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,*Dep. of Chemistry, The Ohio State University, 100 W. 18th Avenue, Columbus, OH 43210
* Corresponding author (chefetz{at}agri.huji.ac.il)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: AL, alkanes/alkenes • CPMAS, cross-polarization magic angle spinning • DAME, dicarboxylic acid dimethyl esters • FAME, fatty acid methyl esters • HA, humic acid • HS, humic substances • IHSS, International Humic Substance Society • LG, lignin-derived compounds • NMR, nuclear magnetic resonance • OM, organic matter • PR, protein-derived compounds • PS, polysaccharide-derived compounds • SOM, soil organic matter • TMAH, tetramethylammonium hydroxide
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Pyrolysis-GC/MS is a powerful analytical tool for obtaining molecular-level information about SOM and HS. In the past, the data from this technique have been used to design two- and three-dimensional model structures of HS (Schulten, 1995; Schulten and Leinweber, 1996). However, problems associated with pyrolysis (e.g., decarboxylation and formation of very polar products) have led to misinterpretations with respect to benzene carboxylic acids and fatty acids in HS (del Rio et al., 1994, 1998). A new technique, which has been recently developed to overcome this limitation, is thermochemolysis with TMAH. This method selectively cleaves ester and certain ether linkages (such as ß-O-4 aliphatic-aryl bonds) in macromolecular organic matter (OM; Hatcher et al., 1995; Hatcher and Minard, 1996) through a chemolytic procedure that hydrolyzes and methylates ester and ether linkages, and assists in the depolymerization and methylation of lignin (Filley et al., 1999). This technique has been employed to characterize HAs, HS, composted OM, and SOM (del Rio et al., 1994, 1998; Hatcher and Clifford, 1994; Fabbri et al., 1996; Chefetz et al., 2000a, b), and is especially useful for detecting polar compounds such as long-chain fatty acids and benzenecarboxylic acids, as part of the HS structure. Another modern technique for the characterization of HS is solid-state NMR (Wilson, 1987; Preston, 1996; Mao et al., 2000). This technique offers a generic description of the major classes of C-containing groups in a sample and has contributed significantly to information regarding the chemical nature of HS.
Information gained from each analytical technique or chemical modification is important because the individual techniques complement each other. When used together, they provide awareness against biases that may evolve because of structural selectivity that each technique harbors. In this study, a combination of two chemical degradation techniques (oxidation using NaClO2 and acid hydrolysis) together with advanced analytical characterization (13C NMR, pyrolysis and TMAH thermochemolysis) were used to obtain data that contributes significantly to the knowledge and understanding of the building blocks in soil and peat Has.
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MATERIALS AND METHODS |
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Chemical Treatments
Subsamples from the isolated HAs were bleached and hydrolyzed. Bleaching was performed according to the method described by Wise et al. (1946) using 10 g of NaClO2, 10 mL of acetic acid, and 100 mL of deionized water per gram of HA. The mixture was stirred overnight, then the supernatant was decanted and replaced by fresh solution. This procedure was repeated three times. Hydrolysis was carried out using 300 mL of 6 M HCl per gram of HA; the mixture was refluxed for 6 h. After each respective treatment, the residues were separated from the supernatant by centrifugation (5000 x g, 15 min), dialyzed (Spectra/Por Membrane, MWCO 6000–8000; Fisher Scientific, Pittsburg, PA) against deionized water, and then freeze-dried.
Elemental Analysis
Elemental (C, H, and N) analysis was conducted in triplicates by Quantitative Technologies Inc. (Whitehouse, NJ).
Solid-State Cross Polarization Magic Angle Spinning Carbon-13 NMR Spectroscopy
Solid-state cross polarization magic angle spinning (CPMAS) 13C NMR spectra, using the ramp-cross polarization technique (Cook and Langford, 1998) were obtained with a Bruker 300 MHz NMR-spectrometer (Bruker Analytic GmbH, Germany). To obtain spectra that would be quantitatively representative of the structural components of the sample, an optimal contact time was chosen to give representative intensities for all types of carbons. This was determined by analyzing the peak intensities at various contact times, ranging from 0.1 to 15 ms, with ramp-CPMAS (Wind et al., 1993). A contact time of 2 ms was chosen to be optimal. The spectrometer operates at a 1H frequency of 300 MHz and a 13C frequency of 75 MHz. The following optimized experimental parameters were used: ramp-cross polariztion contact time of 2 ms; recycle delay time of 1 s; sweep width of 27 kHz (368 ppm) and line broadening of 30 Hz. Freeze-dried samples were placed in a 4-mm rotor and spun at a frequency of 13 kHz at the magic angle (54.7° to the magnetic field). All samples were subjected to the same number of scans (25200).
The 13C NMR spectra were integrated into the following chemical shift regions: 0 to 50 ppm, paraffinic C; 50 to 112 ppm, alkyl-O or C-O, C-N bonds as in carbohydrates, alcohols, ethers, and amines; 112 to 163 ppm, aromatic and phenolic C; and 163 to 190 ppm, carboxyl, ester and amide; 190 to 215 ppm, carbonyl C (Hatcher et al., 1983).
Pyrolysis-Gas Chromatography and Mass Spectroscopy
Pyrolysis-GC/MS was performed using a Mega 500 series gas chromatograph (Carlo Erba, Milan, Italy), equipped with a CDS analytical pyroprobe-2000 controller, a CDS AS-2500 pyrolysis autosampler and a 30-m fused silica capillary column coated with chemically bound DB-1701 (0.25-mm i.d., film thickness 0.25 µm; Restek Corp., Bellefonte, PA). Humic Acid samples (
0.25 mg) were weighed and transferred to quartz tubes. Each tube was dropped into the pyrolysis chamber, which was subsequently heated to 615°C at a rate of 5°C ms-1 and was held at this temperature for 15 s. Detailed information regarding the pyrolysis procedure is described by Chefetz et al. (2000b).
Tetramethylammonium Hydroxide Thermochemolysis-Gas Chromatography and Mass Spectroscopy
Tetramethylammonium hydroxide thermochemolysis was conducted as described in earlier papers (Chefetz et al., 2000a,b). Briefly, HA samples (1 mg) were placed in glass tubes with 200 µL of TMAH (25% [wt./wt.] in methanol). After the methanol was evaporated, the tubes were sealed under vacuum and subsequently placed in an oven at 250°C for 30 min. After cooling, the tubes were cracked open, an internal standard (n-eicosane) was added, and the tubes were extracted with ethyl acetate. The extracts were concentrated and injected (1 µL) into a 6890 series gas chromatograph (Hewlett Packard, Palo Alto, CA), equipped with a 15-m fused silica capillary column coated with chemically bound DB-5 (0.25-mm i.d., film thickness 0.1 mm; Supelco, Bellefonte, PA). The GC was directly coupled to a Pegasus II (Leco Corporation, St. Joseph, MI) time-of-flight mass spectrometer.
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RESULTS |
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The absolute amount (weight units) of the aromatic and paraffinic C functionalities remaining after each treatment was calculated by multiplying the percentage of C functionality, as determined by CPMAS 13C NMR spectra, by the yield of the treatment (the bulk sample containing 100 units of weight). The amount of aromatic C in the peat HA was reduced from 32.8 to 0.83 units after bleaching. Therefore, only 2.5% of the peat HA aromatic structures were resistant to bleaching. The aromatic units of soil HA contained compounds, which were more resistant to the bleaching treatment. Bleaching resulted in a reduction in the aromatic C, from 38 to 4.8 units, i.e., 12.6% of the original weight of the aromatic structure of soil HA. The total amount of paraffinic carbons left after bleaching was 7.7 weight units in the soil HA as compared with 16.8 units in the bulk sample (i.e., 54% of the paraffinic carbons were removed by the procedure). Fewer paraffinic carbons were left after bleaching of the peat HA (4 units), this amount representing only 16.5% of the original paraffinic C content in peat HA.
Acid hydrolysis resulted in a significant reduction of the carbohydrate and amine region (50–112 ppm). The peaks at 72 and 105 ppm were poorly resolved in the two hydrolyzed HA spectra (Fig. 1H), and this is consistent with other observation made from applications of this treatment to HS (Almendros, 1994; Stevenson, 1994). The total amount of polysaccharides (as calculated from the 60–112 ppm region in the CPMAS 13C NMR spectra) left after this treatment was 11.5 and 7 weight units for the peat and soil HAs, respectively. In contrast with the trends observed by bleaching, fewer structural units were removed from the peat HA than the soil HA by the hydrolysis treatment. The nonhydrolyzed polysaccharides represented 55 and 30% of the original amount of polysaccharides for peat and soil HAs, respectively. Of course, it is likely that the peaks in the polysaccharide region of the spectra of hydrolyzed HAs are not polysaccharides at all but other types of alkyl-O carbons (e.g., lignin side-chains, alcohols, esters, etc.). The hydrolysis treatment also resulted in a significant reduction of the carboxyl and amide C peak, and therefore increased the relative intensity of the aromatic C peak in the 13C NMR spectrum. The carboxyl and amide C content of the soil HA was reduced by 33% (from 14.4 to 9.6%). A lesser reduction of the carboxyl and amide peak was exhibited for the peat HA. The CPMAS 13C NMR spectrum of the hydrolyzed peat HA exhibited a shift of the O-substituted aromatic C peak from 152 to 148 ppm. In addition to the reduction of carbohydrate signals in the hydrolyzed soil HA, the methylene C peak was significantly decreased as compared with the 128 ppm peak.
Pyrolysis-Gas Chromatography and Mass Spectroscopy
Total ion current chromatograms obtained by pyrolysis-GC/MS analysis for the bulk and treated soil and peat HAs are presented in Fig. 2 and 3
, respectively. The main compounds identified in the chromatograms were grouped into six major classes: polysaccharide-derived (PS), protein-derived (PR), lignin-derived (LG), alkanes/alkenes (AL), fatty acids, and compounds that could not be assigned to a specific biological source. Peaks are identified in Table 3.
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The major peaks in the untreated peat HA pyrolysis-GC/MS chromatogram (Fig. 3U) were phenol derivatives: phenol (LG1), guaiacol (LG2) and 2,6-dimethoxy phenol (LG5), and 3-methyl phenol (12). The bleaching treatment, which selectively removes O-substituted aromatic components (lignin), resulted in a relative increase in the peak area of alkanes and 1-alkenes, and all homologs from C9 to C27 were observed (Fig. 3B). In addition, several branched alkenes were identified. However, phenol and 2-methoxy phenol peaks were still present in the bleached peat HA chromatogram, suggestive of an incomplete bleaching and removal of lignin. Hydrolysis of the peat HA (Fig. 3H) resulted in little change, mainly a relative enhancement of the LG peaks (LG1, LG2, LG4, and LG6) and AL pairs, because of the removal of polysaccharides and carbonyl groups (such as in esters and amides).
Tetramethylammonium Hydroxide Thermochemolysis-Gas Chromatography and Mass Spectroscopy
Tetramethylammonium hydroxide thermochemolysis of the soil and peat HA samples (Fig. 4 and 5
, respectively) yielded LG compounds (methylated p-hydroxyphenyl, guaiacyl, and syringyl compounds), nonlignin derived aromatic compounds, heterocyclic N compounds, FAMEs, and DAMEs. Peaks are identified in Table 4 and labeled in Fig. 4 and 5.
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Unlike the soil HA, the peat HA treated with TMAH thermochemolysis yielded more LG compounds from syringyl structures (S1, S5, and S6; Fig. 5U). The only LG compound identified in the bleached peat HA chromatogram was G6 and at a significantly reduced intensity (Fig. 5B). The bleached peat HA chromatogram exhibited a well-pronounced series of FAMEs (C8 to C32) with a strong predominance of even over odd C units. In addition to the series of FAMEs, DAMEs (C4 to C10) were also identified. The hydrolysis treatment enhanced the intensity of the LG peaks and FAMEs as compared with the chromatogram of the untreated HA.
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DISCUSSION |
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Bulk Humic Acid Samples
The soil HA was extracted from a surface layer of soil rich in OM, that was developed under grassland vegetation over glacial till. Although both soil and peat samples are considered well humified (Lyon, 1995; Salloum, 1999), the soil HA exhibited a higher level of C-substituted aromatic C and a lower content of phenolic C than the peat HA, as revealed by the 13C NMR spectra. This is consistent with the background and nature of the source samples, as the peat contains more lignin-like matter than the soil. The substantial amount of alkyl-C in both HA samples is consistent with previous reports (Malcolm, 1989; del Rio et al., 1994; Almendros et al., 1998). The peat HA exhibited a sharp peak at 32 ppm which is assigned to carbons in long crystalline (rigid) polymethylenic (CH2)n chains (Kögel-Knabner et al., 1992; Hu et al., 2000). The amorphous (mobile) polymethylenic C peak at 30 ppm was exhibited only as a small shoulder in the peat HA spectrum. However, the mobile alkyl C peak was more intense in the soil HA spectrum. The crystalline polymethylenic domains are expected to be more resistant to microbial degradation and therefore have longer residence times in soils than the amorphous (mobile) region (Hu et al., 2000). The relationship between the chemical nature of the polymethylenic domains in HAs and the degree of humification is not yet well understood and should be further investigated.
Pyrolysis-GC/MS analysis revealed high levels of compounds derived from polysaccharides. The PS peaks were more pronounced in the soil HA than in the peat HA, and this is consistent with the relative intensities of the polysaccharide regions exhibited in the 13C NMR spectra. The pyrolysis-GC/MS data clearly indicate a higher contribution of LG residues in the peat HA structure than in the soil HA. Moreover, the peat HA pyrolysate chromatogram exhibited a relatively higher intensity for syringyl lignin units (such as 2,6-dimethoxy phenol) as compared with its relative intensity in the bulk soil HA chromatogram. This suggests that in the soil HA, the lignin is either derived mainly from gymnosperms, or the syringyl structures are selectively degraded during the humification process. The major LG compound identified in both pyrolysis chromatograms was phenol (Fig. 2 and 3). Phenol, in addition to being a pyrolysis product of lignin, has been reported to be generated as a pyrolysis product of polyphenolic compounds such as melanoidin- and polyphenolic-type components and amino acids, such as Tyr (van Heemst et al., 1999; Peulve et al., 1996).
In addition to the LG compounds, the pyrolysis-GC/MS chromatograms contained N-derived compounds such as pyrroles, indoles, pyridines, amides, and nitriles. The indoles may have originated from tryptophan-containing peptides, whereas the pyrroles can be derived from proline-containing peptides. The soil HA chromatogram exhibited a much higher content of these protein-derived compounds, suggesting a higher content of proteinaceous materials as part of the humic structure. The origin of the alkylated phenols (2- and 3-methyl phenols, and 3-ethyl phenol) that were present as dominant peaks in the peat HA samples, is unknown, and these could be derived from Tyr.
A higher proportion of LG peaks was produced by TMAH thermochemolysis of the bulk peat HA than that of the soil HA. Moreover, the peat HA yielded mainly the more oxidized acid-containing derivatives (such as P6, G6, and S6). The benzenecarboxylic acids in TMAH products are predominantly derived from lignin units where the 
carbon of the side chain has been oxidized to a carboxyl group (Hatcher et al., 1995). This data support the pyrolysis finding, suggesting that both HAs contain lignin at an advanced stage of oxidation. Similar patterns of LG compounds have been reported in peat HA (del Rio and Hatcher, 1998), however a larger abundance of guaiacyl-like structures has been reported for forest soil HAs (Nierop et al., 1999). These data suggest that the sources of lignin (grass vs. wood) and the chemical nature of the HA are strongly related.
Under conditions of TMAH thermochemolysis, both untreated HA samples yielded saturated, unsaturated, and branched FAMEs of varying C chain lengths (from C6 to C32), with the most intense peaks being from C16 FAMEs. In addition, a series of DAMEs (C4 to C28) were also detected. A strong even-over-odd predominance was observed for this series, without a significant difference between the two HA samples. The presence of even-numbered long-chain fatty acids and dicarboxylic acids in both HA samples may be primarily because of an input of aboveground plant aliphatic biopolymers such as cutin and cutan (McKinney et al., 1996; del Rio et al., 1998), and belowground aliphatic plant biopolymers such as suberin and suberan (Nierop, 1998). Short-chain fatty acids and dicarboxylic acids (C4-C9), and the odd-C numbered and branched-chain fatty acids are commonly used as bacterial biomarkers. This set of data suggests that part of the HA macromolecular structure originates from refractory, highly aliphatic plant biopolymers (i.e., cuticular materials) and from microbial remains.
Bleaching
Oxidative degradation of HAs by hypochlorite has been used in the past to analyze soil HAs, coals, and HS from water (Christman et al., 1989). This reaction, in the case of aromatic substrates, involves electrophilic substitution of chlorine, followed by loss of aromatic character and oxidative cleavage of the ring. Chakrabartty and Kretschmer (1974) concluded that chlorine oxidation depends on the presence and reactivity of electronegative ring substituents. Aromatic rings activated by electron-withdrawing substituents such as nitro, hydroxy, methoxy, cyano, and carboxyl substituents can be oxidized, whereas condensed aromatic rings cannot. Therefore, this method is effective for decomposing noncondensed aromatic structures such as lignin-like and polyphenol units found in HAs. In this study, bleaching was conducted under acidic conditions and, therefore, chlorine dioxide or molecular O2 were the bleaching agents (Hebeish et al., 1997).
The 13C NMR spectrum of the bleached peat HA was similar to that of insoluble and nonhydrolyzable organic residue from a forest soil (Augris et al., 1998). Both spectra are characterized by a sharp peak of long polymethylenic chains. These 13C NMR spectra are similar to the spectra of cuticles from spruce needles (Kögel-Knabner et al., 1994). The highly aliphatic nature of the bleached HAs revealed by the 13C NMR spectra supports the conclusion that highly refractory aliphatic plant biopolymers are an important and recalcitrant part of the HA structure. In a similar study by Hatcher et al. (1986) on peat samples from The Everglades, FL, paraffinic structures were found to be the dominant compounds of bleached whole peat. According to the 13C NMR data, the contents of aliphatic bleach-resistant plant biopolymer-like compounds in the soil and peat HAs are 8 and 4% (by weight), respectively. The pyrolyzates of the bleached peat HA exhibited a series of alkane and alkene pairs similar to the pyrolysates of cutan from Agave americana (Kögel-Knabner et al., 1992; McKinney et al., 1996) and nonhydrolyzable organic residue from a forest soil (Augris et al., 1998). Tetramethylammonium hydroxide thermochemolysis of the bleached peat HA yielded methyl derivatives of long-chain fatty acids and dicarboxylic acids. The major FAMEs produced had 16 C atoms, which is the chain length of biopolymers typically found in cutin (del Rio and Hatcher, 1998).
The residual aromatic structures (after bleaching) in the 13C NMR spectra are likely to be from condensed aromatic moieties that are not susceptible to bleaching via NaClO2 or NaClO oxidation. The source of these compounds cannot be lignin or tannins because both are known to have a high degree of O-substituted aromatic carbons. We suspect that charcoal, charred plant materials, or coal fractions could be the origin of these structures, as these have been proposed to be an important pool of C in soils. Charring of plant materials was suggested to be one of the mechanisms of HA formation in volcanic ash soils (Shindo et al., 1986). Skjemstad et al. (1996) used photo-oxidation and NMR analyses to obtain quantitative measures of black C in soils and found that up to 30% of the total soil C can be estimated to be in the form of charcoal. Our data from the NaClO2 oxidation and NMR suggest that materials whose origin may be black C are less likely to contribute to HA structure in our samples. Only 5% (by weight) of the soil HA is likely to originate from condensed aromatic structures. The data for the peat HA suggests less than 1% contribution of condensed aromatic structures to the humic macromolecular structure. These differences could be because of the differences between samples and the different oxidation methods used.
The presence of phenol and methoxybenzene peaks in the bleached chromatograms (pyrolysis and TMAH, respectively) indicates that phenol is not solely a LG compound as already discussed. Nonlignin-derived aromatic compounds, such as methoxymethyl benzene, benzaldehyde, and benzoic acid methyl ester probably represent highly humified lignin structures.
Acid Hydrolysis
The nonhydrolyzable soil HA constituted 60% by weight of the original bulk sample, which is in the typical range for such residues of soil HAs (Orlov, 1995). However in the case of the peat HA, the hydrolysis residue constituted a much higher fraction of the bulk sample (73%), and this is more typical for HAs extracted from brown coals (Orlov, 1995). It was suggested that the degree of hydrolyzability of HS can be used as an indication of their degree of humification. As such, fulvic acid and fresh SOM are subject to much greater hydrolysis than highly humified substances. However, in the current study both HAs can be considered to be well humified, and the relative hydrolyzability does not reflect the maturity of the HA, but rather their constituent building blocks (i.e., origin). It is clear from the 13C NMR spectra (Fig. 1) that the hydrolyzed peat HA contains more lignin-type structures (peaks at 56 and 148 ppm). The disappearance of the peak at 56 ppm in the hydrolyzed soil HA spectrum suggests that it was not from methoxyl C, but was, instead, contributed by other resonances, probably carbons of amino acids (such as Arg, Glu, and Lys). The sharp peak at 128 ppm in the hydrolyzed soil HA spectrum suggests that most of the aromatic carbons in the HA structure are unsubstituted carbons or C-substituted (such as alkylbenzenes). The peak at 128 ppm in the hydrolyzed peat HA can be assigned to C1, C2, and C6 carbons in p-hydroxyphenyl units of lignin (Hatcher, 1987). These units were the main peaks in the hydrolyzed peat HA pyrolysis chromatogram (phenol and 3-methyl phenol).
The hydrolysis procedure resolved a new peak at 148 ppm in the 13C NMR spectrum of the peat HA. The peaks at 153 and 148 ppm can be used to distinguish hardwood from softwood lignin (Hatcher, 1987). The 153 ppm peak is assigned to C3 and C5 of syringyl units (hardwood), whereas the 148 ppm peak is assigned to C3 and C4 of guaiacyl units (softwood). The contribution of softwood lignin units to the humic structures is also supported by the TMAH thermochemolysis analysis. However, this cannot explain the shift of the O-substituted aromatic C peak from 152 ppm in the bulk sample to 148 ppm after hydrolysis. An explanation for this might be that the lignin-related peak in the bulk peat HA spectrum was poorly resolved and the resolution was improved only after removal of nonlignin components by acid hydrolysis. Alternately, cleavage of the ß-O-4 bonds in the lignin structure resulted in changing the resonance of the ring carbons and enhancing the signal at 147 to 148 ppm.
Although acid hydrolysis has been reported to remove most amino acids and carbohydrates from soil HAs (Preston and Schnitzer, 1984), non-hydrolyzable N-containing compounds have been reported to contain 20 to 35% of the total N in soils (Stevenson, 1994). In this study, both TMAH thermochemolysis and pyrolysis of the hydrolyzed HAs yielded N-containing compounds such as 1-methyl-2,5-pyrrolidinedione and pyridine. These compounds could be derived from amino acids bound to phenolic or quinone groups (Stevenson, 1986), proteinaceous materials encapsulated in the humic structures (Knicker and Lüdemann, 1995; Knicker and Hatcher, 1997) or alkyl-substituted heterocyclic N-containing compounds which are structural constituents of the humic structure (Leinweber and Schulten, 1998). Schulten and Schnitzer (1998) reported that 35% of the N in pyrolysis products of HS are heterocyclic N-containing compounds. However, 15N NMR study carried out by Knicker et al. (2000) suggests that most of the N compounds in HS are in the form of proteins that are trapped in the HS macromolecule. The origin of these N-containing compounds is still unknown and should be further investigated.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Current address: Dep. of Soil and Water Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 Israel. 
Received for publication April 30, 2001.
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