Characterization of Sulfur-Containing Functional Groups in

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Characterization of Sulfur-Containing Functional Groups in Sedimentary Humic Substances by X-ray Absorption Near-Edge Structure Spectroscopy Murthy A. Vairavamurthy,*,† Dusan Maletic,† Shenkhe Wang,†,| Bernard Manowitz,† Timothy Eglinton,‡ and Timothy Lyons§ Geochemistry Program, Department of Applied Science, Building 801, Brookhaven National Laboratory, Upton, New York 11973, Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, and Department of Geology, University of Missouri, Columbia, Missouri 65211 Received November 27, 1996. Revised Manuscript Received March 19, 1997X

Humic substances, which are randomly cross-linked heterogeneous organic materials of high molecular weight, comprise a major fraction of the organic matter in soils and sediments. Because humic substances have a variety of functional groups, they play a major role in controlling the physical and chemical characteristics of natural waters, soils, and sediments. Sedimentary humic substances are considered to be the precursors of petroleum-forming kerogens. Sulfur is thought to play a major role in forming humic substances in anoxic marine sediments. Because of their structural heterogeneity, humic substances are not easily amenable to biochemical degradation and, thus, are generally considered to be biologically refractory. The complexity and heterogeneity of humic substances render the elucidation of their chemical structure a major analytical challenge. Degradative techniques, such as pyrolysis GC-MS, can change a sample’s structure during preparation and analysis and, thus, may give inaccurate information. In this study, we investigated the sulfur-containing functional groups in humic acids from various marine sediments using XANES spectroscopy which has proved to be an important, nondestructive tool for analyzing sulfur forms. Humic acids were isolated from near-surface sediments from three locations: a salt marsh in Shelter Island, NY; the Peru Margin; and Florida Bay. We found that organic sulfides, di- and polysulfides, sulfonates, and organic sulfates are the major forms of sulfur in these sedimentary humics. The reduced-sulfur structures, organic sulfides, and di- and polysulfides, are essentially intramolecular as opposed to the highly oxidized forms of sulfur, sulfonates, and ester-bonded sulfates, which can only be present as end groups. The abundance of the reduced-sulfur structures reflects the extent to which sulfur is involved in forming intramolecular cross-links which are crucial for building up the macromolecular structures of humic substances. The significance of the presence of the different sulfur functionalities in humic substances is discussed in relation to early diagenesis and preservation of organic matter in marine sediments.

Introduction Humic substances are considered to be the major components of the organic matter in soils and sediments;1-3 for example, in soils humic matter makes up 60-75% of the total organic matter.4 The original source of the organic matter in soils and sediments is plant and animal residues, which, after extensive post* Corresponding author. Telephone: 516-344-5337. FAX: 516-3445526. E-mail: [email protected]. † Brookhaven National Laboratory. ‡ Woods Hole Oceanographic Institution. § University of Missouri. | Present address: Biophysical Collaborative Access Team (Bio CAT), Center for Synchrotron Radiation Research and Instrumentation, Illinois Institute of Technology, Chicago, IL 60616. X Abstract published in Advance ACS Abstracts, April 15, 1997. (1) Aiken, G. R., McKnight, D. M., Wershaw, R. L., MacCarthy, P., Eds.; Humic Substances in Soil, Sediment, and water: Geochemistry, Isolation, and Characterization; Wiley-Interscience: New York, 1985. (2) Stevenson, F. J. Humus Chemistry; Wiley & Sons: New York, 1994. (3) Senesi, N., Miano, T. M., Eds. Humic Substances in the Global Environment and Implications on Human Health; Elsevier: New York, 1994. (4) Schinitzr, M.; Kahn, S. U. Humic Substances in the Environment; Dekker: New York, 1972.

S0887-0624(96)00212-5 CCC: $14.00

depositional microbiological and chemical transformations, become the dark brown to black polymeric materials, the humic substances. These transformations include depolymerization of biological macromolecules to low-molecular-weight monomers (for example, hydrolysis of proteins to amino acids), redox conversions, and condensation reactions of simple molecules.1-4 Thus, humic substances comprise randomly cross-linked heterogeneous organic material of high molecular weight. The phenomenon of forming humic substances in soils and sediments is known as humification; its mechanisms are not well understood. Because humic substances are random polymers of a variety of degradation products and lack specific functional characteristics normally associated with well-defined organic compounds, there is no simple analytical criterion for isolating and quantifying them. Traditionally, methods to separate humic substances from soil and sedimentary matrices have relied on their water solubility and acidbase property, and this has led to a widely adopted operational definition that recognizes three subsets of humic substances: a base-extractable acid-soluble fulvic © 1997 American Chemical Society

S-Containing Groups in Sedimentary Humic Substances

acid fraction, a base-extractable acid-insoluble humic acid fraction, and a base- and acid-insoluble fraction. Humic substances play a major role in controlling the physical and chemical characteristics of natural waters, soils, and sediments. Because they have a variety of functional groups, humic substances form strong complexes with many heavy metal ions, affecting their speciation, mobilization, and toxicity.5,6 The interaction of humic substances with oxide and clay minerals coat the minerals’ surfaces with organic layers, which modify their physicochemical characteristics, and affect the ways they interact with water molecules, metal ions, and organic molecules.7,8 Low-molecular-weight organic compounds can be adsorbed directly onto humic substances through hydrophobic or hydrophilic interactions which can lower their susceptibility to bacterial degradation. The binding of organic pollutants (for example, pesticides) to humics is of particular environmental concern because pollutants thereby persist longer in subsurface environments. Thus, a more complete knowledge of the chemical structure and physical properties of humic substances is essential to obtain a fuller understanding of the mechanisms by which they interact with inorganic and organic contaminants in the natural environment and to elucidate the environmental fate, bioavailability, and toxicity of the contaminants. From a geochemical viewpoint, humic substances have received much attention because they are thought to be the precursors of petroleum forming kerogen.9,10 Recently, there has been increasing recognition that sulfur plays an important role in forming marine sedimentary humic substances.11-15 Furthermore, sulfur is thought to be a critical element in converting the humic substances to source-rock kerogen and that diand polysulfide linkages are an integral part of the kerogen structure.16-18 The role of sulfur cross-linking in aiding the formation of macromolecular structures in kerogen has been viewed as “natural vulcanization” by analogy with its role in vulcanizing rubber.19 Although investigations on humic substances date back several decades, their chemical structure is still not clear; a major challenge is their heterogeneity. Both structure and composition vary with environmental (5) Schinitzer, M.; Skinner, I. M. Soil Sci. 1965, 4, 278. (6) Cameron, D. F.; Sohn, M. L. Sci. Tot. Environ. 1992, 113, 121. (7) Wershaw, R. L. Environ. Sci. Technol. 1993, 27, 814. (8) Murphy, E. M.; Zachara, J. M. Geoderma 1995, 67, 103. (9) Welte, D. In Advances in Organic Geochemistry 1973; Tissot, B., Bienner, F., Eds.; Editions Technip: Paris, 1974; p 1. (10) Hatcher, P. G.; Spiker, E. C. Szeverenyi, N. M.; Maciel, G. E. Nature 1983, 305, 498. (11) Nissenbaum, A.; Kaplan, I. R. Limnol. Oceanogr. 1972, 17, 570. (12) Francois, R. Geochim. Cosmochim. Acta 1987, 51, 17. (13) Ferdelman, T. G.; Church, T. M.; Luther, G. W. Geochim. Cosmochim. Acta 1991, 55, 979. (14) Vairavamurthy, A.; Zhou, W.; Manowitz, B. Di-and polysulfide cross linking in the formation of humic polymers in marine sediments. Abstracts, ACS Fall National Meeting, Orlando, FL, August 1994; American Chemical Society: Washington, DC, 1994. (15) Vairavamurthy, A.; Wang, S.; Maletic, D.; Chakarian, V. Sulfur and nitrogen speciation in humic substances by X-ray absorption spectroscopy. Abstracts, ACS Fall National Meeting, Orlando, FL, August 1994; American Chemical Society: Washington, DC, 1994. (16) Tegelaar, E. W.; de Leeuw, J. W.; Largeau, C. Geochim. Cosmochim. Acta 1989, 53, 3103. (17) Eglinton, T. I.; Irvine, J. E.; Vairavamurthy, A.; Zhou, W.; Manowitz, B. Org. Geochem. 1994, 22, 781. (18) Aizenshtat, Z.; Krein, E. B.; Vairavamurthy M. A.; Goldstein, T. P. , Wang S. In Geochemical Transformation of Sedimentary Sulfur; Vairavamurthy M. A., Schoonen M. A. A., Eds.; ACS Symposium Series 612; American Chemical Society: Washington, DC, 1995; p 16. (19) Kohnen, M. E. L.; Sinninghe Damste´J. S.; Kock-van Dalen, A. C.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1991, 55, 1375.

Energy & Fuels, Vol. 11, No. 3, 1997 547

factors, in particular, with the source of organic matter and the type of depositional environment. Functional groups have received particular emphasis in structural investigations because their type and abundance directly impact the interactions of humic substances. Most studies have focused on oxygen-containing functional groups which are important in soil and riverine humic material.20 Various oxygen functionalities include carboxyl (-COOH), the dominant group, and phenolic and/or enolic -OH, alcoholic -OH, and carbonyl (-CdO). Groups containing nitrogen and sulfur comprise a significant fraction of the functional sites on humics and play major roles in interacting with inorganic and organic contaminants. Although both have been studied, much of the knowledge was obtained indirectly with degradative methods.21-23 There is concern about the latter results because a sample’s structure can change during preparation and analysis, introducing inaccuracies. On the other hand, spectroscopic methods are nondestructive and, thus, provide direct structural information. 15N NMR spectroscopy is an important technique used recently for nitrogen speciation of humics.24 Unfortunately, sulfur is not amenable to studies with NMR spectroscopy. However, an important nondestructive technique that is becoming popular in speciation studies of environmental and geochemical samples is X-ray absorption fine structure spectroscopy. A form of this technique, X-ray absorption near-edge structure (XANES) spectroscopy, has proved to be a powerful tool for the speciation of sulfur.25-36 XANES provides specific information on the functional groups containing sulfur because of its sensitivity to the electronic structure, oxidation state, and the geometry of its neighboring atoms. In this study, we investigated the speciation of sulfur in humic substances from different marine sediments using XANES spectroscopy. A fuller understanding of the speciation of sulfur in humic substances will shed more light on the interactions between carbon and (20) Leenheer, J. A.; Wershaw, R. L.; Reddy, M. M. Environ. Sci. Technol. 1995, 29, 393. (21) Bettany, J. R.; Stewart, J. W. B.; Saggar, S. Soil Sci. Soc. Am. J. 1979, 43, 981. (22) Lowe, L. E.; Bustin, R. M. Can. J. Soil Sci. 1989, 69, 287. (23) Saiz-Jimenez, C. Environ. Sci. Technol. 1994, 28, 1773. (24) Knicker, H.; Almendros, G.; Gonzalez-Vila, F. J.; Ludemann, H.-D.; Martin, F. Org. Geochem. 1995, 23, 1023. (25) Spiro, C. L.; Wong, J.; Lytle, F. W.; Geegor, R. B.; Maylotte, D. H.; Lamson, S. H. Science 1984, 226, 48. (26) George, G. N.; Gorbaty, M. L. J. Am. Chem. Soc. 1989, 111, 3182. (27) George, G. N.; Gorbaty, M. L.; Kelemen, S. R.; et al. Energy Fuels 1991, 5, 93. (28) Gorbaty, M. L.; Kelemen, S. R.; George, G. N.; Kwiatek, P. J. Fuel 1992, 71, 1255. (29) Waldo, G. S.; Carlson, R. M. K.; Moldowan, J. M.; Peters, K. E.; Penner-Hahn, J. E. Geochim. Cosmochim. Acta 1991, 55, 801. (30) Waldo, G. S.; Mullins, O. C.; Penner-Hahn, J. E.; Cramer, S. P. Fuel 1992, 71, 53. (31) Waldo, G. S.; Penner-Hahn, J. E. Ph.D. Thesis, University of Michigan, 1991. (32) Huffman, G. S.; Mitra, S.; Huggins, F. E.; Shah, N.; Vaidya, S.; Lu F. Energy Fuels 1991, 5, 574. (33) Huffman, G. S.; Shah, N.; Huggins, F. E.; et al. Fuels 1995, 74, 549. (34) Kasrai, M; Bancroft, G. M.; Brunner, R. W.; et al. Geochim. Cosmochim. Acta 1994, 58, 2865. (35) Vairavamurthy, A.; Manowitz, B.; Zhou, W.; Jeon, Y.; Geochim. Cosmochim. Acta 1993, 57, 1619. (36) Vairavamurthy, A.; Manowitz, B.; Zhou, W.; Jeon, Y.; In The Environmental Geochemistry of Sulfide Oxidation; Alpers, C., Blowes, D., Eds.; ACS Symposium Series 412; American Chemical Society: Washington DC, 1994.

548 Energy & Fuels, Vol. 11, No. 3, 1997

sulfur during early diagenesis and sulfur’s role in forming kerogen during late diagenesis. The humic acids used were isolated from near-surface sediments from three contrasting locations: a salt marsh in Shelter Island, NY; the Peru Margin; and Florida Bay, a carbonate platform. Our results show that organic sulfides, di- and polysulfides, sulfonates, and organic sulfates are the major forms of sulfur in these sedimentary humics. The reduced-sulfur structures (organic sulfides, and di- and polysulfides) are essentially intramolecular as opposed to the highly oxidized forms of sulfur (sulfonates and ester-bonded sulfates) which can only be present as end groups. The significance of the presence of these different forms of sulfur in humic substances is discussed, highlighting the role of sulfur in transforming and preserving organic matter in marine sediments. Experimental Section Sediment samples were collected from three different marine locations: (1) a salt marsh in Shelter Island, NY; (2) the Peru Margin; and (3) Florida Bay. These locations were chosen because they represent different depositional settings. The organic matter input to the salt-marsh sediments in Shelter Island is derived mainly from decaying halophytic grasses; two main species inhabit the site (1) the salt-water cordgrass, Spartina alterniflora, and (2) the salt-meadow cordgrass, Spartina patens. The former dominates the lowlying intertidal zones whereas the latter inhabits the high marsh area which is flooded only about once or twice a month. During the summer of 1994, a sediment core was taken using a hand-held polybutyrate tube near a tidal creek with decaying leaves of S. alterniflora at the surface. The Peru Margin sediments are very rich in organics; upwelling of nutrient-rich waters near the coast supports high rates of primary productivity, maintaining a large supply of organic matter to the sediments, mainly planktonic debris.37 These sediments, deposited under an intense oxygen-minimum zone, were collected as a hydraulically damped gravity core (KC 083, Station 1, water depth 106 m, 13°31′09′′ S, 76°28′36′′ W) during a cruise (R/V Seward Johnson) in October 1992. In contrast to the sediments from the Shelter Island salt marsh and the Peru Margin, modern-carbonate sediments from South Florida are strongly depleted in iron. The deposition of significant amounts of organic matter, accompanied by rapid rates of bacterial sulfate reduction in these Fe-poor sediments, allows high levels of dissolved sulfide to accumulate (up to several millimolar). There is significant dissolution of metastable carbonate minerals from the oxidation of H2S by diffused oxygen, mediated by sulfide-oxidizing bacteria.38 The sediments were collected using hand-held corers during July 1993 in the Florida Keys. We isolated humic substances from the core samples at 6-14 cm depths. The samples were water-washed before extracting humics to remove ionic and low-molecular-weight components. Standard procedures used previously were adopted for isolating humic substances.39 Briefly, the samples were extracted with 0.2 M NaOH for 12 h, centrifuged, and acidified with 2 M HCl to separate the humic and fulvic acid fractions. For XANES analyses, samples were prepared by wrapping them in an X-ray film (2.5µm Prolene or Mylar from Chemplex Industries, New York). XANES Spectroscopy. The sulfur K-edge XANES spectra were collected as fluorescence excitation spectra using a Lytle (37) Heinrichs, S. M.; Reeburgh, W. S. Limnol. Oceanogr. 1984, 29, 1. (38) Walter, L. M.; Bischof, S. A.; Patterson, W. P.; Lyons, T. W. Philos. Trans. R. Soc. London A. 1993, 344, 27. (39) Stevenson F. J. Humus Chemistry, Genesis, Composition, Reactions, 2nd ed.; John Wiley & Sons: New York, 1994.

Vairavamurthy et al. fluorescence detector at the National Synchrotron Light Source (NSLS) X-19A beam line at the Brookhaven National Laboratory. In this beam line, the X-ray beam was diffracted by a double-crystal Si(111) monochromator which passes a narrow energy band to the sample. The monochromator was detuned by ca. 80% to minimize higher order harmonics in the X-ray beam. Samples were run in a helium atmosphere to minimize the attenuation of the X-ray beam by air. The spectra were recorded so that the scanning procedure yielded sufficient preedge and post-edge data for precisely determining the background, which is needed for analysis. The X-ray energy was calibrated using XANES spectra of elemental sulfur measured between sample runs, assigning 2472.7 eV to its spectral peak. The uncertainty of the energy calibration was less than (0.15 eV, determined by comparing the spectra of model compounds obtained at different times. A nonlinear least-squares fitting procedure, which uses linear combinations of normalized spectra of model compounds, gave quantitative information on the different forms of sulfur.27,31,36 The fact that the XANES spectra are usually measured as fluorescence excitation spectra presents a problem in quantitative analysis of concentrated samples. With thick samples, the fluorescence signal is considerably distorted (reduced amplitude, especially) due to self-absorption or the thickness effect. Self-absorption is particularly important for elements of low atomic number, such as sulfur, because X-ray absorptions are typically quite large at the low-energy range (2-4 keV). Ideally, the self-absorption effect can be avoided by diluting the sample; however, this can be difficult or impossible for insoluble samples and also degrades the signalto-noise ratio. The numerical method developed by Waldo and Penner-Hahn corrects the fluorescence-excitation spectra for self-absorption before least-squares fitting.31 This algorithm is incorporated into the program that we used for fitting. Essentially, this procedure involves (1) background correction and normalization of the spectrum in the post-edge region to fit a tabulated X-ray-absorption cross-section (McMaster table), (2) correction for attenuation of the fluorescence signal due to the self-absorption effect, and (3) fitting the normalized spectrum with linear combinations of absorption spectra of model compounds. In a typical analysis, the spectrum of a sample is fitted with various linear combinations of the spectra of different sulfur standards and the best fit is taken to indicate the actual composition. The scaling factor applied to each standard spectrum in fitting gives the quantitative amount of that form of sulfur as a fraction of the total sulfur. Using experimental spectra of standard compounds for fitting has a unique advantage over other methods (for example, one using specific mathematical functions to represent the peaks for different forms of sulfur) when a model compound has several major peaks, for example, thiosulfate. Thus, even when two or more compounds have identical peaks at a particular energy position, other differences in spectral features will discriminate the different forms because the entire spectrum is used for fitting.

Results and Discussion Sulfur Speciation with XANES Spectroscopy. The various organic sulfur species present in aquatic, sedimentary, and soil systems can be broadly divided into three groups on the basis of the oxidation level of sulfur in the molecule: (1) reduced organic sulfur, (2) lowly oxidized organic sulfur, and (3) highly oxidized organic sulfur. The reduced sulfur species contain sulfur at a formal oxidation state of -2 (as in H-S-H) or at mixed oxidation states between 0 and -2 (as in H-S-S-S-H); these include thiols (R-SH), organic sulfides (R-S-R), organic polysulfides, and thiophenes. In the lowly oxidized group, sulfur is bonded to one or two oxygen atoms and it includes organic sulfoxides and


Figure 1. Normalized K-edge XANES spectra of various sulfur compounds grouped according to the sulfur oxidation levels.

sulfones. Highly oxidized organic sulfur forms contain three or four oxygen atoms bonded to sulfur and comprise sulfonates and organic sulfates. XANES spectroscopy can distinguish the various forms of sulfur because the energy position of the absorption edge in a XANES spectrum strongly correlates with the oxidation level of sulfur in a molecule, shifting to high energy as the oxidation state increases. The absorption edge occurs when the X-ray photon has sufficient energy to excite a core-level electron in the atom. For sulfur, the edge corresponds to a s f p electronic transition which occurs as an intense, wellresolved transition corresponding to the “white-line” maximum (the peak) of the XANES spectrum. Several studies showed that both the energy and the intensity of the sulfur s f p transition are sensitive to the oxidation state of sulfur.26,29,35,36 This dependence is reflected by the fact that the peak position changes by ca. 10 eV between elemental sulfur and sulfate. Quantitative information also is contained in XANES spectra because a sample’s spectrum is essentially a combination of the spectra of the different sulfur constituents present. As outlined earlier (in the Experimental Section), the method we used involves a nonlinear leastsquares fitting procedure using linear combinations of the normalized spectra of model compounds to deconvolute the sample’s spectrum.29,36 We clarify the term oxidation state here because often it has been used rather loosely. Traditionally, oxidation state is synonymous with the formal oxidation state which represents the electronic charge that an atom


Figure 2. Typical XANES spectra of humic acid extracts from near-surface depths and the corresponding whole sediment samples for the different locations: (I) Shelter Island salt marsh, (II) the Peru margin, and (III) Florida Bay.

would have if the electrons in a compound were assigned to the atoms in a certain way. Simply stated, the formal number reflects charges on atoms assigned by regarding all compounds as totally ionic. In the XANES approach, the shift in the absorption edge reflects the changes in the relative electronic charge density of the absorber. Earlier, Kunzl suggested that there is a linear relationship between an edge shift and oxidation state on the basis of his critical study on the shift of the K-absorption discontinuities of the oxides of several elements.40 Ideally, Kunzl’s law is valid for a monatomic ionic species where the changes in its atomic charge density, represented by edge shifts, can be equated unambiguously to changes in its formal oxidation state. For organic sulfur compounds dominated by covalent bonding, XANES peak-energy correlation directly estimates the relative charge density of sulfur, which may not correlate with the formal oxidation state derived empirically. Figure 1 shows the characteristic XANES spectra for various organic sulfur compounds, indicating the shifts in absorption peaks for different groups. XANES Spectra of Marine Sediments and Humic Acid Extracts. Figure 2 shows the XANES spectra of the water-washed sediment samples and the corresponding humic acids for the three different marine locations. Visual inspection of these spectra shows two major absorption bands mainly corresponding to (a) the reduced sulfur compounds and (b) the highly oxidized sulfur compounds. To gain further information about the different sulfur species involved, we fitted the (40) Kunzl, V. Collect. Czech. Commun. 1932, 4, 213.


Vairavamurthy et al.

Figure 5. Nonlinear least-squares fit of the XANES spectra of the humic acid and whole sediment samples from Florida Bay. Figure 3. Nonlinear least-squares fit of the XANES spectra of the humic acid and whole sediment samples from the Shelter Island salt marsh.

Figure 4. Nonlinear least-squares fit of the XANES spectra of the humic acid and whole sediment samples from the Peru Margin.

spectra with linear combinations of various sulfur standards; Figures 3, 4, and 5 show the best-fit analyses obtained for the various sediment and humic acid samples, and Table 1 summarizes data on sulfur composition from these analyses. As Figures 3 and 4

show for Shelter Island and Peru Margin sediments, a major difference is the presence of pyrite in the whole sediment, but not in the humic-acid isolate. Pyrite is a diagenetic product formed from the reaction of hydrogen sulfide (HS-) with iron minerals.41 In marine sediments, hydrogen sulfide is generated from the anaerobic bacterial reduction of sulfate during early diagenesis.42 For Florida Bay sediments, there was no pyrite, agreeing with previous studies that showed it is not formed in these carbonate-rich sediments due to the lack of iron minerals. On a pyrite-free basis, the percent composition of the organic sulfur forms in whole sediments closely match those of the humic acid isolates for Shelter Island and Peru Margin sediments, but not for Florida Bay sediments because they contain excess sulfate in the latter (Table 2). This excess, which cannot be extracted with water, is probably associated with carbonate minerals, substituting for carbonate in the structural lattice.43 We calculated the percentages of the various sulfur forms after correcting for this inorganic sulfate, assuming that about 50% of the total sulfur is structurally incorporated, and the values obtained agree well with those of the humic acid (Table 2). Thus, in Florida Bay sediments, a major fraction (>70%) of the nondissolvable sulfate probably is incorporated into carbonate minerals. These results suggest that the base-extraction procedure commonly used for isolating humic fractions is nondiscriminative for sulfur compounds and that the composition of organic sulfur species in humic acids represents typically that of the source sediments. Highly Oxidized Forms of Sulfur: Ester-Bonded Sulfate and Sulfonate. As Figures 3-5 show, the second absorption band can be completely fitted with (41) Berner, R. A. Geochim. Cosmochim. Acta 1984, 48, 605. (42) Jørgensen, B. B. Nature 1982, 296, 643. (43) Pingitore, N. E.; Meitzner, G.; Love, K. M. Geochim. Cosmochim. Acta 1995, 59, 2477.



Table 1. XANES Results of Sulfur Composition of Various Sediments and Humic Acids (% of the Total Sulfur) sample

pyrite

organic di- and polysulfide

SI salt marsh sediment Peru Margin sediment Florida bay sediment SI salt marsh humic acid Peru Margin humic acid Florida Bay humic acid IHSS soil humic acid

34 30

46 10 13 72 20 20 15

organic sulfide

thiophene

sulfoxide

sulfonate

sulfate

19

2 2 2 1 5 4 6

9 21 16 14 23 36 37

9 23 63 13 28 25 23

15 6 24 15

Table 2. Comparison of Adjusted Data on Organic Sulfur Composition of Sediments with Those of Humic Acids sample

organic di- and polysulfide (%)

SI salt marsh sediment (adjusted for pyrite) Peru Margin sediment (adjusted for pyrite) Florida Bay sediment (adjusted for mineral sulfate) SI salt marsh humic acid Peru Margin humic acid Florida Bay humic acid

70 14 26 72 20 20

Figure 6. Sulfur K-edge XANES spectrum of the standard soil humic acid from the International Humic Substances Society, Colorado, and its nonlinear least-squares fit of it with model spectra.

absorption spectra of two highly oxidized forms of sulfur: (1) sulfonate sulfur with absorption maximum at 2981.6 ( 0.1 eV and (2) sulfate sulfur with maximum at 2983.1 ( 0.1 eV. The standard spectra used for fitting were those of cysteic acid and chitin sulfate, representing a sulfonate and an organically bound sulfate, respectively. Previous investigations of sulfur speciation in soils using degradative chemical approaches showed that ester-bonded sulfate is a major form of sulfur in soils, accounting for more than 40% of the total sulfur,44,45 although its occurrence in marine sediments has not been clearly documented. We think that an ester sulfate, rather than an inorganic sulfate, causes the absorption peak at 2983.1 ( 0.1 eV because it is unlikely that the sulfate ion could be retained by humic acid after extensive base-acid treatments and water-washing. Further support is derived from comparing these spectra with the spectrum of a soil humic acid known to contain ester-linked sulfate. Figure 6 gives the spectrum of the standard soil humic acid obtained from International Humic Substances Society along with a best fit, which shows a similar spectral feature at 2483.1 eV that could be assigned to ester sulfate. As Table 1 indicates, ester-linked sulfates are a major form of sulfur on marine humics from nearsurface sediments, contributing about 10-25% of the (44) Fitzgerald, J. W. Bacteriol. Rev. 1976, 40, 698. (45) Freney, J. R. In Sulfur in Agriculture; Tabatabai, M. A., Ed.; Agronomy Monograph No. 27, 1986; p 207.

organic sulfide (%) 21 12 24 15

sulfoxide (%)

sulfonate (%)

sulfate (%)

3 3 4 1 5 4

14 30 32 14 23 36

14 33 26 13 28 25

total sulfur. The mechanisms for forming ester-bonded sulfates in marine humics is unclear, although biotic rather than abiotic mechanisms likely are involved. Earlier, ester sulfates were considered unstable in marine sediments;41 however, our study suggests that ester-bonded sulfate is a major form of sulfur in sedimentary humic substances and can survive the exhaustive pH changes involved in isolating the humic acids from the sediment. Sulfonates are the second major oxidized form of organic sulfur in sedimentary humics. As shown in Figures 3-5, the spectral feature at 2981.6 ( 0.1 eV was clearly resolved by fitting with a sulfonate spectrum which contributes to 15-40% of the total sulfur in the near-surface humics. The presence of sulfonates is not surprising because they were recently identified as a major class of organic sulfur compounds in near-surface marine sediments.47 Earlier, it was found that sedimentary sulfonates are mainly associated with sedimentary particulate phase as components of the macromolecular organic matter, and not as low-molecularweight molecules. From our results, we suggest that humic substances are probably such macromolecules harboring sulfonate groups in near-surface marine sediments. The origin of the sulfonate sulfur in marine sediments is not clear. They may represent a direct contribution from the original organic matter because sulfonated compounds (for example, cysteic acid) are present in living organisms.47 However, diagenetic mechanisms may generate the sulfonates; particularly, the reaction of H2S oxidation intermediates (e.g., thiosulfate) with functionalized organic molecules. In a recent study, the diagenetic route seemed to be the dominant one for forming sulfonates in the coastal sediments from the Bay of Concepcion, off Chile.48 Sulfones were not detected among the lowly oxidized forms of sulfur, but the sulfoxide form was found as a minor constituent, at less than 5% of the total sulfur. It is uncertain whether sulfoxides were present originally in the sediments or were formed from the oxida(46) Mossmann, J. R.; Aplin, A. C.; Curtis, C. D.; Coleman, M. L. Geochim. Cosmochim. Acta 1991, 55, 3581. (47) Vairavamurthy, A.; Zhou, W.; Eglinton, T.; Manowitz, B. Geochim. Cosmochim. Acta 1994, 21, 4681. (48) Vairavamurthy, A.; Wang, S. K.; Khandelwal, B.; Manowitz, B.; Ferdelman, T.; Fossing, H. In Geochemical Transformation of Sedimentary Sulfur; Vairavamurthy, M. A., Schoonen, M. A. A., Eds.; ACS Symposium Series 612; American Chemical Society: Washington, DC, 1995; p 38.


tion of certain sulfur compounds while preparing the sample. The latter is more likely because sulfoxides may not persist under reducing conditions. An earlier study suggested that sulfoxides detected in sedimentary outcrops were probably generated from the oxidation in air of some organic sulfides.49 The Reduced-Sulfur Forms. The major forms of reduced organic sulfur detected in the near-surface sedimentary humic acids are organic sulfides and organic polysulfides (which include disulfides). Our fitting results did not reveal thiophenic sulfur in the three marine humics examined, although the thiophenic form was found in the soil humic acid (Figure 6). In general, thiophenes are generated in the deeper layers of the sediment column after considerable diagenesis of the organic matter.17,18,50 Organic polysulfides generate thiophenes when heated (for example, to 160 °C), suggesting that transformation of organic polysulfides is a potential pathway for forming thiophenes in the sediment column.51,52 Thus, not surprisingly, organic sulfides and organic polysulfides, and not thiophenes, are the major functional forms of sulfur in the humics from near-surface sediments. The thiophenic sulfur detected in the soil humic acid could be an “aged product” reflecting extensive transformation of the organic matter, although a recent biological origin cannot be ruled out. The aim in quantitative analysis of an XANES spectrum for organic sulfur compounds is to estimate the various functional groups containing sulfur, rather than individual organic structures. Thus, for the marine humic acids, our finding of an organic sulfide and a polysulfide form of sulfur from fitting implies that different types of sulfur bonding, -C-S-C-, -C-SS-C-, and -C-S-Sn-S-C-, are present in the structure. These structures with reduced sulfur are essentially intramolecular as opposed to the highly oxidized forms of sulfur, sulfonates, and ester-bonded sulfates, which can only be present as end groups. Thus, the reduced-sulfur bonds (sulfide, disulfide, and polysulfide) can be envisioned as an integral part of the intramolecular cross-links essential for forming the randomly polymerized, heterogeneous humic acid. Almost all of the organic sulfur in soils and riverine systems is of biological origin. In contrast, the dominant fraction of the organic sulfur in marine sediments is considered to be of diagenetic origin, generated from the reaction of sulfur nucleophiles with functionalized organic molecules.50 The hydrogen sulfide (generated in bacterial sulfate reduction) and its partial oxidation intermediates, such as polysulfides and thiosulfate, are the active nucleophiles which react, on one hand, with functionalized organic molecules to form organic sulfur compounds and, on the other hand, with iron minerals to form pyrite. Because humic substances coated on clay minerals have many types of reactive functional groups, they present a reactive surface. Sulfur nucleophiles can react with some of those functional sites, (49) Kohnen, M. E. L.; Sinninghe Damste´J. S.; de Leeuw, J. W. Org. Geochem. 1995, 23, 129. (50) Sinninghe Damste, J. S.; de Leeuw, J. W. Org. Geochem. 1989, 16, 1077. (51) Krein, E. B.; Aizenshtat, Z. Org. Geochem. 1994, 21, 1015. (52) Krein, E. B.; Aizenshtat, Z. In Geochemical Transformation of Sedimentary Sulfur; Vairavamurthy, M. A., Schoonen, M. A. A., Eds.; ACS Symposium Series 612; American Chemical Society: Washington, DC, 1995; p 110.

Vairavamurthy et al.

incorporating sulfur into humic substances. Thus, sulfhydryl (thiol) and polysulfide functionalities will be introduced from the reactions involving hydrogen sulfide (HS-) and polysulfide ions, respectively.53,54 Thiol groups can react in a variety of ways. When a thiol group is added across an unsaturated bond, an organic sulfide bond is formed. If such addition occurs intramolecularly, a cyclic sulfide is generated. Intermolecular reactions form a linear chain sulfide. Adding a thiol group to an unsaturated bond represents a diagenetic mechanism for introducing a sulfide bond in humic substances. However, compounds containing sulfide bonds are common in organisms (for example, the amino acid methionine), and therefore, the sulfide bond also could be of biological origin. A disulfide bond is formed from oxidation involving two thiol molecules, either from the same compound or from two different ones (the resultant product is a mixed disulfide). Intermolecular disulfide cross-linking between two simple thiols will form a simple disulfide molecule. However, for sedimentary humic substances which probably contain many thiol sites, such intermolecular cross-linking will form a network of disulfide bonds, with a concomitant increase in molecular weight. For the Shelter Island humic acid, the first absorption band can be completely fitted with the standard spectrum of an organic polysulfide, suggesting that reduced sulfur mainly is contained in polysulfide and disulfide linkages. If so, then treating the humic acid with tributyl phosphine (a reducing agent) should cleave the poly- and disulfide bonds to generate thiols. In fact, the shape of the first absorption band changed considerably upon treatment, and because of that change, we had to use an additional model, thiol, to obtain a best fit of the spectrum (Figure 7); the contribution of thiols was ca. 45%. These results further support the presence of polyand disulfides in the humic acid. A major diagenetic pathway for introducing polysulfide bonds into humic substances is through the reaction with inorganic polysulfides. Francois12 and Ferdelman et al.13 showed that humic substances extracted from near-surface coastal and salt-marsh sediments were highly enriched in sulfur, with a maximum in the top sediment column near the oxic-anoxic interface, thus demonstrating the importance of polysulfide-humicsubstance interactions in the early incorporation of sulfur into sedimentary organic matter. Mossman et al.46 suggested that inorganic polysulfides probably are the most active sulfurization agents in Peru margin sediments, causing organic polysulfides to form in the uppermost surficial layer of the sediment column. Recent studies by Vairavamurthy et al. of sulfur transformation in organic-rich sediments from the Bay of Concepcion, off Chile, also suggest that organic polysulfides are generated in the near-surface layers of the sediment column.48 In an earlier study, Vairavamurthy et al. suggested that a major fraction of reactive polysulfides in nearsurface sediments is bound to the particle phase.55 Recently, it was suggested that such particle-bound reactive polysulfides are formed by the reaction of (53) Vairavamurthy, A.; Mopper, K. Nature 1987, 329, 623. (54) Kohnen, M. E. L.; Sinninghe, Damste J. S.; ten Haven, H. L.; de Leeuw, J. W. Nature 1989, 341, 640. (55) Vairavamurthy, A.; Mopper, K.; Taylor, B. F. Geophys. Res. Lett. 1992, 19, 2043.


Figure 7. XANES spectra of samples of the humic acid from the Shelter Island salt marsh showing changes after treating with tributylphosphine, a di-and polysulfide cleaving reagent.55 Note that the shape of the first absorption band changed considerably upon treatment, requiring an additional model, a thiol, for fitting.

inorganic polysulfides with active sites on humics coated on mineral particles.56 Thus, particle-bound polysulfides could also be considered as humic-bound polysulfides. In this view, the reactivity of the particle-bound polysulfides can be ascribed to the fact that the free end of the polysulfide chain is available for reactions with other reactive molecules (such as thiols and compounds with activated unsaturated bonds), while the other end of the chain is attached to the humic macromolecule. Such reactions involving humic-bound polysulfides would increase the complexity and molecular size of the humic macromolecule with an extensive framework of polysulfide linkages. Because of the structural heterogeneity caused by random polymerization, humic substances, in general, are considered to be biologically refractory. Hence, their incorporation into humic substances helps to preserve the sedimentary organic matter.


organic molecules enrich the sulfur in sedimentary organic matter relative to the source organic matter of biological origin. We used XANES spectroscopy to examine the chemical form of sulfur in humic substances which comprise the major fraction of the sedimentary organic matter. XANES is particularly advantageous for sulfur because the k-edge spectra of the variety of forms each are richly endowed with characteristic features, including the edge energy, which facilitates qualitative recognition among various oxidation states and structures. Quantitative information on the various sulfur forms can be derived by deconvoluting the spectra appropriately because a sample’s spectrum is essentially a combination of the spectra of the different sulfur constituents present. From our results, the types of sulfur in humic substances can be broadly categorized into two major groups: (1) the reduced sulfur forms comprising organic sulfides and di- and polysulfides and (2) the highly oxidized sulfur forms, sulfonates and ester-bonded sulfates. While the reducedsulfur structures occur essentially as intramolecular bridges, the oxidized forms of sulfur (i.e., sulfonates and ester-bonded sulfates) can be present only as terminal groups. Although the reduced forms of sulfur and sulfonates may be formed by both biochemical and geochemical pathways, there is no known geochemical route for forming ester-bonded sulfates. Thus, they should represent either those originally present in the source organic matter or those formed by bacterial mediation. Contrary to the earlier view that ester sulfates are unstable in marine sediments,46 our results suggest that ester-bonded sulfate is a major form of sulfur in sedimentary humic substances and can survive the exhaustive pH changes involved in isolating the humic acids from the sediment. Most reduced sulfur existing as mono-, di-, and polysulfides is probably derived through diagenetic incorporation. These reducedsulfur bridges probably play a crucial role in crosslinking between molecules and, thus, in building up the macromolecular structures of humic substances. Since sulfur cross-linking is a key factor in forming humic polymers in sediments, we suggest that it plays a critical role in preserving organic matter in anoxic sediments. Recent studies recognize an extensive framework of diand polysulfide cross-linking in source-rock kerogens of marine origin, which is thought to play a crucial role in kerogen formation.16,18 This study shows that such sulfur linkages are generated during humification in the early stages of diagenesis and strengthens the argument that humic substances are the precursors of the sourcerock kerogens.

Conclusions Sulfur plays a critical role in the diagenesis of organic matter in marine sediments. Diagenetic reactions betwen sulfur nucleophiles (such as the hydrogen sulfide and polysulfides generated during microbial sulfate reduction under anoxic conditions) and functionalizied (56) Vairavamurthy, A; Manowitz, B.; Maletic, D.; Wolfe, H. Org. Geochem. 1997, in press.

Acknowledgment. This research was performed under the auspices of the U.S. Department of Energy Division of Engineering and Geosciences of the Office of the Basic Energy Sciences under Contract No. DEAC02-76CH00016 (KC-04). We thank A. Woodhead for valuable comments. EF960212A

Characterization of Sulfur-Containing Functional Groups in

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