also receives some inputs from the minor river Gosthani occurring 15 km towards north. During southwest monsoon season whenthe rivers experience flood, the fresh water input can reach high valuesWith an objective to make a fluorescence-based identification of these different water (external) sources as well as the in situ contributions to FDOM, we made a seasonal study during the year October 2010 to October 2011.From the water column, water
samples (n = 116) were collected on three occasions (Premonsoon, Monsoon and Postmonsoon seasons) each representing one season viz.from different depths ca., 0, 10, 20, 30, 50, 75 and 100 m at six stations located at 10 m, 20 m, 30 m, 50 m, 75 m and 100 m isobaths in the coastal region off Visakhapatnam (Fig. 1).. Fluorescence Excitation Emission Matrix spectra were run for the water samples filtered through 0.22 µ membrane filters on a Horiba Jobin Yvon Specrofluorimeter.
The fluorophore components extracted from these
spectra by parallel factor (PARAFAC) analysis (Stedmon et al., 2008) were examined in relation to chlorophyll a determined fluorimetrically and salinity measured by CTD. Five fluorophoric components (Fig. 2) two protein (B: Tyrosine and T: Tryptophan) like and three humic (A: UV humic, M: Marine humic and C: Visible humic) like resulted (Table. 1). The tyrosine protein like component showed seasonally higher intensity in the premonsoon season (March to May) compared to other seasons in surface waters. The remaining Components showed more or less the same intensities year-round. The enrichment of tyrosine protein like component during premonsoon may be due to a stable and enhanced bacterial decay of phytoplankton and of photosynthetically produced organic matter. The humic like components (C1, C3 and C4) showed conservative behaviour at salinity < 31.06 in the coastal surface waters during monsoon and postmonsoon season. But during the premonsoon season showed less values compared to the conservative line attributed to photo bleaching. The humic like fluorophores, during the premonsoon season, had significant positive correlation with Chlorophyll a inferring
361
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
that the humic like fluorophores are likely formed from chlorophyll a containing phytoplankton during the premonsoon season. Humification and fluorescence indices (Zsolnay et al., 1999;
McKnight et al., 2001) showed significant seasonal trend. The humic like fluorophores showed significant vertical trend where as the protein like fluorophores did not, at the offshore stations (100 m isobaths). The maxima of the humic fluorophores occurred at subsurface in the premonsoon (Fig. 3a) and at surface in the monsoon season (Fig. 3b) attributed to losses (as explained above) and surface freshening by the monsoon driven riverine influx respectively.
Figure: 1
362
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Figure: 2
363
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Table: 1 Components Ex/Em (nm)
Characterization
Alphabet designation
C1
250, 306/404
Marine and terrestrial humic like
M
C2
278/330
Tryptophan Protein like
T
C3
282(370)/466
Visible Humic like (C)
A1, C1
C4
262 (366)/ 464 Terrestrial humic like
C5
274/292
A2, C2
Protein like (Tyrosine or Phenyle alanine)
B
Figure. 3 A
b R.U. 0
0.001
0.002
0.003
0.004
0.005
0
0.002
R.U. 0.004
0.006
0.008
0
0
20
20 C1
C3
C4
C1
C3
C4
40
Depth (m)
Depth (m)
40
60
60
80
80
100
100
C1
References:
C3
C1
C4
C3
C4
Stedmon C A and Bro R 2008 Characterizing dissolved organic matter fluorescence with parallel factor analysis: a tutorial; Limnol. Oceanogr. Methods. 6 1-6. McKnight, B.E.W., Westerhoff, P.K., Doran, P.T., Andersen, D.T., 2001. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnology and Oceanography 46, 38e48. Zsolnay, A., Baigar, E., Jimenez, M., Steinweg, B., Saccomandi, F., 1999. Differentiating with fluorescence spectroscopy the sources of dissolved organic matter in soils subjected to drying. Chemosphere 38, 45-50.
364
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Use of FTIR and UV-Vis spectroscopy for analyze structural changes of humic acids after hummus addition (filter cake and orange peel) in soil contaminated with deltamethrin F. Benetti (a)*, L.B.F. Pigatin(a), M.M. Kanashiro(a), R.N. Rodrigues(a), M.O.O. Rezende(a) (a) Instituto de Química de São Carlos, Universidade de São Paulo, Trabalhador Sãocarlense Ave, 400, São Carlos, São Paulo, Brazil * [email protected] Keywords: FTIR, UV-Vis, humic acid, commercial deltamethrin, filter cake hummus, orange peel hummus
Abstract: The spectroscopic studies of FTIR and UV-Vis involving the interaction between humic acids and commercial formulation of deltamethrin, a pyretroid used in the assessment of toxicity tests on Eisenia foetida earthworms, are presented. The presence of filter cake and orange peel hummus were also available. After toxicity tests finalization, the humic acids were extracted according to IHSS method, followed by dialysis and lyophilization. The spectra showed that there are differences in humic acids after the addition of the pyretroid and hummus.
Introduction Humic acids (HA) have the ability to interact with minerals clay and organic compounds. AH are the main contituents of natural hummus, and are avaliable in larger quantity. They can interact with pesticides in diferents ways, such as ionic adsorption, hydrogen bond, covalent bond and hydrophobic adsorption.1 The understanding of the chemical nature, properties and reactivity of humic substances as the main materials that interact with pesticides in the soil is important to determine the way and extension of their interaction with the system.1 Spectroscopic analyzes have brought utility in the study of soil organic matter. Techniques such as ultraviolet-visible spectroscopy (UV-Vis) and Fourier transform infrared (FTIR) have produced excellent results with respect to possible interactions of the functional groups constituent of organic matter and pesticides, for example. The infrared spectrum analyzes the molecule as a whole, but there are certain functional groups with bands that occur more or less at the same frequency regardless of the molecule structure. Therefore, it is possible to elucidate the structural features of the studied molecule. According to Stevenson2, the FTIR provides information on the nature, reactivity and structural arrangement of the functional groups present in humic substances. FTIR analyzes have been used to identify functional groups such as carboxyl, amine, hydroxyl, carbonyl and others. The spectrum of humic acids is relatively simple, with few absorption extended bands.3 The comprehension of light absorption by matter is the most usual way of determining the
365
concentration of compounds present in solution. The UV visible spectroscopy (UV-Vis) involves photons. It uses light in the visible, ultraviolet (UV) near and near infrared. These energy bands molecules undergo molecular electronic 4 transitions. In the case of humic substances, which contains many aromatic groups absorbing light in the UV spectrum and leaving the low resolution, hence with little information about the structural characteristics of humic substances, although Kononova5 found that the ratio between the absorbance at 465 and 665 nm is related to the humification degree of humic substances. For humic acids, this ratio ranges from 2 to 5. Experimental The samples used for spectroscopic studies were derived from toxicological tests involving Eisenia foetida earthworms. Humic acids were extracted after completion of the mortality test, which lasted 56 days. To better evaluate the effect of deltamethrin in humic acids, commercial formulations of deltamethrin have been used with and without the active ingredient. The formulation without the active ingredient was called dispersant. Figure 1 presents the molecular structure of deltamethrin.
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Figure 1 – Molecular structure of deltamethrin. For all samples, the formulation concentration used was 500 mg kg-1. In total, seven samples were subjected to a process of extraction and purification by differential solubility, according to the methodology proposed by IHSS6:
vermicomposting also has a high percentage of colloidal material, thereby improving the soil quality. Therefore, it is mandatory to make spectroscopic analysis in order to chemically evaluate the changes occured during this process. It is known that humic substances in organic matter is able to adsorb pesticides, due to the presence of distinct funtional groups (carboxyls hidroxyls, phenols, carbonyls, etc), consequently, the addition of hummus was needed, in order to verify a possible complexation of the deltamethrin with humic acid. It can be observed in Figure 2 that FTIR spectra for the four humic acid studied present the same profile (with only dispersant and entire formulation). The bands were allocated according to Stevenson 2 and are characteristcs of humic substances in soils.
Soil Soil Soil Soil
1) soil (reference); 2) soil + dispersant; 3) soil + dispersant + hummus (filter cake); 4) soil + dispersant + hummus (orange peel); 5) soil + deltamethrin; 6) soil + deltamethrin + hummus (filter cake); 7) soil + deltamethrin + hummus (orange peel). Dialysis proceeded until no chloride was determined in dialysis water. For this, the procedure was maintained for approximately seven days, with a daily deionized water exchange. After dialysis, the samples were frozen in liquid nitrogen and lyophilized. The FTIR spectra were performed using the method suggested by Stevenson 2. The tablets were prepared on a 1.0 mg sample of humic acid per 100 mg of KBr (dried at 105 ⁰C). The tablets were pressed for 2 minutes with a load equivalent to about 10 tons. The spectra were obtained from 32 scans in the range 4000 – 400 cm-1 with spectral resolution of 4 cm -1. For UV-Vis, absorption spectra were obtained at a solution of 200 mg L-1 of humic acid in NaHCO 3 -1 0.05 mol L in the region of 800-400 nm. The scanning of the spectrum was taken with a pitch of 0.1 nm.
4000
3500
3000
(reference) + dispersant + dispersant + hummus (filter cake) + dispersant + hummus (orange peel)
2500
2000
1500
1000
500
0
-1
wave number (cm )
Soil (reference) Soil + deltamethrin Soil + deltamethrin + hummus (filter cake) Soil + deltamethrin + hummus (orange peel)
4000
3500
3000
2500
2000
1500
1000
500
0
-1
wave number (cm )
Results and Discussion
Figure 2 – FTIR spectra of the humic acid (with only dispersants and entire formulation).
Vermicomposting is the mineralization process of organic matter executed by earthworms, and during this process, there is the liberation of some nutrientes (nitrogen, phosphorus, potassium, among others) for plants. The hummus produced in the
For the experiment with the dispersant only, the bands at 3620, 3527 and 3448 cm-1 are characteristic of inorganic impurities, in these cases
366
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
they are O – H stretches in gibbsite. In 1633 cm-1 it is found a band related to the asymmetric stretch of C–O carboxylate ions (COO-) C=C stretch in aromatic rings, C=O stretch deformation and also N–H primary amides. In 1419 cm-1 it is found a characteristic band of carbonyl adjacent CH2 groups, from angular deformation. At 1265 cm-1 there is a band of C-O related to carboxylic acids, esters, aliphatic and aromatic deformation N–H. At 1085 cm-1 there is a band stretch corresponding to Si–O– Si (quartz) and 1033 cm-1 C–O there is a stretch corresponding to a polysaccharide band. There are also bands at 912 cm-1 that correspond to O–H stretch (angular carbonyls deformation outside the plane) at 802 cm-1, groups deformation outside the plane RCH2=CHR, and 744 cm -1 (aromatic ring). For all the mentioned bands it can be observed that the samples exhibit almost the same characteristics, regardless of the addition of humus. The relative intensities of the bands at 1033 and 1633 cm-1 were: Soil ( reference): 1.27 Soil dispersant: 1.46 Soil + dispersant + hummus (filter cake): 1.45 Soil + dispersant + hummus (orange peel ): 1.26 It can be seen that, compared to the reference only soil + dispersant and soil + dispersant + hummus (filter cake) samples were increased (regarding the reason between the bands at 1033 and 1633 cm-1). The soil + dispersant + hummus (orange peel) sample showed a similar behavior as the reference sample. For the experiments involving the entire formulation, the bands in 3695, 3618, 3525 and 3365 cm-1 are characteristic of inorganic impurities in these cases been O–H stretches in gibbsite. At 1612cm-1, it is found a band related to the asymmetric C-O carboxylate ions (COO-) stretch, C=C in aromatic rings stretch, C=O stretch deformation and also N–H primary amides. At 1400 cm- 1 it is found a characteristic band of CH 2 groups adjacent to the carbonyl, from angular deformation. At 1261 cm-1 there is a band of C-O related to carboxylic acids and esters and aliphatic and N–H aromatic deformation. At 1085 cm-1 there is a band corresponding to Si–O–Si (quartz) stretch and 1031 cm-1 there is a band corresponding to C–O stretch of polysaccharides. There are also bands at 912cm-1 that corresponds to O-H stretch (angular carbonyls deformation outside the plane), at 808 cm-1, there is a deformation outside the plane RCH2=CHR groups, and 538 cm-1 there is a band corresponding to C–Br stretch.
For all the bands mentioned, it can be seen that this time the samples have different characteristics. The relative intensities of the bands at 1033 and at 1633 cm-1 were: Soil (reference): 1.27 Soil + deltamethrin: 0.94 Soil + deltamethrin + hummus (filter cake): 1.08 Soil + deltamethrin + hummus (orange peel): 1.02 For all the above mentioned bands an increase in the intensity of the same order Soil + deltamethrin < Soil + deltamethrin + hummus (orange peel ) < Soil + deltamethrin + hummus (filter cake) < soil (reference) was observed. Table 1 shows the aromaticity and hydrophobicity index, obtained by FTIR. Table 1 – Aromaticity and hydrophobicity index obtained in FTIR analysis Sample Hydrophobicity Aromaticity index index Soil (reference) 0.88 1.27 Soil + 0.82 1.05 deltamethrin Soil + 0.98 1.03 deltamethrin + hummus (filter cake) Soil + 0.86 1.06 deltamethrin + hummus (orange peel) Soil + 0.84 1.44 dispersant Soil + 0.86 1.30 dispersant + hummus (filter cake) Soil + 0.82 1.25 dispersant + hummus (orange peel) When evaluating the hydrophobicity index (ratio of intensities at 2927 and 1050 cm-1 - which governs the relationship between polar and nonpolar groups – (the higher this ratio, the greater resistance to microbial degradation), the addition of hummus causes a slight increase in the relationship between polar and nonpolar structures. It is possible to infere that the dispersants have aromatic structures, since the aromaticity index was higher in samples with no deltamethrin. Even so, the changes found by the two indexes are very discrete (even with the addition of deltamethrin in soil and vermicompost). It is unable to reach any conclusion based on FTIR analyzes because the experimental period was only
367
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
two months, which is a very short time so that there could be reflect significant changes in the spectrum of humic acids. In Figure 3, the absorption spectra of UV-Vis spectroscopy of humic acids are presented.
Soil + dispersant Soil + dispersant + hummus (filter cake) Soil + dispersant + hummus (orange pill)
0,20
Absorbance
0,15
0,10
0,05
0,00
400
500
600
700
800
Wavelength (nm)
Soil (reference) Soil + deltamethrin Soil + deltamethrin + hummus (filter cake) Soil + deltamethrin + hummus (orange pill)
0,50 0,45 0,40
Absorbance
0,35
Table 2 – Humification degree by UV-Vis Sample Humification degree (UV-Vis) Soil (reference) 5.24 ± 0.98 Soil + dispersant 4.82 ± 0.88 Soil + dispersant + 4.77 ± 0.05 hummus (filter cake) Soil + dispersant + 4.64 ± 0.13 hummus (orange peel) Soil + deltamethrin 4.61 ± 0.11 Soil + deltamethrin + 4.45 ± 0.14 hummus (filter cake) Soil + deltamethrin + 4.73 ± 0.01 hummus (orange peel) Humic acids from the soil reference shows higher E4/E6 (5.24), while the humic acid extracted from soil + deltamethrin + hummus (filter cake) has the lowest ratio E4/E6 (4.45) and therefore the highest degree of humification. The addition of deltamethrin in soil causes a stress (due to the high toxicity of the pyretroid) in the earthworms body. In search of better living conditions, earthworms ingest more substrate (soil + dispersants or soil + deltamethrin), swallowing it in a more degraded degree and therefore more humified.
0,30 0,25
REFERENCES
0,20 0,15
1.
0,10 0,05 0,00 -0,05 400
500
600
700
800
Wavelength (nm)
Figure 3 – UV-Vis spectra of the humic acids (soil samples with only dispersant and with the entire formulation). Table 2 shows the degree of humification of all samples, obtained by UV-Vis.
Senesi N. Binding mechanisms of pesticides to soil humic substances. Sci Total Environ. 1992;123124:63–76. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1439745. 2. Stevenson JF. Humus chemistry: genesis, composition, reactions. New York: John Wiley; 1994:496. 3. Cotta JAO. Aplicação de vermicompostagem para a biorremediaçao de solos contaminados por hidrocarbonetos policíclicos aromáticos. 2008:199. 4. Skoog DA, Holler FJ, Nieman TA. Princípios de Análise Instrumental. São Paulo: Bookman; 2006:836. 5. Kononova MM. Soil Organic Matter. Oxford: Pergamon Press; 1966:103–106. 6. Sparks DL, Page AL, Helmke PA, et al. Methods of Soil Analysis - Part 3: Chemical Methods. Madison, Wis.: Soil Science Society of America: American Society of Agronomy; 1996:1264. doi:10.1097/00010694196511000-00020. 7. Mc Phie P. Enzyme purification and related techniques: dialysis. In: Methods in Enzymology. New York: Academic Press; 1971:23–32.
Acknowledgments: FAPESP (Projects 2011/22651-8, 2011/13294-7, 2011/13918-0 e 2012/08709-6), CNPq and IQSC
368
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Assessment of the vermicomposting dynamic by pyrolysis gas chromatography mass Spectrometry (py-gc/ms) and 13C nuclar magnetic Resonance (13C-NMR) L.B.F. Pigatin(a)*, F. Benetti Rezende(a)
(a)
, R.N. Rodrigues(a), A.V. Borsato(b), M.D. Landgraf(a), M.O.O.
(a) Instituto de Química de São Carlos, Universidade de São Paulo, Avenida Trabalhador Sãocarlense, 400, São Carlos, São Paulo, Brazil (b) Embrapa Pantanal, 21 de Setembro St, 1880, Corumbá, MS, Brazil. * [email protected] Keywords: Py-GC/MS, NMR, humic acid, vermicomposting, characterization Abstract The successful use of vermicomposting of agroindustrial and municipal solid waste depends on the establishment of reliable criteria to evaluate the quality of the vermicompost produced as well as to understand the transformations of organic matter during the process. Properly assessment of the maturity of vermicompost is crucial to establish such criteria. For this purpose, the transformations of organic matter during vermicomposting of agroindustrial solid waste correlating the spectra obtained by 13C-NMR and the pyrograms obtained by Py-GC/MS were studied. The results indicated that these organic wastes could be converted into good quality humus by vermicomposting. Introduction The carbon applied through organic wastes can lead to inadequate soil-atmosphere dynamics and favor mechanisms that negatively influence climate change. The organic material applied to the soil may find conditions that favor its decomposition, emitting a large amount of CO2 into the atmosphere and, along with other gases, increasing the greenhouse effect. The successful use of vermicomposting of agroindustrial and municipal solid waste depends on the establishment of reliable criteria to evaluate the quality of vermicompost produced as well as to understand the transformations of organic matter during the process. Properly assessment of the maturity of vermicompost is crucial to establish such criteria. This is the general direction of this study. Experiments with humic substances using NMR studies on solid samples usually use the technique of CP-MAS (Cross Polarization and Magic Angle Spinning) to monitor the 13C isotopes nuclei. The information for the humic substances obtained by NMR analyzes are: aromaticity and aliphaticity degree and structural characterization of samples by identification of compounds such as lignins, tannins, carbohydrates, alkyl groups, methoxyl, phenolic and carboxylic.1,2 Pyrolysis is characterized by the thermal decomposition of materials in an inert atmosphere, different from the combustion field where it is burned in the presence of O2 atmospheric air. In the pyrolysis technique, a macromolecule absorbs thermal energy
by rapid transfer of heat which makes the distribution of energy throughout the molecule occurs, promoting the shaft vibration of links. The vibration relaxation happens with homolytic or heterolytic breaking of the weakest links. The fragments are quickly removed, separated by gas chromatography and identified by mass spectrometry.3 The use of Py-GC/MS is a powerful technique to identifying structural units of humic substances. The results obtained by Py-GC/MS allies to NMR caracterization have placed the problem of structural determination of humic substances in a sphere beyond the imagined before the advent of spectroscopy.4 For this purpose, the transformations of organic matter during vermicomposting of agroindustrial solid waste correlating the spectra obtained by 13C-NMR and the chromatograms obtained by Py-GC/MS were studied. Experimental The experiment was conducted on an outdoor concrete platform on a farm located in the city of São Carlos, São Paulo state. The experiment was initiated in September 2011 and lasted 6 months. Approximately 300 kg of each residue (orange peel, filter cake and cattle manure) were used. The wet residues were dried at room temperature, ground in a knife mill. The experiment was conducted in triplicate. Nine mixtures (piles) were prepared using different organic residues with the following dry proportions (the residues proportion was calculated to obtain an initial C/N ratio between 25 and 40):
369
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Pile 1, 2 and 3 (P1, P2 and P3): cattle manure + orange peel (2:1); Pile 4, 5 and 6 (P4, P5 and P6): cattle manure + filter cake (3:1);
Figure 1. 13C NMR spectra (VACP/MAS) of HA extracted from vermicomposts of orange peel + cattle manure at different time (0, 30, 75, 90 and 135 days) of the vermicomposting process.
Pile 7, 8 and 9 (P7, P8 and P9): cattle manure (1) Sawdust was used as structuring agent and to assist in the aeration of these piles. Each pile presents the dimensions: 1.5 m (base) and 1.0 m (height). The piles were assembled layer by layer (approximately 20 cm each layer) in the following order: sawdust - cattle manure - orange peel or filter cake and so on and so forth. Water was added to each layer, so that the end piles were already humid. At the end the residues were mixed and the piles were molded into a conical shape. The mixtures were composted and their moisture adjusted near to 60% once a week during the composting process. The piles were manually turned each week until the 6th week. At the 6th week the composting process returned to mesophilic phase, as observed by temperature measurements and so the organic composts were moved to boxes. Five hundred earthworms were added in each box. The mixtures were vermicomposted and their moisture adjusted near to 60% once a week during the vermicomposting process. The boxes were manually turned each 15 days until the end of the process. Samples were collected at five different points of each pile. These samples were mixed and homogenized obtaining approximately 1.0 kg of composite sample of each pile. The compost piles were sampled at 0, 7, 15, 30, 45, 60, 75, 90, 105, 120 and 135 days. The samples were dried at 60 oC, then ground and sieved until particles of 0.5 mm maximum in diameter. Humic acids (HAs) were extracted according to the IHSS methodology for soils and characterized by Py-GC/MS and 13C-NMR.
Table 1. Distribution of 13C in HA extracted from samples at different times during the vermicomposting process, determined by 13C NMR, and aromaticity and aliphaticity degrees (OP+CM: orange peel + cattle manure; FC+CM: filter cake + cattle manure; CM: cattle manure)
Results and Discussion The 13C NMR spectra of HA materials are shown in Figure 1. Chemical shifts were used to identify chemical components as follows: non-substituted alkyl groups (0–45 ppm), methoxyl and N- alkyl (45–60 ppm), O-alkyl, carbohydrates and polysaccharides (60– 110 ppm), aromatics (110–140 ppm), phenolics (140– 160 ppm) and carboxyls (160–185 ppm).5 The relative distributions of 13C spectral intensity were determined by integration of the chemical shift areas (Table 1).
Table 2 shows the results of aromaticity and aliphaticity degrees from HA calculated on data of Table 1.
370
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Table 2. Aromaticity and aliphaticity degrees from HA extracted from samples at different times during the vermicomposting process (OP+CM: orange peel + cattle manure; FC+CM: filter cake + cattle manure; CM: cattle manure)
Figure 3. Pyrolysis products chromatogram of humic acids extracted from samples of orange peel + manure vermicompost (OP+CM) collected after 135 days of vermicomposting.
The 13C NMR spectra indicated the decomposition of carbohydrate type structures, polysaccharides and alkyl groups and the increase in aromatic structures during vermicomposting process. For the HA extracted from orange peel + cattle manure (OP+CM) and cattle manure (CM) vermicomposts, an increase in aromaticity degree was observed, while for the HA from filter cake + cattle manure it was not (FC+CM). After 135 days of vermicomposting the aromaticity degree was: 31, 30 and 30 for OP+CM, FC+CM and CM, respectively. Earlier studies in the literature described lower aromaticity degrees for orange peel, filter cake and cattle manure composts, such as: 24, 30 and 25, respectively, after 210 days of conventional composting.6 The Figures 2, 3, 4, 5, 6 and 7 show the chromatograms of volatiles separated after pyrolysis of both humic acids studied. In general, the chromatograms showed the same profile. The identification and possible structural composition of the products were performed by analysis of the fragmentation pattern by mass spectrometry and comparison with the database. Evaluating the results of comparison with NIRST library, some interesting differences between the humic acids extracted from the beginning and end of the vermicomposting process could be observed for the different treatments.
Figure 4. Chromatogram of the pyrolysis products of humic acids extracted from samples of filter cake + cattle manure vermicompost (FC+CM) collected at the beginning of vermicomposting (time 0).
Figure 5. Pyrolysis products chromatogram of humic acids extracted from samples of filter cake + manure vermicompost (FC+CM) collected after 135 days of vermicomposting.
Figure 6. Chromatogram of the pyrolysis products of humic acids extracted from samples of cattle manure vermicompost (CM) collected at the beginning of vermicomposting (time 0). Figure 2. Chromatogram of the pyrolysis products of humic acids extracted from samples of orange peel + cattle manure vermicompost (OP+CM) collected at the beginning of vermicomposting (time 0).
371
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Figure 7. Pyrolysis products chromatogram of humic acids extracted from samples of cattle manure vermicompost (CM) collected after 135 days of vermicomposting. As noted by Canellas et al. (2000)7 in the region of C alkyl, compounds with long alkyl chains were identified and the intensity of these signals is greater in HAs extracted from the beginning of the vermicomposting process, which is in agreement with the largest integrated area in the region between δC 0 and δC 45 observed by 13C. Compounds with fragments of higher m/z ratio were predominant in HAs extracted after 135 days of vermicomposting for all treatments, and correspond to the fragmentation patterns of mono and diaryl esters with long alkyl chain. Intense peaks attributed to fatty acids were found in the studied HAs. The presence of volatile fatty acids is an indicative of some degree of anaerobic conditions in the initial stages of composting waste.8 Another important indicator that HA studied is in a relatively early stage of development is the presence of a large proportion of esterified fatty acids and the trend with the maturation of HA7, it is the disappearance of fatty acids and presence of alkanes in the region of C alkyl. For the HA extracted after 135 days of vermicomposting, it were identified, in region C of aromatic, benzene derivatives in larger quantities. The compounds derived from benzene present in the pyrolysis of HA from OP+CM (orange peel + cattle manure) and CM (cattle manure) showed lower mass fragments, such as toluene, which were not found in HA from FC+CM (filter cake + cattle manure). These compounds were identified by Canellas et al. (2000)7, as part of aromatic compounds of humic acids extracted from vermicomposting of sludge from sewage treatment plant and municipal solid waste. In all pyrograms obtained characteristic peaks of substituted phenols were found. In oxygenated carbons, substituted furans were observed and this is characteristic of carbohydrate degradation. As observed by Canellas et al. (2000)7, the peaks obtained from the pyrolysis of nitrogenous groups revealed the predominance of nitrogen heterocyclic compounds in the structural units of humic acids, mainly derived pyrrole, substituted pyrroles and pyridines. According to the authors these compounds
represent an important source of nitrogen, when added to the soil. Benzocarboxylics acids were found in the structure of the HA extracted after 135 days of vermicomposting, which showed ketone function. These fragments were also identified in humic acids from CM at time 0. The presence of these groups may be an evidence that can justify the acidity integrated area corresponding to the region of δC 160-185 ppm in 13C-NMR spectroscopy
REFERENCES (1) STEVENSON, F. J. Humus chemistry: genesis, composition, reactions. 2. ed. New York: J. Wiley, 1994. 496p. (2) PRESTON, C. M. Applications of NMR to soil organic matter analysis: history and prospects. Soil Science, v. 161, n. 3, p. 144-166, 1996. (3) CANELLAS L. P.; OLIVARES F. L.; RUNJANEK V. M.; SANTOS G. A. Métodos Complemetares. In: SANTOS, G. A.; SILVA, L.; CANELLAS, L. P.; CAMARGO F. A. O. Fundamentos da Matéria Orgânica do Solo: Ecossistemas Tropicais e Subtropicais. 2 ed. Porto Alegre: Metropole, 2008. p. 277. (4) CANELLAS, L. P.; SANTOS, G. A. Humosfera: tratado preliminar sobre a química das substâncias húmicas. Campos dos Goytacazes: 2005. p. 309. (5) STEVENSON, F. J. Humus chemistry: genesis, composition, reactions. 2. ed. New York: J. Wiley, 1994. 496p. (6) FIALHO, L. L.; SILVA, W. T. L.; MILORI, D. M. B. P.; SIMÕES, M. L.; MARTIN-NETO, L. Characterization of organic matter from composting of different residues by physicochemical and spectroscopic methods. Bioresource Technology, v. 101, p. 1927- 1934, 2010. (7) CANELLAS L. P.; SANTOS G. A.; MORAES A. A.; RUMJANEK V. M.; OLIVARES F. L. Avaliaação de características de ácidos húmicos de resíduos de origem urbana: métodos espectroscópicos (UV-Vis, IV,RMN 13 C-CP/MAS) e microscopia eletrônica e microscopia eletrônica de varredura. Revista Brasileira de Ciência do Solo, 24:741-750, 2000. (8) KEELING, A.A., MULLET, J.A.J; PATON, I.K. CGmass spectrometry of refuse-derived composts. Soil Biology Biochemistry., 26: 773-776, 1994.
Acknowledgments: FAPESP (Projects 2011/13294-7, 2011/22651-8,2011/13918-0 e 2012/08709-6), CNPq and IQSC/USP.
372
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
Pyrolysis-compound specific isotopic analysis (Py-CSIA) of the Senonian (Upper Cretaceous) conifer Frenelopsis oligostomata J.A. González-Pérez(a)*, N.T. Jiménez-Morillo(a), J.M. de la Rosa(a), G. Almendros(b), F.J. González-Vila(a) (a) IRNAS-CSIC. Avda. Reina Mercedes, 10. Sevilla, E-41012 (Spain). (b) MNCN-CSIC. Serrano 115bis, Madrid, E-41080 (Spain). * Corresponding author e-mail: [email protected] Keywords: Paleoecology, Fossil organic matter, Analytical pyrolysis, Stable isotopes, Py-CSIA Abstract. Bulk δ13C indicate that Frenelopsis oligostomata was adapted to highly xeric and saline conditions. This is the first report of δD, δ15N and δ18O values for Frenelopsis organic fossil remains. and its humic fractions in bulk and also for δD, δ13C for specific compounds. The δ13C Py-CSIA values are given for a number of biogenic molecules and for probable diagenetic compounds. Py-CSIA gives better approach to the real δ13C (c. -22 ‰). Values for δD Py-CSIA of lipids in the humin fraction is assumed to be representative of the original hydrogen isotopic signal of water in the area in the Upper Cretaceous (δD= -124.44±5.21). Shifts towards depleted δD Py-CSIA values of other compounds may reflect different rates of hydrogen exchange mainly with the environment water during diagenesis. Then Py-CSIA provides valuable clues to reconstruct palaeoecosystems. Introduction Frenelopsis (Schenk) emend. Watson (Cheirolepidiaceae) is a frequently found genus of the Cretaceous floras. The morphology include features to prevent water loss and is indicative of adaptation to dry and saline environments i.e. thick cuticles, stomata deeply sunken in pits and strengthened by papillae in the succulent, photosynthetic stems. Diversity found in Iberian Peninsula fossils has been related with adaptation to diverse ecological constraints always characterized by water stress [1]. Stable isotope analysis of fossil organic materials can be used to infer palaeoenvironmental variables helpful to reconstruct plant paleohabitats [2]. In this study stable isotope analysis of well-preserved organic fossil remains (FR) of Frenelopsis oligiostomata (in average 776 g kg-1 C) and humic extracts (humic acid (HA), fulvic acid (FA) and humin (HU)) are studied. The analyses were done in bulk (C, H, O, N IRMS) and in specific compounds released by pyrolysis of the fossil remains and its humic fractions (C, H, Py-CSIA). Experimental The fossil, geological setting and extraction of humic substances: well preserved fossil plant remains were taken from a limestone included in compacted marls from Upper Cretaceous (Senonian c. 72 Ma) in Guadalix de la Sierra (Madrid, Spain) [3]. The fossil presented features indicative of adaptation to a xeric and saline habitat i.e. thick cuticles and cell walls and cryptoporous stomata. The limestone rock was cracked and Frenelopsis remains handpicked. The rest was dissolved with 6M HCl and sieved to collect fossils and includes in a coarse fraction. Finally a light fraction mainly of Frenelopsis remains was separated using an ethanol-bromoform mixture (δ=1.8 g cm-3). Humic substances were extracted from finely ground fossil remains (FR) by successive treatments with 0.1M Na4P2O7 + NaOH [4]. The extract was acidified resulting into insoluble HA and soluble FA fractions. The HA and FA were purified as in [5] and [6] respectively.
Bulk stable isotopic analysis (δ13C, δD, δ18O, δ15N IRMS): was done in a Flash 2000 HT elemental microanalyser coupled, via a ConFlo IV, to a continuous flow Delta V Advantage isotope ratio mass spectrometer (IRMS). Isotopic ratios are reported as ‰ deviations from International Atomic Energy Agency (IAEA) standards. Standard deviations of bulk δ13C, δD, δ15N and δ18O were less than ± 0.05‰, ± 2‰, ± 0.2‰, ± 0.5‰ respectively. Pyrolysis compound specific isotopic analysis PyCSIA (δ13C, δD): was done by coupling a double-shot pyrolyzer to a chromatograph connected to an IRMS. Samples (1–2 mg) were introduced in a micro-furnace (500 °C) for 1 min. Evolved gases were injected into the gas chromatograph (GC) fitted with a low polarity HP-5ms-UI column using He as carrier gas. To locate specific peaks the flux was divided 10% to a flame ionization detector (GC/FID) and 90% to a GC-Isolink with micro-furnaces for combustion (C) and pyrolysis (TC) coupled via ConFlo IV interface to the Delta V IRMS (Py-GC-(FID)-C\TC-IRMS). Isotopic ratios are reported as ‰ deviations from IAEA standards. Standard deviations of compound specific δ13C and δD, was less than ± 0.1‰ and ± 5‰ respectively. Structural features of specific peaks were inferred by comparing/matching mass spectra from conventional Py-GC/MS (data not shown) with the PyGC/FID and Py-GC/IRMS chromatograms obtained using the same chromatographic conditions. Results and Discussion Bulk isotopic signatures: (Table 1) C isotopic signatures are in accordance with previous studies [2, 7–9]. The heavy isotopic δ13C indicate a depleted stomatal conductance and paleoenvironmental growth conditions of water and salt stress. This is in line with the morphological and depositional characteristics [3] indicating that F. oligostomata was adapted to highly xeric and saline habitats being a component of saltmarsh vegetation. To the best of our knowledge, this is the first time that δD, δ15N and δ18O have been analysed in plant (bulk) and humic fractions and, although the values found lay within those previously
373
17th Meeting of the International Humic Substances Society Ioannina, Greece 1-5 September 2014
reported for fossil floras [10] the interpretation is still on its way. Compound specific isotopic signatures: (Table 2, Fig. 1) for the first time δD, δ13C is reported in specific compounds of Frenelopsis remains and humic extracts. δ13C Py-CSIA was recorded for biogenic compound; polysaccharides, lipidic series, lignin (coumarin) and for probable diagenetic S contining compounds. Values for δ13C Py-CSIA are more depleted that the bulk ones and can be considered a better approach to the real value that for our fossil plant δ13C will be c. -22 ‰.; the marl, rich in heavy C (carbonates), may contribute to the bulk heavy δ13C. Table 1. Bulk stable isotope ratios (‰) of F. oligostomata fossil and humic fractions. δ13C δD δ18O δ15N FR -20.54±0.02 -101.9±2.16 20.94±0.39 10.66±0.18 HA -20.52±0.38 -113.46±0.52 14.03±0.31 14.85±0.71 FA -19.83±0.09 -138.35±0.02 25.39±0.50 22.22±1.41 HU -21.19±0.10 -130.37±5.18 13.42±0.04 10.06±5.41
Values for δD CSIA of lipid compounds such as nalkanes with C chain lengths, C23–C31 are believed to derive exclusively from leaf waxes of higher plants. Plant δD carries information isotope information of environmental water that is particularly preserved during the geological record in n-alkyl structures, whereas other structures i.e. isoprenoids are most prone to hydrogen exchange [11–12]. Therefore the values of δD Py-CSIA shown in Fig. 1 for alkane/alkene pairs in the HU fraction, at least the series C24–C29, indicate the original hydrogen isotopic signal of water in the area in the Upper Cretaceous (δD = -124.44±5.21). Shifts towards depleted δD values in isoprenoid structure (16) and in high molecular weight alkanes (C30–C31) may reflect hydrogen exchange during the geological record. The variations observed in δD Py-CSIA of other compounds probably indicate different rates of hydrogen exchange mainly with the environment water during diagenesis.
Figure 1. Compound specific isotopic analysis of alkane/alkene pairs released by direct pyrolysis (Py-CSIA) of F. oligostomata humin fraction. The void circle represents isoprenoid Ref. 16 (Table 2). Vertical bars are STD (n≥3).
REFERENCES
(1) Gómez, B.; Martín-Closas C.; Brale G.; Solé de Porta N.; Thévenard F.; Guignard G. Paleontology 2002 45, 997–1036. (2) Nguyen Tu, T.T.; Kvaček, J.; Uličnỷ, D.; Bocherens, H.; Mariotti, A.; Broutin, J. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2002 183, 43–70. (3) Almendros, G.; Álvarez-Ramis, C.; Polo, A. Revista de la Real Academia de Ciencias Exactas, Físicas y Naturales 1982 76, 285–302. (4) Dabin, B. Chah. ORSTOM Ser. Pedol. 1976 4, 287– 297. (5) Schnitzer, M.; Khan, S.U. Humic Substances in the Environment. Marcel Dekker Inc. 1972, New York, N.Y. (6) Dorado, E.; Polo. A. An. Edafol. Agrobiol. 1976 55, 723–732. (7) Bocherens, H.; Friis, E.M.; Mariotti, A.; Pedersen, K.R. Lethaia 1993 26, 347–358. (8) Nguyen Tu, T.T.; Bocherens, H.; Mariotti, A.; Baudin, F.; Pons, D.; Broutin, J.; Derenne, S.; Largeau C. Palaeogeogr. Palaeoclimatol. Palaeoecol. 1999 145, 79–93. (9) Aucour, A-.M.; Gomez, B.; Sheppard, S.M.F., Thévenard, F. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2008 257, 462–473. (10) Michener, N.; Lajtha K. (Eds). Stable Isotopes in Ecology and Environmental Science (2nd Ed) 2007 Blackwell Publishing. (11) Pedentchouk, N.; Freeman, K.H.; Harris, N.B. Geochim. Cosmochim. Acta 2006 70, 2063–2072. (12) Radke, J.; Bechtel, A.; Gaupp, R.; Püttmann, W.; Schwark, L.; Sachse D.; Gleixner, G. Geochim. Cosmochim. Acta 2005 69, 5517–5530.
Table 2. Compound specific isotope analysis (‰) of molecules released by direct pyrolysis (Py-CSIA) of F. oligostomata and its derived humic fractions. Ref*
Compound
1 2 3 5 9 12 13 18 27 33
Benzene, methyl (Toluene) Benzene, dimethyl (Xylene) 2-Cyclohexen-1-one Phenol Phenol, methyl Phenol, ethyl Phenol, dimethyl Phenol, trimethyl Decanoic acid 2H-1-Benzopyran-2-one (Coumarin)
1 2 3 4 5 6 9 14 16 20 22
Benzene, methyl (Toluene) Benzene, dimethyl (Xylene) Phenol Benzene, propenyl (Allylbenzene) Benzene, trimethyl Phenol, methyl Phenol, ethyl Benzofuran, 4,7-dimethyl Naphthalene, methyl Naphthalene, dimethyl Naphthalene, trimethyl
5 6 11 16 20 26
Phenol 2,5-Furandione, dihydro3-Thiophenecarboxylic acid 3,4-Dicyanothiophene 3H-1,2-Dithiole-3-thione, 4-methyl3H-1,2-Benzodithiol-3-one
1 2 6 9 16
Methylthiophene Benzene, dimethyl (Xylene) Phenol, methyl Phenol, ethyl1-Heptene, 2-isohexyl-6-methyl-
δ13C FR -22.24±0.07 -22.16±0.08 -21.76±0.25 -22.23±0.17 -22.33±0.27 -22.04±0.19 -22.48±0.31 -22.33±0.40 -22.08±0.20 -21.36±0.85 HA -23.36±0.32 -23.36±0.34 -22.86±0.23 -22.73±0.32 -22.38±0.15 -21.93±0.20 -21.38±0.61 -21.44±0.69 -21.85±0.26 -18.34±0.65 -19.37±0.10 FA -19.50±0.17 -18.51±0.26 -21.34±0.15 -19.16±0.23 -21.36±0.26 -20.79±0.18 HU -23.77±0.20 -23.90±0.42 -22.86±0.01 -23.72±0.62 -27.99±0.99
δD
Class/Origin
-122.66±2.52 -122.94±1.03 -119.82±1.90 -120.22±1.45 -120.24±2.06 -117.46±1.06 -121.27±2.09 -114.57±2.01 -112.06±1.43 -113.50±0.57
Benzenes Benzenes Polysacch. Phenols Phenols Phenols Phenols Phenols Lipids Lignin
---127.62 -129.64 -130.43 -134.81 -134.36 -141.86 -145.04 -150.27 -167.47 ---
Benzenes Benzenes Phenols Benzenes Benzenes Phenols Phenols Polysacch. PAH PAH PAH
-95.43±8.85 -115.48±4.97 -87.04±2.16 -------
Phenols Polysacch. S compound S & N comp. S compound S compound
-142.50±3.19 -150.14±1.07 -152.34±2.72 -154.70±1.21 -218.26±6.82
S compound Benzenes Phenols Phenols Lipids
Acknowledgements Projects CGL2012-38655-C04-01 and CGL200804296 and fellowship BES-2013-062573 given by the Spanish Ministry for Economy and Competitiveness to N.T.J.M.
374
A Abaalkheel I. Abaker M. Abakumov E. Abiz E. G. Adamo P. Adepetu J. A. Adesanwo K. J. Adesanwo O. O. Adu A.A. Agar G. Aguiar N. O.
Akinoğlu G. Alfantokh S. Alidoost M. Almendros G. Alrefaie J. Alsewailem F. Angeletti C. Antilén M. Antonopoulou M. Aoyama M. Appiani E. Araújo B.R. Arbelo C.D. Aristilde L. Arslan E. Asakawa D. Atanassova I. D. Auccaise R. Azeez M. O.
B
Bacaicoa E. Badalikova B. Baigorri R.
Balieiro F. C. Barančíková G. Bekier J. Belik A.A. Bell N. G. A.
140 5 17 9 7 110 299 110 299 208 335 311 144 220 140 355 373 140 140 74 88 329 60 319 150 224 262 208 254 130 144 110 94 92 19 94 92 90 78 281 15 264 256 234
Bellera C. Benetti F Berbara R.L.L Berns A.E. Beznosikov V.A. Bieluczyk W. Bikiaris D. N. Blackburn J.W.T. Bletsa E. Bloom P.R. Bogdanov M. Bognola I. A. Bogolitsyn K. Borggaard O.K. Borsato A.V. Botero W.G. Brezonik P. L. . Brocchi E.A. Brookes P. Brown A. Bueno C. C.
C
Cabrera JM. Campos L. Cancian A. Canellas L.P.
Cao X. Caricasole P. Carleer R. Casanova E. Catoni M. Cavagnaro T. R
140 365 369 156 186 32 108 281 345 234 289 3 118 287 341 124 341 339 116 365 369 307 309 120 118 122 357 46 84 222 327 230 250 295 335 311 144 29 246 80 92 333 210 222 194
Celi L. Ceretta C.A. Cerqueira W.V. Chanton J. Chen Y. Chen H.
Chilom G. Cho J. Clark J.M Čokeša Dj. Comans R.N.J. Contin M. Cooper W. Cora J. Cory R.M. Cozzolino V. Cwielag – Piasecka I. Czech J.
D
D’Orazio V. Da Silva W.T.L. Dai Zhongmin Daikopoulos C. de Almeida V. R. De la Rosa J. M.
De Nobili M. De Paula B.S. Debicka M. Delay M. Deligiannakis Y.
Demachi T. Diaz M. Dick D. P .
333 295 192 104 168 114 248 158 178 164 154 228 325 46 104 250 118 331 264 80 295 7 301 84 329 102 202 204 186 373 46 301 54 31 329 317 314 289 3 44 230 126 357 172 124
DiDonato N. Dieckow J. Dijkstra J.J. Dinnes D.L. Disko U. Dittmar T. Dittz L.A. Dizman M. Doerr S. H. Domeizel M. Domingues M. T. dos Reis D. A. dos Santos C. H. Doskočil L. Drosos M.
Duarte A.C. Duarte R.M.B.O. Dudare D. Dumontet S. Dvorski S. Dzheldybaeva I.
E
Einschlag F.S. Enev V. Erro J. Esteves V. I.
F
Fadeev V. Faundéz M. Fernandes M. L. Ferreira T. M. Filcheva E.
Filipiak A. Finlay J. C. Fiori A.F. Fracácio R.
122 295 158 124 325 291 76 240 323 72 220 130 5 327 200 200 343 70 148 52 29 176 176 11 297 170 349 230 275 226 92 78 190 266 88 200 136 272 132 98 142 120 333 252
Fraceto L. F. Francisco S San Freeman C. Friese K. Frimmel F. H. Fuentes M.
Fujitake N.
G Gan H. M. Ganjali A. Gao Yuan García A.C García-Mina J.M.
Garnica M Gatselou V. Gaupp R. Giokas D. Gladkova M. Glaser B. Gleixner G. Glowacki M. Goldt A. Gonsalvesh L. Gontijo E. S. J.
González-Pérez J. A.
González-Vila F. J.
Goodilin E. Gorczyca B.
327 92 154 200 31 94 92 90 254 214 194 7 321 156 94 90 78 92 92 94 96 240 96 212 256 242 240 142 266 80 200 252 216 206 172 224 373 206 224 204 373 266 138 40 38
Goto A. Graham M. C. Graham N.J.D Green N. W. Guitoku M. Gulluce M. Gutsche A
268 234 154 238 303 208 180
H Habova M. Haderlein S. Hanke D. Hanson T. Harir M. Hasegawa T. Hatcher P.G.
Hayes M. H. B. He Yan Hertkorn N. Hodgkins S. Hoffman L. Hofmann D. Holm P.E. Horuz A. Hybler V.
I
Ilieva R.
J Jackson W. R. Jacundino J.S. Jamroz E. Jandak J. Jensen J.K. Jerzykiewicz M.
19 48 124 246 170 106 268 246 158 118 248 293 283 114 112 106 170 104 178 76 116 220 72 21
98 132 222 194 307 264 54 21 116 264
Jiménez-Morillo N. T.
Johnson-Edler C. Jovanović U. Jung J.
56 54 206 373 172 178 228 218
Konstantinov A.I. Koopal L. K. Koper R. Korak J.A. Kordi A. Korkmaz A. Korshin G. V. Kostka J. Kostyukevich Y.
349 256 360 275 152 268 365 314 210 44 168 160 160 118 268 11 1 275 226 152 146 36 13 204 202 186 172 214 162 54 232 360 32 188 236 329
Koukoulakis P.H. Kraemer V. Krasnova E. Kusakabe T. Kussow W.R. Kuzyakov Y. Kyzas G. Z.
K Kairbekov J. Kalabin G.A . Kalavrouziotis I.K. Kalina M. Kanamori M. Kanashiro M.M. Kappa S. Kargosha A. Katayama A. Katz S. Kharcheva A. Khundzhua D. Khwaja A.R. Kirishima A. Klavins M. Klučáková M.
Klymenko N. Knicker H.
Kobayashi T. Kobierski M. Kocowicz A. Kodama H. Kokkinos P. Kondratenok B. Kononikhin A. Konstantinou I.K.
L
Lambropoulou D. A. Landgraf M.D. Leal O. A. Leal J. F. Lebedev V. Lee E. Leenheer J. Leite A.B. Lepane V. Levshina S. Li Aimin Li Y. Li Zhiling Lima L.S. Lin X. Lira V.S. Litavec T. Little K. R. Lobianco D. Lodygin E.
Lombardi K. C. Lopéz-Martín M.
236 196 325 174 7 220 321 104 236 188 360 126 160 260 291 242 345 345 365 369 206 172 190 266 164 29 122 34 68 86 170 44 311 104 252 15 194 222 7 32 108 17 172 258 202
Louloudi M. Lourenzi C.R. Lucas Y. Lucio M. Ludtke A. C. Luneva A. Lv Y. Z.
M
Macháčková M. Maciel V. G. Maia C. M. B. F. MaltsevaE. Mangrich A.S. Mao J.-D. Maorofdoost M.M. Marinov S. P. Marković M. Martin M. Martinez-Balmori D. Matiushkina L.A Matos J.T.V. Mätzler C. Mazzei P. McNeill K. Medynska – Juraszek A. Melo V. F. Mendes O.T. Merino A. Mezzapesa G.N. Miano T.
Michalchuk A. Miller A. Z. Milori D. M. B. P.
Miyajima T. Miyata Y. Molina-Velasco M. Monaci E. Monda H. Mondelli D. Monteiro A. S. C.
186 3 295 301 170 126 244 82 70 172 303 339 150 29 353 80 228 333 335 279 176 76 52 319 264 124 258 182 297 295 297 7 234 204 305 250 343 301 232 268 186 74 331 297 252
Montes C. R.
Mora V. Morosino L. Mouloubou O. R. Mounier S.
Murray L. Mylotte R.
N
Nagao S. Nahas E. Naisse C. Najwer K. Nakata K. Nankova M. Naydenova L. Nebbioso A. Nebenzahl A. D. B. Neizvestnaya NG Nicolodelli G. Nikolaev E.N. Novák F. Novotny E. H.
Ntzala G.
O
Ochi Y. Ochiai S. Odu B.O. Ogutcu H. Okuyama Y. Olaetxea M.
200 216 343 305 301 94 92 126 285 285 5 343 234 293 268 27 242 142 232 134 272 148 52 172 122 256 343 305 236 188 226 335 323 277 192 144 360 214 268 299 208 214 92 94
Olivares F.L. . Oliveira D. Oliveira L.C. Oliveira A. F. Oliveira R.R. Olk D.C. Olmanson L. G. Orlov A. Orsetti S. Oufqir Sofia A. Özcan C.
311 335 250 307 136 277 291 120 339 48 287 220
Pirahouskaya H. Pisarek I. Piscitelli L. Polyakov A. Ponomarenko SA Popov A. Popov I. Pospisilova L.
Pan Bo Paneque M. Papageorgiou A. Parfenova L.
50 204 42 341 339 164 160 222 210 194 78 357 70 341 238 258 281 192 236 188 180 266 337 331 148 52 281 365 369 309 309 216 192
Purmalis O.
P
Park J. Patsaeva S. Patti A. F.
Pazos-Pérez N. Pechoa Renzon Cosme Pekař M. Pekhtereva V. Perdue E. M. Pereira F.B. Pereira M.G. Pereira Netto A.D. Perminova I.V.
Piccolo A.
Piccolo M. C. Pigatin L.B.F. Pineau M.A Pinheiro J.P. Pinheiro M.A.
Preuss G. Proctor M Prudent P.
Q R
Radmanović S. Raeke J. Rambo M.K.D. Ramos A.C. Rapetti N. Rasmussen S.B. Rauen C. C.M. Reemtsma T. Reis J.O.M. Reis L. C. Rezende E.I.P. Rezende M.O.O. Ribeiro B.R. Rice J. Rice C. Richard C. Richards K.M. Ritson, J.P. Rittl T.F. Rocha L.S. Rodrigues A.F. Rodrigues R.N. Rodríguez-Eugenio N. Rodríguez-Rodríguez A. Roeser H. M. P. Renan Arnon R. Romão L.P.C.
23 142 297 266 180 244 188 21 19 76 90 285 5 1
228 184 144 311 5 116 303 184 150 281 150 365 369 323 178 312 114 64 246 154 192 309 277 365 369 224 224 200 343 150
Rosa A. H.
Rosa E. Rosario-Ortiz F.L. Rose M. T.
Roth V.-N. Rovira P. Rovira P. Rumpel C.
S
Sá T.R. Sadrnourmohamadi M. Sahin F. Said-Pullicino D. Saito T. Sala A. Samsoni-Todorova O. Santos E. B. H. Santos O.S. Sarah E. Sardashti A.R.
Sasnow S. S. Savchyna L. Savić A. Schmidt-Rohr K. Schmitt-Kopplin P. Schwegmann H. Sedláček P. Selyanina S. Seyedbagheri Mir-M Shaaban A. Shimizu Y. Shirshin E. Shotyk W. Signor D.
327 200 252 216 162 174 210 222 194 240 182 140 242 252 40 38 208 333 198 182 13 190 307 319 355 353 9 7 262 13 228 29 170 106 31 146 152 339 341 128 297 260 266 7 303
Silva T.S. Silva W. T. L. Sirotsky S. Skic K. Skoutelis C.G. Sleighter R.L. Sleighter R.L. Smedley K. Smilek J. Smith A. Sodano M. Sokolova T. Sokołowska Z. Soleimani M. Song G.X. Soni M. Souza S.O. Spaccini R.
Stathi P. Stefaniak S. Stefanova M. Steffen B. Steinnes E. Stepanov A. Strigutskiy V. Stylianou S. Suursööt K. Suzuki D. Suzuki T. Swift R. S. Szpoganicz B.
T Tadini A. M. Tarchitzky J. Taspinar M.S. Tavares R.L.M
323 305 68 166 329 289 246 118 270 152 146 312 114 333 341 166 116 82 270 307 337 335 331 52 3 66 80 76 66 58 339 42 34 44 268 283 136 102 100 136 305 343 168 208 27
Taylor R. Templeton, M.R. Terekhova V. Tfaily M. Theraulaz F. Tomashunas V. Tomihara S. Tomson A. Tonello P. S. Toner B. M. Traversa A. Troncoso M. Trubetskaya O.E. Trubetskoj O.A. Trufanova M. Tsyganov A. Turan M. Tutar A. Twardowska I.
U
Uemura T. Uhrín D. Urrutia O.
V Väli A.-L. van Zomeren A. Vashishta A. Vasilevich R. Vaz D.O. Velasco-Molina M. Vetvicka V Vianna M.S. Vidali M.S. Vinci G. Vischetti C. Visser A.-N. Vlastos D. Vlcek V. Vlessidis A.
270 154 212 256 104 285 17 268 339 200 287 295 88 64 62 64 62 341 339 341 208 72 66 268 234 92 78 34 325 90 32 108 100 204 90 323 289 148 52 74 48 289 21 96
Volikov A.B. Voronov D. Voutsa D.
180 160 42
Wada N. Waggoner D.
260 158 248 76 200 252 327 216 264 54 262 351 196 196 86 174 312 114 242 248 138 162
W
Wagner G. Watanabe C. H.
Weber J. Wei H. Weidlich T. WenFeng Tan Weng LiPing Wentao Li Wert E.C. White B. Wiedner K. Willoughby A.S. Winning L. Wojewódzki P.
X
Xing B. Xiong J. Xu Jianming
Xu Yan
Y
Yakimenko O.S.
Yamamoto M. Yamashita Y. Yanagi Y. Yankov P. Ye Lizhen Yermoldina E. Yılmaz R. F. Yoo J.
50 196 114 112 84 46 112 291 256 58 268 198 214 134 44 349 72 218
Yperman J. Yuzhakov V. Yvin J.C.
80 160 90
Zaccone C. Zacharias I.
7 317 314 94 92 90 317 314 44 50 112 82 349 236 188 272 114 42
Z
Zamarreño A.M
Zamparas M. . Zhang C. Zhang Di Zhang Qian Zhao N. Zharmagambetova A. Zherebker A. Zhiyanski M. Zhu Xinyu Zouboulis A.I.