| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Dep. of Soil Science, North Carolina State Univ., Raleigh, NC 27695
b Dep. of Physics, North Carolina State Univ., Raleigh, NC 27695
Corresponding author (kim_hutchison{at}ncsu.edu)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
70% (mol/mol) reduced S (modeled as benzyl disulfide) and 30% (mol/mol) oxidized S (modeled as sulfonate and ester sulfate). For humic acid exposed to aeration for 4 h at pH levels between 11.5 and 13.0, reduced organic S was oxidized only at pH 13.0 (15% [mol/mol] of total S). Samples exposed to aeration for 44 h between pH 3.5 and 12.4 showed no detectable change in reduced organic S.
Abbreviations: CV, coefficient of variation • IHSS, International Humic Substance Society • LDPE, low-density polyethylene • OM, organic matter • XANES, x-ray absorption near edge structure • UV, ultra-violet
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Organic S in OM exists across a range of oxidation states. Using XANES spectroscopy, Morra et al. (1997) reported electronic oxidation states of +6 (ester-bonded sulfates), +5 (sulfonates), and 0 to +3 (low-valent) S species. Differences between formal and electronic oxidation states occur for lower valence organic S compounds because of changes in electron distribution resulting from covalent bonding (Waldo et al., 1991; Vairavamurthy et al., 1993, 1997). Reduced organic S compounds with lower formal oxidation states ranging from S(-I) to S(0) (as in thiols, organic sulfides, organic polysulfides, and thiophenes) exhibit electronic oxidation states in the range of 0.5 to +1 (Xia et al., 1998). Using XANES spectroscopy, Xia et al. (1998) identified four major S groups in humic acids having average electronic oxidation states of +0.2 to +0.6 (sulfide and thiol), +1.8 (sulfoxide), +5 (sulfonate), and +6 (sulfate). Waldo et al. (1991) reported electronic oxidation states of 0 to +3 for organic sulfides and disulfides, thiols, methionine, thiophenes, and sulfoxides. Vairavamurthy et al. (1997) reported that the major forms of S in sedimentary humic substances were organic sulfides, organic polysulfides, sulfonates, and organic sulfates. The amount of reduced S ranged from 15% (mol/mol) of total S for soil humic acids (Vairavamurthy et al., 1997; Xia et al., 1998) up to 72% (mol/mol) of total S for a salt marsh humic acid (Vairavamurthy et al., 1997).
Reduced S-containing functional groups have been found to play a significant role in the binding of soft metal cations such as Hg(II). For example, recent x-ray absorption spectroscopy studies showed the preferential binding of Hg(II) to S rather than O2-containing functional groups (Hesterberg et al., 1999; Xia et al., 1999). Because the complexation of metals with humic acids is often studied to understand reactions occurring in OM, soil sampling techniques and humic acid extractions should ideally inhibit the oxidation of reduced S groups such as thiols (R-SH). A general oxidation reaction of a thiol compound would be
 | (1) |
Because it is difficult to compare data on humic acids extracted by different methods, the International Humic Substance Society (IHSS) proposed a standard extraction method (Swift, 1996). This method uses extraction solutions ranging from high pH to solubilize humic acid, to pH 1 for precipitation of the humic acid. It is recommended that alkaline extractions be performed under an inert atmosphere because autooxidation and humification by condensation reactions of some organic constituents increases with increasing alkalinity in the presence of O2 (Stevenson, 1994; Ziechman, 1994; Bremner, 1950). However, it has not been established how changes in pH affect the stability of reduced S groups in humic acid, or how sensitive these reduced groups are to O2 exposure. Although the yield of humic acid typically increases with increasing extraction pH (Stevenson, 1994), it is not known how extraction pH affects S speciation in the humic acid. In this study we determined yields of humic acid extracted at different pH levels, and used XANES spectroscopy to study changes in S oxidation states. The objectives of this study were (i) to determine how extraction pH affects humic acid yield and oxidation states of organic S in humic acid and (ii) to determine the effect of pH and aeration on S oxidation states.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Humic Acid Extraction at Varying pH
The humic acid extractions followed the IHSS method (Swift, 1996), with some exceptions. Essentially all work was performed under O2-scrubbed Ar(g) and under a red-filtered safe light. All solutions were made using deoxygenated, deionized water prepared by boiling for 15 min and purging with N2(g) while cooling. Deoxygenated solutions were stored in glass bottles for no longer than 48 h. Twelve 20-g (dry weight basis) subsamples of thawed soil were transferred to 250-mL high-density polyethylene centrifuge bottles and acidified to pH 2 with 1.0 M HCl. After shaking for 1 h, the samples were centrifuged at 1500 g for 10 min and the clear supernatant solution was decanted. Four samples, each comprising three combined subsamples, were extracted with base. These were initially adjusted to pH 11.5, 12.2, 12.5, and 13.0 using 0.10 or 2.0 M NaOH, then brought to 610 mL with deionized H2O. Soils were shaken for 4 h and allowed to stand under Ar(g) for 16 h at room temperature (Swift, 1996), then centrifuged at 1500 g for 10 min. The pH of each sample was checked after shaking (pH varied by 
0.3 pH units), and readjusted to achieve the target pH values after the 16-h standing period. The supernatant solution was collected, and dissolved humic and fulvic acids were separated by acidifying the solution to pH 1 using 6.0 M HCl, and allowing the samples to stand for 16 h at room temperature. After centrifuging the samples at 1500 g for 10 min, the supernatant solution containing fulvic acid was decanted.
The ash content of the humic acid was reduced by dissolving the humic acid at pH 11.0 (adding 1.0 M KOH) and adding KCl to bring the solution to 0.3 M KCl (Swift, 1996). These solutions were shaken for 30 min and centrifuged in 50-mL polypropylene tubes for 4 h at 43000 g. To further reduce the ash content, the humic acid suspensions were decanted and syringe filtered two times through a 0.2-µm filter (Gelman Supor, Gelman Sciences, Ann Arbor, MI). The filtered solutions were acidified to pH 1 with 6.0 M HCl and centrifuged at 9000 g. The humic acid extracts were washed two times with 20 mL of a 1:1 solution of 0.10 M HCl/0.30 M HF. Only the shaking steps, filtration steps, and the HCl–HF wash were not performed in a glove box. To minimize exposure to O2 during shaking, the headspace of each sample tube was flushed with Ar(g). The tubes were also tightly covered with aluminum foil to limit exposure to UV radiation and to reduce diffusion of air into the samples. To remove hydrofluoric acid residues, the humic acid was washed two times with 30 mL of deionized water. The centrifuged samples (at pH 2.3) were saturated with Na+ and NO-3 by adding 0.3 M NaNO3, shaking for 30 min, and centrifuging. Excess salts were removed by washing five times, each with 30 mL of deionized water. The humic acid was freeze-dried and stored in sealed containers at -14°C until XANES analyses were performed.
Humic Acid Aeration Experiments
Preparation of Humic Acids 1 and 2
Because there were no differences in XANES results from humic acids extracted at different pH levels (Table 2, discussed below), 28 mg of each humic acid sample extracted at a different pH were combined as one sample for the 4-h aeration experiment. This sample will be referred to as Humic Acid 1. Humic Acid 2 (44-h aeration experiment) was extracted as outlined above, with the following exception. Three samples, each comprising six combined subsamples, were adjusted to pH 12.8 with 2.0 M NaOH for the initial base extraction. The composite sample was brought to 1200 mL with deionized H2O before shaking for 4 h.
|
0.25 M NaOH added to bring to pH 11.5. After shaking for 2 h, the stock solutions were adjusted to a 0.10 M NaNO3 background by adding 0.35 M NaNO3 solution. For aeration experiments, 4 mL (7.1 mg humic acid g-1) of solution for the 4-h experiment or 12 mL (6.7 mg humic acid g-1) of solution for the 44-h experiment were pipetted into glass Erlenmeyer flasks and adjusted to various pH levels using 0.10 M HNO3 or various concentrations of NaOH solution. Typically, 0.10 or 0.25 M NaOH was used, but to achieve the highest pH (13.0), we added relatively minor amounts of 6 M NaOH. These solutions were brought to a volume of 15 mL (4 h experiment) or 60 mL (44 h experiment) using 0.1 M NaNO3 solution to maintain a 0.1 M Na background electrolyte when possible. The samples were purged with O2-scrubbed Ar(g) for 2 h before aerating for 4 or 44 h. Ambient air was passed through a 1.0 M NaOH solution to trap CO2 and aspirated into the humic acid samples at 0.2 L min-1. The pH of the samples was periodically measured and adjusted as needed to the target levels using 0.10 to 0.25 M HNO3 or 0.10 to 6 M NaOH. Most samples were kept at the same volume by adding NaNO3 solution to compensate for acid or base additions to some samples. Due to the high pH buffering capacity of the humic acid at pH >12, the final volume of the pH 13.0 sample (4-h experiment) was about twofold greater than for other samples in the set, and the volume of the pH 12.4 sample (44-h experiment) was 1.3-fold greater. Because more concentrated base solution was needed to achieve the higher pH levels, these samples also had higher final Na+ concentrations, 0.2 M (pH 13.0 sample) and 0.11 M (pH 12.4 sample). After the aeration period, solutions were purged with O2-scrubbed Ar(g) for 2 h. Each solution was adjusted to pH 2.0 with 0.10 or 0.24 M HNO3 and centrifuged at 11953 g for 15 min. The humic acid samples were washed two times with deionized water to remove most of the background electrolyte, then freeze-dried and stored at -14°C until XANES analyses were performed.
Sulfur K-XANES Analysis
High purity standards (
98%) for XANES analysis were purchased from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO) chemical companies. Standards were prepared at 650 mmol S kg-1 in boron nitride and pressed into disks having a 1.3-cm diameter and a 1-mm thickness. Freeze-dried humic acid samples were sealed in 3-µm-thick mylar film bags (Spex Industries, Edison, NJ) and mounted in the plexiglass holders.
Sulfur K-XANES data were collected in fluorescence mode at beamline X-19A at the National Synchrotron Light Source, Brookhaven National Laboratory. The electron beam energy was 2.5 GeV and the maximum beam current was 300 mA. Samples were held in a He atmosphere during data collection and fluorescence yield was measured using a passivated implanted planar silicon detector (Canberra Industries, Meriden, CT). The beamline was equipped with a double-crystal Si (111) monochromator, which was detuned 80% during data collection. The x-ray energy was calibrated to the K-edge of elemental S, 2472 eV, using a sample that was diluted in boron nitride to 650 mmol S kg-1.
For most samples, multiple scans were collected and averaged, and the data were baseline corrected using a linear regression between -50 (or -15 eV) and -5 eV relative to the S K-edge, and background corrected using a linear regression between 35 and 60 eV (Sayers and Bunker, 1988). Because of time constraints during data collection, only one scan was collected on some samples. However, multiple scans on samples showed that the data were always well reproduced. For example, when two to five scans of samples were normalized individually, the coefficients of variation (CV) on peak intensities for the 2 and 10 eV peaks were typically 1%. A CV of 4% was observed for duplicate spectra of one sample.
Linear combination fitting (Vairavamurthy et al., 1994) of XANES spectra for humic acid samples was done using binary and ternary combinations of elemental S, benzyl disulfide, cysteic acid, benzyl sulfoxide, methionine, and chitin sulfate for the relative energy range from -5 to 20 eV. The goodness of fit was judged by 
2 values. The computer program Kaleidagraph-PC (version 3.0, Synergy Software Co., Reading, PA) was used to fit the data. The general equation for a ternary fit was
 | (2) |
Chemical Analysis
Total S was determined in humic acids by digesting 13 mg of freeze-dried humic acid in 10 mL of 30% H2O2 and 12.5 mL of deionized water (Douek and Ing, 1989). Digests were analyzed for sulfate on a Dionex Ion Chromatograph (Model DX-500, Dionex, Sunnyvale, CA) using suppressed conductivity with a detection limit of 0.19 g kg-1 of humic acid. Concentrations of Fe, Al, and silica were determined by dissolving 35 mg of freeze-dried humic acid in deionized water adjusted to pH 11.0 with NaOH, shaking for 2 h, acidifying to pH 4, and analyzing metals on a Perkin Elmer atomic absorption spectrometer (Model 5000, Perkin Elmer, Norwalk, CT). Freeze-dried samples of humic acid were analyzed for organic C and N on a Perkin Elmer CHN analyzer (Model 2400).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
2 eV (relative energy) and a doublet at 10 eV. These peaks arise from S of different formal oxidation states in the sample, with a K-edge for reduced S groups [S(-I) to S(0)] near 0 eV and for oxidized forms of S [S(IV)] and [S(VI)] in the 9- to 11-eV range (Morra et al., 1997). The overall best fit for Humic Acid 2 was obtained using a combination of benzyl disulfide at a fraction of 0.721 ± 0.005, cysteic acid (0.037 ± 0.005), and chitin sulfate (0.242 ± 0.007) (see Table 2). Benzyl disulfide was used as a model compound for reduced S groups such as thiol and sulfides (mono-, di-, and polysulfides), which cannot be easily distinguished using S K-XANES (Morra et al., 1997; Xia et al., 1998). Chitin sulfate (ester sulfate) and cysteic acid (sulfonate) were the model compounds yielding satisfactory fits for highly oxidized forms of S, which was consistent with results of Vairavamurthy et al. (1997).
|
|
Aeration Experiments
Sulfur K-XANES spectra for humic acid samples from the 4-h aeration experiment (Fig. 2)
were very similar to the spectrum in Fig. 1, as were spectra for the 44-h aeration experiment (not shown). However, for the spectrum of humic acid aerated for 4 h at pH 13, the intensity of the doublet peak between 9 and 11 eV was increased relative to the intensity of the peak at 2 eV. These differences in relative peak intensities indicate an increase in oxidized forms of S relative to reduced forms.
|
6% (mol/mol) lower than that calculated for these samples from the aeration experiments. For the humic acid sample aerated at pH 13.0 for 4 h, the fractions of standard S spectra that gave the best fit to the sample spectrum were 0.630 ± 0.006 benzyl disulfide, 0.113 ± 0.006 cysteic acid, and 0.257 ± 0.008 chitin sulfate (Fig. 3). The decrease in the benzyl disulfide type component and the corresponding increase in fractions of oxidized forms of organic S indicate that the 4 h aeration treatment at pH 13 (also having higher ionic strength) oxidized about 15% (mol/mol) of the reduced organic S.
|
Thiol groups (R-SH) may be oxidized to other reduced organic S forms such as disulfides and polysulfides without our detecting it in our XANES spectra. Redox potentials for thiols are generally less than those for organic mono-, di-, and polysulfides (when comparing similar structures), indicating that thiols are more easily oxidized (Bard and Lund, 1978). In fact, diagenesis of humic substance involves the oxidation of thiols to form disulfide and polysulfide inter and intramolecular bridges within the humic structure (Stevenson, 1994; Vairavamurthy et al., 1997). Quinones, which may be present at high pH or produced during oxidation of phenolic OH, are highly reactive oxidants (Patai, 1974) that could promote oxidation of thiol species to monosulfides (Stevenson, 1994). For humic acid that was aerated for 4 h at pH 13.0, the apparent oxidation of reduced S to sulfonate and ester-sulfate type groups (Fig. 3) might be explained by alkaline autooxidation of thiol groups. Alkaline autooxidation of thiol groups to sulfonate groups may occur by the mechanism proposed by Berger (1962) and Oae (1991).
Whereas results for the 4-h aeration experiment showed that some reduced organic S was oxidized at pH 13.0 (Fig. 3), S K-XANES spectra did not show a difference in reduced organic S between the humic acid sample that was initially extracted at pH 13 (in the absence of O2) or samples extracted at lower pH levels. These results suggest that the elimination of O2 and UV radiation may have prevented some oxidation of reduced organic S during extraction. However, we could not determine from our data the extent of organic S oxidation during the extraction. Considering the levels of reduced organic S in the humic acid samples [72 ± 2% (mol/mol) of total S; Table 2], <30% (mol/mol) of the total organic S was converted to sulfonate and ester-sulfate types species. Based on results showing that reduced organic S persisted at pH 12.4, even when exposed to O2 for up to 44 h, we recommend that the pH of solutions used in humic acid extractions not exceed pH 12.4 if there is a possibility of exposure to O2.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
15% (mol/mol) of reduced organic S to sulfonate or ester sulfate-like structures. XANES spectra indicated that organic S resisted oxidation during exposure of humic acid to O2 for up to a 44-h duration at pH levels between 3.5 and 12.4. However, we could not detect changes in thiol, disulfide, and polysulfide components. We recommend that the pH of humic acid solutions be maintained at pH 12.4, ideally under O2-free conditions, for studies where organic S may be important. |
ACKNOWLEDGMENTS |
|---|
Received for publication June 26, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  N. Khare, D. Hesterberg, S. Beauchemin, and S.-L. Wang XANES Determination of Adsorbed Phosphate Distribution between Ferrihydrite and Boehmite in Mixtures Soil Sci. Soc. Am. J., March 1, 2004; 68(2): 460 - 469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Solomon, J. Lehmann, and C. E. Martinez Sulfur K-edge XANES Spectroscopy as a Tool for Understanding Sulfur Dynamics in Soil Organic Matter Soil Sci. Soc. Am. J., November 1, 2003; 67(6): 1721 - 1731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. DeVolder, S. L. Brown, D. Hesterberg, and K. Pandya Metal Bioavailability and Speciation in a Wetland Tailings Repository Amended with Biosolids Compost, Wood Ash, and Sulfate J. Environ. Qual., May 1, 2003; 32(3): 851 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Beauchemin, D. Hesterberg, and M. Beauchemin Principal Component Analysis Approach for Modeling Sulfur K-XANES Spectra of Humic Acids Soil Sci. Soc. Am. J., January 1, 2002; 66(1): 83 - 91. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| The SCI Journals | Agronomy Journal | Crop Science | |||
| Journal of Natural Resources and Life Sciences Education |
Vadose Zone Journal | ||||
| Journal of Plant Registrations | Journal of Environmental Quality |
The Plant Genome | |||
