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DIVISION S-2-SOIL CHEMISTRY

Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)

^a Dep. of Chemistry, Ohio State Univ., 100 W. 18th Ave. Columbus, OH 43210 USA
^b Dep. of Soil and Water Sciences, Faculty of Agriculture, Food and Environ. Qual. Sci., Rehovot P.O.B. 12 Israel 76100
^c USDA–ARS, Univ. of Minnesota, St. Paul, MN 55108 USA

bchefetz{at}chemistry.ohio-state.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusion REFERENCES

 
Tetramethylammonium hydroxide (TMAH) thermochemolysis–gas chromatography/mass spectrometry (GC/MS) was employed to study the chemical structure of soil organic matter sampled from a soil plot in which corn (Zea mays L.) was farmed continuously for 15 yr. The chromatograms exhibited peaks related to compounds derived from lignin, fatty acid methyl esters (FAMEs), non-lignin aromatic structures, and heterocyclic N compounds. The dominant lignin-derived peaks in the TMAH thermochemolysis–GC/MS chromatograms were mainly derivatives of p-hydroxyphenyl and guaiacyl structures, suggesting a non-woody (grass) lignin type. With depth, the ratio of syringyl to guaiacyl compounds (S/G) decreased, suggesting a preferential degradation of the syringyl units by microorganisms. Fatty acid methyl esters of varying C-chain length (C₇ to C₂₇) were identified in the soil chromatograms. Both TMAH–GC/MS and ¹³C-NMR (nuclear magnetic resonance) data suggested a relative increase of long-chain fatty acids with soil depth (or degree of humification), suggesting a refractory nature for these compounds. The heterocyclic N compounds yielded from the TMAH thermochemolysis were mainly pyrroles, pyridines, and pyrazoles. In addition, low levels of methylated amino acids (phenylalanine, leucine, and valine) were detected. The presence of the amino acids in the bottom layer of the soil suggests a preservation mechanism. The changes identified in the chemical components provide clues as to the nature of the humification processes in the soil profile and also yield information on the nature of the sources of soil organic matter.

Abbreviations: C/G, cinnamyl phenol to guaiacyl ratio • CPMAS, cross polarization magic angle spinning • FA, fulvic acid • FAMEs, fatty acid methyl esters • G, lignin derived guaiacyl compounds (methylated) • GC, gas chromatograph • HA, humic acid • HS, humic substances • MS, mass spectrometry • NMR, nuclear magnetic resonance • P, lignin-derived p-hydroxyphenyl compounds (methylated) • S, lignin-derived syringyl compounds (methylated) • S/G, syringyl to guaiacyl ratio • SOM, soil organic matter • TMAH, tetramethylammonium hydroxide

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusion REFERENCES

 
THERMOCHEMOLYSIS in the presence of tetramethylammonium hydroxide (TMAH; off-line thermochemolysis) is a novel technique that provides new detailed information on the structure and composition of soil organic matter (SOM). This technique and pyrolysis in the presence of TMAH (in situ methylation) have been introduced as effective techniques for analyzing a large variety of natural macromolecules. These techniques were used to characterize lignin (Hatcher et al., 1995; Clifford et al., 1995; Hatcher and Minard, 1996), humic substances (Hatcher and Clifford, 1994; Martin et al., 1994, 1995; del Rio and Hatcher, 1996), coalified woods (McKinney and Hatcher, 1996), carbohydrates (Fabbri and Helleur, 1999), lipids (Challinor, 1996; Ishida et al., 1999), and cutan (McKinney et al., 1996). It has been demonstrated that the TMAH technique is a chemolytic procedure that hydrolyzes and methylates esters and ether linkages, assisting depolymerization and methylation. Methylation makes the polar products of the reaction volatile enough for gas chromatographic analysis. The TMAH thermochemolysis was found to be effective in cleaving the major linkage in native lignin, the ß-O-4 ester bond (Hatcher and Minard, 1996). A detailed study of the TMAH reaction mechanism using ¹³C-labeled TMAH suggests that the cleavage of the ß-O-4 ester bond in lignin proceeds through a base-catalyzed intermolecular epoxidation, with the or hydroxyl acting as a nucleophile, resulting in a loss of the phenoxide group (Filley et al., 1999).

Soil organic matter–extracted fractions such as humic acid (HA), fulvic acid (FA), and humin have been analyzed by TMAH thermochemolysis. Hatcher and Clifford (1994) and del Rio et al. (1994) have shown that the TMAH thermochemolysis of HA yields monomethoxy, dimethoxy, and trimethoxy benzecarboxylic acid methyl esters and fatty acid methyl esters (FAMEs) originating from structural components of the HA.

Soil organic matter in agricultural soils consists of a mixture of plant, microbial, and insect residues; dissolved organic matter; and humic substances (HS). Humic substances are formed during microbial decomposition of plant litter (Stevenson, 1994). With soil depth, SOM is mineralized and humified, resulting in a mixture of structurally identifiable materials as plant residue macromolecules and HS. Therefore, knowing the chemical structure of plant tissues and their transformation in soils is essential to understanding the humification processes that occur in soils. In this study, we applied the off-line TMAH thermochemolysis–GC/MS together with solid-state ¹³C-NMR, using the cross polarization magic angle spinning (CPMAS) with the ramp-CP technique, for the analysis of bulk (untreated) agricultural soil profile. Our goal is to use these chemical approaches on untreated samples to develop a better understanding of how humification occurs in a well-defined profile from a cultivated soil.

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusion REFERENCES

 
Soil Profile Samples
The soil samples used in this study were provided from a field experiment at Waseca, MN. In short, soil samples (Webster clay loam) from four depths, 0 to 7.5, 7.5 to 15, 15 to 30, and 30 to 45 cm, were collected from a 9.9- by 9.1-m plot in which only corn was grown continuously for 15 yr. The samples were air-dried, sieved through a 2-mm sieve, and oven-dried (65°C) before analysis. Total SOM content was measured by loss of weight on ignition at 550°C for 8 h.

Tetramethylammonium Hydroxide Thermochemolysis–Gas Chromatography/Mass Spectrometry
For the TMAH analysis, 1 to 2 mg of soil organic matter was used. Therefore, 50 mg of oven-dried soil samples from the 0- to 7.5-cm and 7.5- to 15-cm layers (3–4.5% organic matter), and 100 mg of samples from the 15- to 30-cm and 30- to 45-cm layers (1.5–2% organic matter) were weighed and placed in glass tubes (three replicates). Two hundred milliliters of TMAH (25% in methanol; Aldrich, Milwaukee, WI) was added to the tubes containing the soil samples and gently mixed before the methanol was evaporated under a stream of N₂. Then, 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, internal standard (1951 ng of n-eicosane) was added, and the inside surfaces were extracted (three times) using ethyl acetate. The combined extracts were reduced to 50 mL under a stream of N₂. Gas chromatographic analyses were performed using a Hewlett-Packard 6890 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 of 0.1 mm; Supelco, Bellefonte, PA). Samples (1 mL) were injected using an autoinjector (Hewlett-Packard 7683 series), with a split ratio of 5 and a front inlet temperature of 310°C. Helium was used as a carrier gas with a flow rate of 1 mL min^-1; electronic flow control was set for constant flow. The GC oven temperature was programmed from 40 to 300°C at a rate of 8°C min^-1. The GC was directly coupled to a Pegasus II (Leco, St. Joseph, MI) time-of-flight mass spectrometer by a deactivated fused silica transfer line heated to 300°C. Mass spectra, from 33 to 700 m/z, accumulated at a rate of 9 scans s^-1. Most peaks were identified by comparison with the NIST (version 1.6) library.

For quantitative purposes, a single response factor was used for all syringyl lignin–derived compounds and another for all guaiacyl lignin–derived compounds. The response factors were calculated by taking the average of the response factors of the commercial standards dimethoxybenzaldehyde and trimethoxybenzaldehyde (G4 and S4, respectively) and dimethoxybenzoic and trimethoxybenzoic acid methyl ester (G6 and S6, respectively) relative to the standard (n-eicosane). The syringyl to guaiacyl ratios (S/G) were calculated by summing the responses for amounts of the compounds containing a syringyl structure divided by the sum of responses for compounds containing a guaiacyl structure.

Carbon-13 Nuclear Magnetic Resonance Spectroscopy
Solid-state CPMAS ¹³C-NMR spectra were obtained using a Bruker DPX 300-MHz NMR-spectrometer (Bruker Analytic GmbH, Germany). The spectrometer operates at a ¹H frequency of 75 MHz and a ¹³C frequency of 300 MHz. The experimental parameters were the following: contact time of 3 ms; recycle delay time of 1 s; sweep width of 27 kHz (368 ppm), and line broadening of 100 Hz. Freeze-dried samples were placed into a 4-mm rotor and spun at a frequency of 13 kHz at the magic angle (54.7° to the magnetic field). The CP method described by Cook and Langford (1998) was used to obtain quantitative CPMAS spectra at this field. The two-pulse phase-modulation procedure was also used.

	Results and discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusion REFERENCES

 
The TMAH thermochemolysis is highly selective for specific polar groups such as esters, phenols, and acids. It provides information concerning the molecular fragments comprising the SOM. Therefore, to elaborate the overall structural changes in the SOM and the associated humification process in soils, this technique was complemented with ¹³C-NMR, which provides a more global perspective on structural information from bulk SOM. The TMAH thermochemolysis of the four soil layers (Fig. 1 and 2) yielded methylated p-hydroxyphenyl, guaiacyl and syringyl compounds (lignin-derived structures), aromatic (non-lignin–derived) structures, heterocyclic N (protein-derived), and FAMEs. Peak identifications are presented in Table 1 .

View larger version (26K): [in this window] [in a new window]   Fig. 1 (A) Tetramethylammonium hydroxide (TMAH) thermochemolysis–gas chromatography/mass spectrometry chromatogram of 0- to 7.5-cm soil layer, and (B) 7.5–15 cm soil layer. For peak identification refer to Table 1

View larger version (24K): [in this window] [in a new window]   Fig. 2 (A) Tetramethylammonium hydroxide (TMAH) thermochemolysis–gas chromatography/mass spectrometry chromatogram of 15–30 cm soil layer, and (B) 30–45 cm soil layer. For peak identification refer to Table 1

With depth, a few changes in relative intensity of the grass lignin–derived peaks were observed (Fig. 1 and 2). The relative intensity of the all lignin–derived peaks was reduced, but the S compounds (S1 and S5) exhibited the sharpest decrease. In addition, the peak of G3 decreased, compared with a relative increase of the peaks for P4, P5, and P6. The intensity of S5, which was one of the major peaks in the chromatogram of the top soil layer, decreased significantly with depth and could not be observed in the deepest layer (30–45 cm). This phenomenon indicates a decomposition and transformation mechanism of the lignin building blocks as humification processes with increasing depth. Lignin decomposition and its structural changes can be monitored by the S/G (syringyl to guaiacyl) and C/G (cinnamyl phenol to guaiacyl) ratios. The use of the S/G ratio as a parameter for lignin degradation is based on the assumption that condensed lignin G-type units are more resistant to biological degradation than lignin with a larger proportion of methoxyl groups (S-type; Kögel-Knabner, 1997). With increasing depth (Fig. 3) , the S/G ratio decreased, suggesting a preferential degradation of the syringyl units by microorganisms. The decrease of the S/G ratio resulted mainly from the sharp decrease of the S1 and S5 peaks. However, the C/G ratio exhibited no changes with soil depth and was stable at a level of 0.2. In sedimentary systems, the C/G ratio increases with increasing contributions of non-woody plants, while increases of the S/G ratio suggest input of woody plant (Hedges and Mann, 1979). In our study, we can assume that there were no changes to the vegetation over the course of humification; thus, the structural changes of lignin recorded by the S/G ratio are solely related to biodegradation and humification processes.

View larger version (12K): [in this window] [in a new window]   Fig. 3 Syringyl to guaiacyl ratio (S/G) as calculated from the tetramethylammonium hydroxide (TMAH) thermochemolysis–gas chromatography/mass spectrometry chromatograms vs. soil depth

In support of this view, the carbohydrate peaks in the ¹³C-NMR spectra (72 and 105 ppm; Fig. 4) exhibited a sharp decrease with depth, suggesting decomposition of this carbohydrate component of SOM. The polysaccharide level, calculated from the ¹³C-NMR spectra (60–112 ppm), decreased 21% (from 38–30% of the total C) with depth. A similar trend was reported by Zech et al. (1997) and Kögel-Knabner (1997) for the cellulose content in a forest soil profile. These studies have reported a decrease in the carbohydrate content with humification. The lack of carbohydrate-derived peaks in the TMAH chromatogram is due to poor sensitivity of this technique to carbohydrates (Clifford et al., 1995). Thus, we conclude on the basis of the above findings that the origin of the 1,2,4-trimethoxybenzene peak in the soil chromatogram was not cellulose but potentially refractory organic-matter compounds of soil HS.

View larger version (17K): [in this window] [in a new window]   Fig. 4 Cross polarization magic angle spinning (CPMAS) 13C-nuclear magnetic resonance (NMR) spectra of the soil layers at (A) 0–7.5 cm, (B) 7.5–15 cm, (C) 15–30 cm, and (D) 30–45 cm

Under TMAH thermochemolysis conditions, the soil sample throughout the profile yielded saturated, unsaturated, and branched FAMEs of varying C-chain length (from C₇ to C₂₇). The presence of long-chain fatty acids in the soil may be primarily due to the input of aboveground plant aliphatic biopolymers such as cutin (McKinney et al., 1996), plant root aliphatic biopolymers such as suberin, and microbial activity byproducts. The presence of FAMEs originating from aliphatic biopolymers in soil is supported by the characteristic 32-ppm peak in the solid-state ¹³C-NMR (Fig. 4). Several studies have shown that aliphatic biopolymers (e.g., cutan) are non-hydrolyzable and can be preserved in sediments with minor alteration (Nip et al., 1986; Hatcher et al., 1983). Moreover, FAMEs were produced by TMAH from low-rank coal and soil HAs, and the fatty acids were suggested to be linked to the HA macromolecule network via ester bonds (del Rio et al., 1994; Hatcher and Clifford, 1994). A similar series of FAMEs was observed from milled beech leaf litter (Hermosin and Saiz-Jimenez, 1999) and a soil humin fraction (Fabbri et al., 1996). The humin and beech leaf yielded long-chain saturated and unsaturated FAMEs that probably originated from cutin and cutan. The product 1,3,5-trimethoxybenzene, which was found to be a major part of cutan's structure (McKinney et al., 1996), was observed only in the upper 15-cm layer and was not present in the chromatographs from the 30- to 45-cm layer. This suggests that cutan might not be the only source for aliphatic biopolymers in the soil that might yield long-chain FAMEs.

In agricultural soils, where most of the aboveground biomass is removed, the contribution of the underground biomass (roots) to the SOM increases. Thus, the origin of the fatty acids (FAMEs, produced by the TMAH thermochemolysis) could be the corn root system. Nierop (1998) reported that pyrolysis of roots revealed alkane and alkene patterns, which can be attributed to suberin present in the roots. In addition, the unsaturated and branched fatty acids could originate from microbial activity. The solid-state NMR spectra exhibited a relative increase of the paraffinic-C region (0–50 ppm) with depth (from 21–25% of the total C). The increasing level of aliphatic C recorded from the NMR spectra and the unchanged levels of fatty acids with depth, suggest that the structures yielding these aliphatic compounds in the soil are highly refractory. Thus, their relative concentration increased with humification. A similar trend for the aliphatic C structures was reported for forest soil horizons using NMR analyses (Zech et al., 1997).

Tetramethylammonium hydroxide thermochemolysis of the studied soil samples yielded heterocyclic N products as pyrroles, pyridines, and pyrazoles (Table 1). These peaks were present in all four soil layers, suggesting low bioavailability of these N forms. A series of N-heterocyclic structures were identified in pyrolysis GC/MS chromatogram of several SOM samples (Leinweber and Schulten, 1999). These authors suggested that these structures are original N structures present in the soil. But ¹⁵N-NMR spectroscopic investigation of soils revealed that the major part of organic N is bound in amide-N fanctional groups, while heterocyclic-N compounds were not detected (Kögel-Knabner, 1997). Moreover, it was observed that during pyrolysis, dipeptides have been formed to cyclic dipeptides and amino acids were transformed by intermolecular condensation (Chiavari and Galletti, 1992; Hendricker and Voorhees, 1998). Thus, the heterocyclic N compounds identified in the TMAH thermochemolysis chromatograms originate from peptides and proteins in refractory forms of the SOM (Knicker and Hatcher, 1997). The TMAH thermochemolysis analysis of the soil profile samples studied yielded, in addition to the N-heterocyclic structures, methylated amino acids (phenylalanine, leucine, and valine). These amino acids were present in relatively high levels in the higher top layer (0–7.5 cm) than in the deep layers. The dominant source for amino acids in soils is cell walls of microorganisms and plant roots exudates (Stevenson, 1994). Therefore, the presence of amino acids in the deep soil layers suggests a preservation mechanism, which protects the labile proteins from biodegradation. Encapsulation of organic N (mainly proteins) in refractory SOM has been suggested as a possible mechanism (Knicker and Hatcher, 1997).

	Conclusion

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion Conclusion REFERENCES

 
In this research, we studied chemical structures in SOM using TMAH thermochemolysis: a novel, advanced technique that allows analysis without extraction of the SOM. The research aim was to elaborate SOM structural changes with soil depth. The TMAH thermochemolysis was shown to be a useful technique for characterizing non-extracted SOM. The main lignin-derived products were monomethoxyphenyl (P) and dimethoxyphenyl (G), suggesting that the main source of organic-matter inputs to the soil was grass-type lignin litter. With depth, the S/G ratio decreased, suggesting preferential degradation processes of the syringyl units by microorganisms. The decrease of the S/G ratio and the relative increase of the aromatic and carboxylic C–containing groups exhibited in the ¹³C-NMR spectra, support the theory that side-chain oxidation of lignin structures is one of the major humification processes occurring in soils.

In addition to lignin-derived compounds, the TMAH thermochemolysis indicated the presence of a relatively large fraction of long-chain fatty acids. The long-chain fatty acids, which were also found in TMAH analyses of HAs, seem to be highly resistant to biological degradation. This hypothesis was also supported by the ¹³C-NMR spectra, which exhibited an increase of the paraffinic C with depth.

Refractory and highly aged organic matter dominates the soil C storage. However, surface soil layers seem to contain mostly organic materials exhibiting pattern characteristics of plant litter. With humification and/or soil depth, the resemblance of both the ¹³C-NMR spectra and TMAH thermochemolysis chromatograms to those of plant components decreases as a result of mineralization, decomposition, and repolymerization processes.

	ACKNOWLEDGMENTS

 
This research was supported by postdoctoral award no. FI-275-98 from BARD, The United States–Israel Binational Agricultural Research and Development Fund. We thank Dr. Johnie Brown (Dep. of Chemistry, Ohio State Univ.) for helping with the GC/MS analyses. We thank Dr. R.R. Allmaras (USDA–ARS, Univ. of Minnesota) for assisting in the selection of the soil samples.

Received for publication June 22, 1999.

	REFERENCES

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