Analytical approaches for characterizing soil organic matter
Ingrid KoÈgel-Knabner *
Lehrstuhl fuÈr Bodenkunde, Technische UniversitaÈt MuÈnchen, 85350 Freising-Weihenstephan, Germany
Abstract
Structural information on soil organic matter (SOM) at the molecular level can be obtained on diverse structural units that are amenable to degradation techniques. Chemolytic techniques in combination with colorimetric analyses or GC MS are used to determine amino acids (proteins), sugars (polysaccharides), lipids, or aromatic oxidation products from lignin or charred organic matter. Microbial markers (amino sugars, muramic acid) are analyzed after hydrolysis and gas chromatographic separation. Macromolecular structures can also be subjected to thermochemolytic degrada-tion or pyrolysis and subsequent analysis of the fragments by GC MS. Alternative techniques for the examinadegrada-tion of organic matter in heterogeneous macromolecular mixtures are non-destructive spectroscopic methods, such as nuclear magnetic resonance (NMR) spectroscopy. Although this technique can give good results concerning the gross chemical composition, speci®c compounds are hardly identi®ed. The combination of spectroscopic techniques with thermolytic and chemolytic methods will add substantially to the understanding of the nature of refractory soil organic matter. Physical fractionation prior to analysis provides a means to dierentiate between distinct SOM pools that can be fur-ther characterized by the methods described above. Studies on SOM structural characteristics have focused mainly on the A horizons of soils under agriculture and litter biodegradation in forest soils and need to be extended to a wider variety of soil types and the subsoil.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Soil organic matter; Pyrolysis;13C NMR spectroscopy;15N NMR spectroscopy; Chemolysis; TMAH; Humic substances
1. Introduction
A number of techniques are now used for the struc-tural characterization of soil organic matter (SOM) or SOM components. From these results it has become clear that C cycling and stabilization in soils is inti-mately associated with C structure. The intention of this review is to summarize the recent developments in the structural characterization of SOM components with respect to the biodegradation and stabilization of organic carbon in soils.
First an overview of recent developments in the tech-niques for structural characterization of SOM is given. In combination with new fractionation approaches, these applications have led to major achievements in the understanding of C sequestration and cycling in soils.
Examples are given for tracing the fate of individual plant or microbial components in soils, and for analyz-ing the structural composition of organic nitrogen-con-taining compounds in soils. The presence of charred organic matter from burning events or atmospheric organic contamination may drastically aect the chemical composition of soil organic matter. Finally, a personal view on promising research perspectives for structural chemical investigation of organic matter in soils is pre-sented.
2. Analytical techniques
For the characterization of the chemical composition of organic matter in soils, humic fractions and organic material associated with particle size separates, several approaches using modern analytical techniques are available. Most of the organic matter in soils is present in macromolecular structures that cannot be investigated at
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the molecular level without a degradative step. In general, their investigation involves thermolytic (pyrolysis) and/or chemolytic degradation of the macromolecule into small fragments that are separated and analyzed by colorimetric or chromatographic means. Such techniques comprise the determination of the amino acids content, the amount of carbohydrates or lipids, nucleic acids and amino sugars. Because secondary reactions (rearrange-ment, cracking, hydrogenation and polymerization) in a heterogeneous mixture cannot be excluded, it is obvious that conclusions regarding the original structure of SOM in the macromolecular phase have to be drawn with caution.
2.1. Chemolytic techniques
The chemolytic techniques that have been recently applied successfully in soils are mainly techniques to analyze plant or microbial biopolymers (KoÈgel-Knab-ner, 1995). Hydrolysis is used to obtain hydrolyzable amino acids from proteins. Carbohydrates are released from polysaccharides after dierent types of acid hydrolysis. Solvent extraction has been used extensively to separate extractable lipids in soils (Dinel et al., 1990). Saponi®cation (KoÈgel-Knabner et al., 1989) and CuO oxidation (Goni and Hedges, 1990) have been described as chemolytic methods for cutin and suberin compo-nents in soils, but have not been used for soils any more in recent years. With these techniques, only a part of the soil organic matter present in speci®c structures as determined by CPMAS 13C NMR can be identi®ed. Most of the O-alkyl carbon found in soils is hydrolyz-able, although only part of the hydrolyzable material can be identi®ed as individual carbohydrates. In a number of arable soils investigated by Kiem et al. (2000), the contents of carbohydrate C were signi®cantly correlated with the signal intensities ofO-alkyl C in the CPMAS13C NMR spectra (60 to 110 ppm). But on the average carbohydrate C accounted for only 47% of the relative signal intensity in the O-alkyl-C chemical shift area. It is not yet clear if these results are due to the presence of other components, that contribute to the signal intensity in theO-alkyl-C chemical shift region, but are not carbohydrates, or due to losses of carbohydrates during the hydrolysis procedure. NMR spectroscopy of the hydrolysis residue shows that most of the signal intensity in the O-alkyl-C region of the NMR spectra disappears upon hydrolysis. This is not the case for the proteinaceous structures in SOM. It has been shown in a number of investigations that only a part of the organic nitrogen is hydrolyzable (Schulten and Schnitzer, 1998). The proportion of organic nitrogen that is hydrolyzable is lower in the subsoil than in the A horizon (Schmidt et al., 2000b). However, the hydrolysis residue from soils still shows signal intensity for organic N in amide structures (Siebert et al., 1998).
2.2. Analytical pyrolysis
Various pyrolysis techniques hold considerable pro-mise for assessing SOM composition. The techniques that have been used extensively for soils include Py-GC MS and Py-FIMS (Saiz-Jimenez, 1994a ; Leinweber and Schulten, 1998). Py-GC MS involves chromatographic separation of pyrolysis products into single components and mass spectral data obtained for each component. Py-FIMS does not involve separation of the pyrolysis products, but uses soft ionization to produce pre-dominantly molecular ions of the pyrolysis products.
The interpretation of pyrolysis data requires a detailed knowledge of the pyrolysis behavior of the compounds under study. Many pyrolysis products can originate from chemically diverse SOM components (Saiz-Jimenez, 1994b). Thermal secondary reaction can cause considerable modi®cation of the original compound, which may bias the pyrolysis data. For example, pyr-olysis of cellulose results in carbonyl compounds, acids, furans, pyranones, anhydrosugars and phenols. Besides other compounds, the pyrolysate of proteins revealed alkylpyrrolediones and pyrrolidinediones. Fatty acids may be decarboxylated under the pyrolysis procedure, especially in the presence of mineral soil that may have a catalytic eect on such reactions. Thus mainly alkanes and alkenes can be identi®ed in the pyrolysates obtained from soils, with only minor occurrence of fatty acids (Saiz-Jimenez, 1994a). Nitriles found in soil pyrolysis could originate from the reaction of long chain fatty acid with some nitrogen derivatives present in the soil (van Bergen et al., 1998). The given examples illustrate the complexity of pyrolysates obtained from macro-molecular structures and the diculties involved in the interpretation of data obtained from pyrolytic studies of soil organic material (Saiz-Jimenez, 1994b). Fig. 1 and Table 1 give an example for the total ion current (TIC) obtained from pyrolysis of a forest ¯oor and mineral soil horizon.
2.3. Thermochemolysis with TMAH
Sub-pyrolysis temperatures of 300C in the presence
of TMAH was found to produce a suite of products similar to that observed at higher pyrolysis temperatures (Hatcher and Cliord, 1994; Cliord et al., 1995). It was suggested that the reaction involved in the TMAH/ pyrolysis scheme is one of chemolysis rather than pyrolysis. Performing chemolysis in sealed glass ampoules prior to gas chromatographic analysis, internal standards can be used allowing a quantitative determination of the products generated. Applying this technique, lignin degradation can be analyzed much in the same way as the CuO oxi-dation does (Hatcher et al., 1995). A signi®cant feature of this method is that it may be able to trace lignin where extensive degradation has occurred and resulted in sucient alteration of lignin to render it undetected by the conventional CuO oxidation method or pyrolysis procedure. The mechanism of TMAH thermochemolysis
reactions can be studied using13C-labeled TMAH (Filley et al., 1999).
2.4. Compound-speci®c stable isotope and radiocarbon analysis
Further information on SOM composition and turnover may be obtained by combining structural information from chemolysis or pyrolysis with compound-speci®c stable C or N isotope data (Goni and Eglinton, 1996; Gleixner and Schmidt, 1998; Macko et al., 1998) or AMS radiocarbon dating (Eglinton et al., 1996). These techniques have up to now mainly been applied to sedi-mentary systems (e.g. Eglinton et al., 1997). Gleixner et al. (1999) investigated the turnover of carbohydrates, lignin, lipids and N-containing compounds in an arable soil cropped with a C3 plant (wheat) compared to a soil
Table 1
Nitrogen-containing pyrolysis products detected in the pyrolysates of the three dierent soil horizons (from van Bergen et al., 1998)
Pyrolysis products Origina Mw Leaf litterb Humic layerb Mineral soilb
1 2 3 1 2 3 1 2 3
1 Pyrrole Pro, Hyp, Glu 67 + + + ++ ++ + +++ +++ ++
2 N-methylpyrrole AA/unknown 81 + + + + + + + + +++
3 2-Methylpyrrole Pro, Hyp 81 ÿ + ÿ + + + + + +
4 3-Methylpyrrole Pro, Hyp 81 ÿ + ÿ + + + + + +
5 C2-pyrroles Hyp 95 ÿ ÿ ÿ ÿ + + + + +
6 Pyridine AS/Ala 79 + + + + + + ++ ++ +
7 C1-pyridines AS/Ala 93 ÿ ÿ ÿ ++ + + + + +
8 Indole Trp 117 + + ÿ + ++ + ++ ++ +
9 3-Methylindole Trp 131 + + ÿ + + + + + ÿ
10 C1-Indole Unknown 131 ÿ ÿ ÿ ÿ ÿ + + + ÿ
11 Quinoline? Unknown 129 ÿ ÿ ÿ ÿ + + + + ÿ
12 Isoquinoline? Unknown 129 ÿ ÿ ÿ ÿ + + + + ÿ
13 Benzenamine Unknown 93 ÿ ÿ ÿ ÿ ÿ ÿ ? ÿ ÿ
14 Benzonitrile Unknown 103 + ÿ ÿ + + + ++ ++ ++
15 C1-Benzonitriles Unknown 117 ÿ ÿ ÿ ÿ ÿ + + + +
16 Benzenacetonitrile Phe 117 + + ÿ + + + + + +
17 Benzenepropanenitrile Phe 131 + ÿ ÿ + + + + + ÿ
18 Acetamide AS 59 + + ÿ + + ÿ + + ÿ
19 Acetylpyrrolidone AS 127 ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ
20 3-Acetamido-5-methylfuran AS 139 ÿ ÿ ÿ + + ÿ ÿ ÿ ÿ
21 3-Acetamido-4-pyrone or 3-Acetamido-2-pyrone
AS 153 ÿ ÿ ÿ ÿ + ÿ ÿ ÿ ÿ
22 Oxazoline structures AS 185 ÿ ? ÿ + + ÿ ÿ ÿ ÿ
23 Diketodipyrrole Hyp-Hyp 186 + + + ++ ++ + ++ ++ ?
24 2,5-Diketopiperazines der. Pro-Val, Pro-Arg 154 ÿ + ÿ + + + ÿ + ?
25 2,5-Diketopiperazines der Pro-Ala 168 ÿ ÿ ÿ + + ÿ ÿ ÿ ÿ
26 2,5-Diketopiperazine Pro-Pro 194 ÿ ÿ ÿ + + ÿ + + ÿ
27 Tetradecanenitrile Unknown 209 ÿ ÿ ÿ ÿ ÿ ÿ + + ÿ
28 Hexadecanenitrile Unknown 237 ÿ ÿ ÿ ÿ ÿ ÿ + + ÿ
29 Octadecanenitrile Unknown 265 ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ
30 Eicosanenitrile Unknown 293 ÿ ÿ ÿ ÿ ÿ ÿ + ÿ ÿ
a AA, amino acid; AS, amino sugar; Pro, proline; Hyp, hydroxyproline; Glu, glutamine; Ala, alanine; Trp, tryptophan; Phe, p,
phenylalanine; Val, valine; Arg, arginine. b
transferred to a C4 plant (maize) with pyrolysis-gas chromatography and isotope ratio mass spectrometry (Py-GC/IRMS). They found pyrolysis products with mainly C4 signal, especially lignin degradation products, pyrolysis products with intermediate isotopic enrichment, that were attributed to physically protected plant fragments from both plant precursors and pyrolysis products with a mainly C3 signature. These were mainly pyrolysis products of proteinaceous origin, con®rming that proteins or peptides are preserved during biodegradation and stabilized in soils.
Bol et al. (1996) reported that the radiocarbon age of the aliphatic hydrocarbon fractions of a stagnohumic gley soil were generally older than the bulk soil and mostly older than the residues after acid hydrolysis. Radiocarbon age of the aliphatic hydrocarbon fraction also increased with soil depth in this peat soil. In other soil types, results from such investigations may be complicated by pedogenetic processes, such as the downward move-ment and selective sorption of dissolved organic matter and bioturbation eects of the soil fauna. Nonetheless, these techniques hold great promise to investigate the structural composition of SOM components at the molecular level with simultaneous information on the turnover rates or the speci®c origin of the individual SOM components.
2.5. Spectroscopic techniques
Alternative techniques for the examination of organic matter in heterogeneous macromolecular mixtures are non-destructive spectroscopic methods, which include nuclear magnetic resonance (NMR) spectroscopy, infra-red (IR) spectroscopy and electron spin resonance (ESR) spectroscopy. The big advantage of such techniques lies in the fact that the sample can be analyzed without major pretreatment and extraction. The sample can be examined as a whole and secondary reactions can be avoided. Most of these methods, however, are relatively insensitive and reveal low resolution. Although these techniques can give good results concerning the gross chemical composition, speci®c compounds are hardly identi®ed.
2.5.1. 13C and15N NMR spectroscopy
13C and15N NMR spectroscopy is now widely used
for the characterization of SOM composition. Applying an appropriate instrument setup, the intensity of a NMR signal is proportional to the concentration of the nuclei creating the signal (Knicker and Nanny, 1997). Application of13C and15N NMR to soils has, for a long time, been con®ned to the study of bulk soils or humic extracts for structural characterization using the CPMAS (cross-polarization magic angle spinning) technique (Preston, 1996; KoÈgel-Knabner, 1997). The solid-state 13C NMR spectra are generally recorded as free induction
decay (FID) and integrated using the integration routine of the spectrometer. The chemical shift regions 0±45, 45±110, 110±160 and 160±220 ppm are assigned to alkyl C,O-alkyl C, aromatic C and carboxylic C, respectively (Wilson, 1987). The variation of integration data of sig-nals due to the treatment of a well resolved FID (fourier transformation, phasing and baseline correction) is 5% (Knicker, 1993).
Mineral soils with low C contents have not been investigated in the same number as C-rich soils, although the latter represent only a small percentage of the total soil cover. Almost no information is available for organic carbon composition in the subsoil, except for rather C-rich soil horizons, such as spodic B hor-izons. This was attributed to the fact that spectra from solid samples low in organic carbon are more dicult to obtain because of sensitivity problems (Mahieu et al., 1999). The review of Mahieu et al. (1999) shows that13C NMR spectra from bulk soils are remarkably similar, dominated by signals fromO-alkyl C (45%), followed by alkyl (25%) and aromatic C (20%) and carboxyl and amide C (10%). This may be due to the fact that the SOM in top soils is dominated by high proportions of plant residues with a relatively uniform composition. Additionally, the soil types investigated up to now are rather limited, and may not represent the variability of pedogenetic environments.
Speci®c pulse techniques are now emerging, that may provide signi®cant progress in our knowledge on soil organic matter. Using speci®c pulse techniques in com-bination with 13C- and 15N-labeled parent materials added to the soil, the evolution of these C and N labels can be followed in dierent C and N pools. Dipolar dephasing or interrupted decoupling is a pulse sequence that can be used to dierentiate carbons at the same chemical shift but with dierences of molecular motions or dierences in substitution level (quaternary vs. non-quaternary carbons). In this experiment, the high power decoupling of the conventional CPMAS 13C NMR experiment is turned o for a certain dipolar dephasing time (Tdd), such that the signals from carbons in solids are diminished. During this time, the signals aected by strong proton dipolar coupling are preferentially lost. The loss of13C signal intensity with increasing T
technique to investigate a soil that had been amended with 13C-labeled glucose and 13C-labeled glycin. After incubation, most of the added 13C-label was found in mobile alkyl-C structures assigned to microbial C, whereas the non-living background alkyl-C of this soil was present in more rigid structures.
PSRE (proton spin relaxation editing) spectra are generated by linear combinations of two or more spectra obtained with the inversion recovery pulse sequence at dierent delay times. The subspectra thus generated correspond to signals from domains with dierent proton spin-lattice relaxation time constants. By doing so it is possible to ``fractionate'' OM in dierent structural components. These dierences in proton spin relaxation time constants have been applied to edit CPMAS NMR spectra of dierent types of soil organic matter, by gen-erating subspectra associated with the fast and slow relaxing pools of protons. The PSRE technique was used recently to dierentiate between dierent SOM pools in soils. Golchin et al. (1997b) were able to dier-entiate between partly decomposed plant residues and charcoal. Clinton et al. (1996) identi®ed dierent SOM pools in forest litter using PSRE. Fig. 2 shows the dif-ferentiation between the slow and fast relaxing sub-components of a grassland soil, associated with plant fragments, partly decomposed plant residues, and more recalcitrant organic matter (Condron and Newman, 1998). Sub-spectrum A was assigned to highly ordered plant fragments, dominated by crystalline cellulose. Sub-spectrum B originates from structures with higher molecular disorder and was assigned to partly degraded plant residues, with major contributions of O-alkyl-C, carboxylic acids and proteins. Sub-spectrum C is domi-nated by mobile polymethylene structures (30 ppm). It resembles SOM associated with the ®ne clay fractions of soils and was thus considered as recalcitrant fraction.
The so-called Bloch decay does not require the proximity of protons as in the cross polarization experiment. Solid-state 13C NMR spectra are thus obtained after direct excitation of the 13C spins. This becomes important for the detection of highly con-densed aromatic structures in soils, such as charred organic matter, bituminous coal residues or soot (KoÈgel-Knabner and Knicker, 2000). The CPMAS13C NMR spectrum of the Oh horizon of a mine soil devel-oped from forest-remediated Tertiary sandy overburden material aected by lignite dust from nearby briquette-producing factories and power plants emitting thermally altered lignite combustion products. (Fig. 3) shows the typical pattern of forest-¯oor organic matter, with signals mainly from plant litter components (polysaccharides, lignin, aliphatic biopolymers). In the Ai horizon, a decrease in signal intensity in the chemical shift region ofO-alkyl carbon is observed. It occurs simultaneously to an increase in relative signal intensity in all other regions and can be explained by the preferred degradation
of carbohydrates and the accumulation of more refractory organic material. The NMR spectrum of the reference, lignite-derived dust material consists mainly of aromatic and aliphatic carbon species (chemical shift regions 110± 160 and 0±45 ppm). The Bloch decay spectrum of the Ai horizon reveals a high proportion of aromatic carbon, most tentatively derived from lignite combustion products, that is only partly observed in CPMAS13C NMR spectrum of the same soil material.
Fig. 2. Proton spin relaxation edited subspectra A±C from the 13C NMR spectrum of a grassland (top) and a forest soil
2.5.2. IR and ESR spectroscopy
IR spectroscopy in the DRIFT mode may provide rapid and reliable information on SOM composition. Capriel (1997) used this technique to obtain quantitative information on the amount of aliphatic C±H units in soil under dierent management practice. The decrease of C content due to management was accompanied by a decrease of the aliphatic C±H components within SOM. ESR spectroscopy gives information on species that contain unpaired electrons, for SOM especially free radicals (Cheshire and Senesi, 1998). This technique has only scarcely been used in recent years for SOM studies. Generally a single unstructured signal is obtained for humic substances. Cheshire and McPhail (1996) showed that the resolution of ESR spectroscopy can be improved and that by using appropriate instrument set-tings hyper®ne structure information could be obtained for a number of humic acids. An important ®nding from this work is that these humic acids did not give spectra resembling those of semiquinone monomer radicals generated from catechol or protocatechuic acid.
3. Characterization of SOM fractions
3.1. Fractionation procedures
Plant residues, microbial residues, and their transfor-mation products (= humic substances?) form the organic matter in soils. Thus, in contrast to sediments,
each soil horizon represents a mixture of these materials in dierent stages of degradation. Therefore, analysis of SOM has bene®ted tremendously from physical fraction-ation according to size and/or density (Baldock and Skjemstad, 2000). These methods are designed to frac-tionate SOM into pools of dierent turnover times as they dierentiate between free particulate organic materials and organic materials associated with soil minerals (Tiessen et al. 1984; Christensen, 1992; Golchin et al., 1997a). Although there is not a single fractionation procedure that is applicable to all soils and gives a complete separation of OM with dierent turnover times, a combination of methods is available to obtain proximate fractions (Trumbore and Zheng, 1996; Baldock and Skjemstad, 2000).
They are supposed to disperse soils into dierent types of substructures, that have been designated primary (organo-mineral associates) and secondary organo-mineral compounds, i.e. soil aggregates (Chris-tensen, 1996). In this simpli®ed conception of the soil structural arrangements, primary organo-mineral associates are formed by adsorption of organic matter to soil mineral surfaces, mainly clay minerals and iron and aluminium oxides. They are isolated after complete dispersion of the soil. The primary particles, in turn, are held together in larger soil aggregates. These secondary organo-mineral associates are obtained after limited dispersion, and consist of aggregates of smaller primary organo-mineral compounds and particulate organic matter.
The development of modern physical fractionation methods allows to process large amounts of soil samples (Hedges and Oades, 1997). The physical fractionation procedures as described above mostly rely on dierent types of ultrasonic treatments for dispersion. A number of publications have been produced without giving details of the isolation procedure for primary organo-mineral associates. Thus, lack of standardization of isolation procedures and ultrasonic energies used for dispersion is a major concern (Schmidt et al., 1999b; Baldock and Skjemstad, 2000). Schmidt et al. (1999b) found high discrepancies between dierent ultrasonic isolation pro-cedures. The amount of ultrasonic energy input applied to soils has to be calibrated and cannot be compared between dierent laboratories without calibration. Another problem associated with this type of fractionation is redistribution of organic matter between fractions during the isolation procedure. By using low dispersion energies between 450 and 500 J mlÿ1or a sequence of
dispersion energies, it is possible to completely disperse soil samples and avoiding or minimizing formation of artefacts (Amelung and Zech, 1999; Schmidt et al., 1999b).
More work is necessary to standardize and compare the procedures used for the isolation of secondary organo-mineral associates or aggregates. A number of complex fractionation schemes have been developed (e.g. Six et al., 1998), but the information on the com-position of the organic matter in these fractions often remains limited. Such an approach should especially include a comparison of soils developed under dierent pedogenetic environments, with special emphasis on dierent mineral composition.
3.2. Composition of SOM fractions
With the use of methods that can be applied to solid samples, such as analytical pyrolysis, (thermo)chemolysis, and especially 13C NMR spectroscopy, it has become possible to characterize these physical fractions with respect to the chemical composition of their organic matter component. The use of13C NMR spectroscopy is often limited because of the low content of organic carbon in such samples, especially in mineral soils, and the presence of high concentration of paramagnetic com-pounds (Fe, Cu). This can be overcome by selectively removing the mineral fraction and thus concentrating the organic carbon content (Skjemstad et al., 1994, Schmidt et al., 1997).
Based on such fractionation procedures, Golchin et al. (1997a) developed a model linking organic matter decomposition, chemistry and aggregate dynamics in soils. They describe a hierarchy of aggregates in soils where organic matter is an important agent binding soil mineral particles together. The model assumes several fractions of SOM, free particulate OM (POM), POM
occluded in aggregates, and OM associated in organo-mineral associations (microaggregates). As shown by solid-state13C NMR spectroscopy, the microaggregates contain highly altered OM derived from plant residues that is enriched in alkyl and lignin structures. At the same time, microbial residues and products are stabi-lized by adsorption to mineral particles within these microaggregates. A detailed review on the protection mechanisms of organic matter by the soil mineral matrix is given by Baldock and Skjemstad (2000).
The ®ne fractions of soils are more dierent from the bulk soil in their OM composition than the coarse fractions. Clay-size fractions generally show a higher content of alkyl carbon than the whole soils, as revealed by13C NMR spectroscopy (Mahieu et al., 1999). The sand-sized fractions are dominated by high proportions of O-alkyl carbon, followed by alkyl C, indicating the plant fragment origin of this fraction. Much larger dierences between soils are found, if the clay fractions from dierent soils are analyzed instead of the bulk soil material (Fig. 4). The Phaeozem has a chernozemic pedogenesis and is characterized by about similar contributions of alkyl,O -alkyl, aromatic and carboxyl/amide carbon. The Luvisol and Alisol have Lessive dynamics (migration of clay and associated OM) and both show high proportions ofO -alkyl and -alkyl C, but smaller proportions of aromatic C components. In contrast, the OM in the clay fraction of a sandy Podzol is strongly dominated by alkyl C. Obviously, the dierent pedogenetic environments have
a strong eect on the composition of the organic matter in the clay fraction. The presence of plant fragments, that are rather similar in composition, probably mask the eect of dierent pedogenetic environments in the bulk soils.
4. Individual plant or microbial components
4.1. Polysaccharides
Polysaccharides enter the soil from both plant and micro-bial residues. Polysaccharides are commonly analyzed after hydrolysis, which also allows to dierentiate between crystalline (cellulose) and non-cellulosic polysaccharides (hemicelluloses from plants and microbial poly-saccharides). Plant-derived celluloses and hemicelluloses can be almost completely decomposed in soils. The largely non-cellulosic polysaccharides found in soils have a microbial signature, as indicated by analysis of individual carbohydrates after hydrolysis or pyrolysis. A number of dierent studies show that plant polysaccharides are decomposed and microbial polysaccharides accumulate during biodegradation in the forest ¯oor or mineral soils (e.g. Dijkstra et al., 1998; Huang et al., 1998).
Huang et al. (1998) compared the dissolved organic matter (DOM) fraction to the parent organic matter in a grass upland soil by analytical pyrolysis. The DOM had considerably more oxidized lignin and aromatic struc-tures and the polysaccharides showed more diverse polymeric structures, more modi®cation, as indicated from the greater abundance of furan structures in pyr-olysis products, and lower molecular weight than the parent soil material (Table 2).
4.2. Lipids
Free and bound lipids of plant origin are partly pre-served in soils. The total lipid extracts of a soil from Rothamsted under dierent vegetative cover were markedly in¯uenced by the vegetation type (van Bergen et al., 1997). Leaf-derived lipids in a wooded area could be clearly distinguished from grazed and stubbed areas, which were dominated by grass-derived lipids. Nierop
(1998) showed that the organic matter in the B horizons of young Podzols is dominated by aliphatic materials of plant origin. They are mostly derived from root biopolymers (suberin, suberan), as determined by Curie-point pyrolysis in the presence of TMAH. He concludes that the con-tribution of root litter to the formation of organic matter in these Podzol B horizons is considerable. This con®rms earlier work by Riederer et al. (1993), who found high contributions of cutin acids in forest soils after saponi-®cation, with increasing proportions of suberin-derived hydroxy fatty acids in the subsoil of podzolic soils. The work of Lichtfouse et al. (1998) shows that some of the aliphatic material in soil humin is made of straight-chain saturated hydrocarbons, as indicated from 13C NMR spectroscopy and pyrolysis. Additional information from the 13C-isotopic composition of the pyrolysate suggested that the material is a selectively preserved highly aliphatic biopolymer of microbial origin. Augris et al. (1998) described the occurrence of an insoluble, non-hydrolyzable macromolecular component in a forest soil that was considered to originate from higher plant cutans or suberans. In other forest soils, cutan or sub-eran components could not be detected by Curie-point pyrolysis (KoÈgel-Knabner et al., 1992).
Lipids may be trapped in the macromolecular net-work of SOM and thus are not directly extractable with organic solvents. Grasset and AmbleÁs (1998) provide evidence for the release of trapped lipids, mainly fatty acids, fatty acid methyl esters,n-alkanes andn-alkenes from soil humin after enzymatic hydrolysis of cellulose. The free lipids in soils have a plant origin, whereas the trapped lipids released after this enzymatic treatment from humin were found by Grasset and AmbleÁs (1998) to originate from bacterial sources, as concluded from the absence of any odd/even predominance in the n -alkane distribution.
These results on the free and bound lipid component in soils show that a number of dierent types of lipids and aliphatic compounds, of both plant and microbial origin, can contribute to SOM. Future work should try to combine information on the qualitative composition of these compounds with information on the quantita-tive relevance of the individual compounds for stable SOM.
Table 2
Percentages of polysaccharides in soil and DOM samples, calculated by integration of sum of major fragments of polysaccharide pyrolysis products in Py-(NH3)CIMS analyses (from Huang et al., 1998)
Samples Anhydrohexose
(m/z182, 162)
Anhydropentose (m/z150, 132)
Anhydrodeoxyhexose (m/z146, 164)
Furans
(m/z114, 116, 128, 144)
Soil Lf 69.2 15.9 4.7 10.3
Soil Oh 62.7 13.3 7.3 16.9
Mineral soil 55 9.9 8.8 26.4
DOM A 20.8 12.9 16.1 50.2
4.3. Lignin
CuO oxidation is now extensively used to characterize the lignin component of SOM and SOM fractions. Oxi-dation with CuO transforms the lignin network to phe-nol units with aldehyde, carboxylic acid and ketone functionality. Depending on the type of lignin present in dierent tissues of gymnosperms, angiosperms or grasses, monomers with a vanillyl or syringyl substitu-tion pattern are released. After extracsubstitu-tion, puri®casubstitu-tion and addition of internal standards, the amount of the individual monomers can be determined by quantitative gas chromatography (Hedges and Ertel, 1982). Total carbon-normalized yields of the predominant lignin monomers (the so-calledlparameter) have been used to estimate the relative amount of lignin in soils (KoÈgel-Knabner, 1993; Shevchenko and Bailey, 1996). The state of lignin degradation can be recognized by the ratios of the oxidized derivatives (carboxylic acids) versus the corre-sponding aldehyde (Ad/Al) of the vanillyl and syringyl unit (Ertel and Hedges, 1985). This ratio has been shown to systematically increase as the lignin is degraded in soils.
Lignin is decomposed via side-chain oxidation and ring opening (Haider, 1992). A number of publications have completed the picture on changes in the lignin molecule during biodegradation in soils. Dijkstra et al. (1998) reported that the oxidative degradation of Scots pine needle litter in the forest ¯oor results in a short-ening of the guaiacyl lignin side-chains and an increase of carbonyl and carboxyl groups. The lignin pyrolysis products observed from the forest ¯oor materials were similar to those obtained from controlled laboratory degradation experiments with model compounds or wood lignin. Chemical modi®cations of the lignin bio-polymer composition during degradation in soils have been observed also by Kuder and Kruge (1998) with Py-GC/MS. Major changes observed in a peat bog pro®le include loss of ester-bond ferulic and coumaric acids, increased oxidation at theCa, shortening of alkyl side-chains, and demethylation.
The lignin signature of forest soils can be dier-entiated from the lignin in agricultural soils (Fig. 5). In the forest soils, relative contributions of lignin-derived CuO oxidation products increase with increasing soil carbon contents. In agricultural soils, contents of lignin-derived compounds are higher and are not related to organic matter content. This re¯ects the complex OM input pattern to agricultural soils and the presence of lignin in dierent degree of biodegradation in A horizons of ploughed soils. The progressive decomposition of lignin in the forest ¯oor to the mineral soil leads to a distinct relationship between organic matter content and lignin yield. At the same time, the forest soil A horizons have higher acid-aldehyde ratios, i.e. a higher degree of lignin alteration, as compared to A horizons of agri-cultural soils (Schmidt, 1998).
The lignin signatures obtained from CuO oxidation are now also used to dierentiate between organic matter derived from dierent vegetation types. Hetherington and Anderson (1998) identi®ed organic layers derived from bracken (Pteridium aquilinium) from below-ground layers that were derived from the original heather (Calluna vulgaris) in British moorland soils. Sanger et al. (1997) used CuO oxidation to investigate the lignin signature of soils under beech and spruce woodland, pasture and arable cropping. They consider this technique useful for monitoring the eects of vegetation and land manage-ment changes on soil C cycling, but at the same time indicate that a more extensive data base for soils with known land-use history is necessary to calibrate the results and establish a time-scale for the translocation and biodegradation of lignin in dierent soils.
The relevance of a number of other plant and micro-bial precursors for the formation of SOM is still not clear. Little is known about the amount and fate of tannins in plant litter and soils. Preston et al. (1997) used the proanthocyanidin assay for condensed tannins, a colorimetric procedure, in combination with 13C NMR spectroscopy to investigate the fate of tannins in forest soils. Tannins are probably attacked rapidly in soils, as indicated from the colorimetric analyses (Lor-enz et al., 2000). However, they may have undergone only slight changes during microbial attack and thus escape from the analytical window, thus slight changes in the structural composition of the tannins during bio-degradation may make them unavailable for the colori-metric analysis. Other techniques should be used additionally to follow the fate of these compounds in soils. A wide range of soil fungi produce melanins, that may be decomposed by lignin peroxidases (Butler and Day, 1998). Knicker et al. (1995) investigated the com-position of fungal melanins by13C and15N NMR spectro-scopy in comparison to SOM from representative soils and to composts. They found large dierences within the structural composition of the melanins, but also in comparison to the soils and composts. Gomes et al. (1996) investigated the melanins from dierent actino-mycetes from Brazilian soils in comparison to humic acids from these soils. As indicated by IR spectroscopy the melanins had a higher aliphaticity. There was also evidence for high contents of proteinaceous materials and varying amounts of polysaccharides. No more recent data are available on the composition, biodegradation pathway and quantitative importance of fungal melanins in soils.
4.4. Microbial biomarkers
as markers after hydrolysis and gas chromatographic determination (Knabner et al., 1990; KoÈgel-Knabner, 1995). Chantigny et al. (1997) used glucosamine and muramic acid to dierentiate between bacterial and fungal contributions to soil aggregation and concluded that fungi play a predominant role in soil macro-aggregate formation. Zhang et al. (1998) found that the major part of the hexosamines (glucosamine, galactosa-mine, mannosamine) and muramic acid is attached to the clay fraction in native prairie soils of North America. The association of these markers in the cell wall remains may aect their degradative behavior, as the dynamics of the amino sugars were dierent to that of muramic acid. Guggenberger et al. (1999) examined the eect of conventional and no-tillage management on the contents of bacterial and fungal cell wall residues in soils also by measuring amino sugar contents and muramic acid. Soils under no-tillage had higher contents of fungal residues, consistent with a higher aggregate stability which in turn was associated with a higher SOM storage in particulate organic matter (Table 3).
5. Organic nitrogen in soils
Plant proteins undergo rapid biodegradation when entering the soil, and at the same time ecient recycling through the microbial biomass. Solid-state 15N NMR spectroscopy provides insight in the nature of refractory nitrogen in soils or soil fractions (Knicker and KoÈgel-Knabner, 1998). The solid-state15N NMR spectroscopic
investigation of the medium silt, ®ne silt and clay fraction of a Haplic Podzol revealed that the major part of the organic nitrogen is bound in amide-N functional groups, most probably as part of proteinaceous material (Knicker et al., 1999). This con®rms previous observations on dierent bulk soils and composts (Knicker et al., 1997). Hydrolysis with 6 N HCl could only release less than 43% of this total-N. Therefore at least some of the organic nitrogen in these samples, identi®ed as amide-N, must be present in a form protected from microbial
Table 3
Correlation coecients between soil properties of four soil pairs (conventional tillage±no tillage) and parameters for microbial cell wall residues (from G. Guggenberger, unpu-blished)a
Soil property Glucosamine mg kgÿ1 gluN/murb
pH NS NS
clay content (g kgÿ1) NS NS
water content (g gÿ1) 0.59* 0.68**
fungal biomass (mg gÿ1) 0.52* 0.76**
POMc-C (g kgÿ1) 0.80*** 0.73***
MWD (mm)d 0.64** 0.77***
a n=24.
b Ratio glucosamine/muramic acid.
c Particulate organic matter.
d Mean weight diameter of water stable aggregates,
log-transformed.
*, **, *** statistically signi®cant at theP<0.05, 0.01, and 0.001 probability levels, respectively.
degradation and resistant to drastic chemical treatment. This resistance may explain to some degree the diculties in identifying such structures with common wet chemical degradative methods. Derenne et al. (1993) observed the presence of amide-N resistant to drastic hydrolysis con-ditions and resistant to microbial degradation in the algaenan of green microalgae. Such compounds could be selectively preserved in soils. However, algae are not a major group of soil microorganisms and the occurrence of these components in other soil microorganisms has to be investigated further.
Thermochemolysis with TMAH in sealed ampoules was recently performed to investigate the nature of refractory organic nitrogen in the hydrolysis residues of sediments (Knicker and Hatcher, 1997), a soil (Knicker and KoÈgel-Knabner, 1998) and soils after addition of biowaste composts (Siebert et al., 1998). Several products characteristic for protein TMAH degradation products (Knicker and Hatcher, 1997) were detected. N-Containing aromatic compounds could not be identi®ed in major proportions. From these studies, it was assumed that in soils some peptides can resist harsh acid hydrolysis. The recognizable nitrogen-containing products from ¯ash pyrolysis-GC MS of a Rothamsted soil were identi®ed as derived from amino acids or amino sugars in the litter and forest ¯oor horizons (van Bergen et al., 1998). In contrast, the pyrolysate from the mineral soil at Rothamsted was dominated by products not related to known biomolecules. More data are needed on the molecular composition of the unknown soil nitrogen to resolve these discrepancies. Major dierences are often observed between litter materials or humic horizons and the mineral soil. Further studies should therefore also include the eect of the mineral matrix on chemolysis or pyrolysis products.
Cheshire et al. (1999) used15N NMR spectroscopy to follow the incorporation of labeled15N fertilizer during the decomposition of wheat straw. They found that the organic N after incubation was mainly present in fungal tissue, and only to a small extent in bacterial tissue. Together with data on microbial biomass and microbial biomarkers (ergosterol, glucosamine) this led to the conclusion that fungi are predominantly involved in the immobilization of N during straw decomposition in soils.
6. Charcoal, anthropogenic C components
Burning vegetation produces partly charred plant material which subsequently could contribute to the highly refractory proportion of soil organic matter (Schmidt and Noack, 2000). The content of charred organic carbon in soils can be determined by the tech-nique of high energy ultra violet photo-oxidation in combination with13C nuclear magnetic resonance spectro-scopy (Skjemstad et al., 1996). A number of materials
found in soils, including wood, lignin, and humic acids, can all be destroyed by the high energy photo-oxidation process, provided they are exposed to ultraviolet radia-tion in the presence of excess oxygen. Thus it is possible to dierentiate between natural soil organic matter and charred organic carbon, which remains in the residue from UV photo-oxidation.
Schmidt et al. (1999a) investigated soils from Germany and The Netherlands with high energy ultraviolet photo-oxidation, scanning electron microscopy, solid-state13C NMR, and lignin analysis by CuO oxidation for the presence of charred organic matter. Charred organic carbon could not be detected in the A horizons of an Alisol and a Gleysol, but it contributed up to 45% of the organic carbon and up to about 8 g kgÿ1of the
soil in a range of soils with chernozemic soil properties (dark color, A±C pro®le, high base saturation, bio-turbation). A strong relation between color and the content of charred organic carbon was observed. This suggests that ®nely divided charred organic matter may be a major constituent of many chernozemic soils in Germany.
Glaser et al. (1998) use the benzenecarboxylic acids obtained from digestion with HCl followed by HNO3as speci®c markers for the presence of black carbon in soils. They found that nitric acid oxidation was better suited to completely digest black carbon, compared to alkaline permanganate and acid dichromate, because it degrades the highly aromatic core of charred organic components in soils. At the same time, the nitric acid digestion yields only aromatic degradation products with mellitic acid as major product, so that GC±FID is sucient for the identi®cation and quanti®cation of the major degradation products.
Solid-state 13C and 15N NMR spectroscopy was applied for characterizing the chemical nature of the organic fraction remaining from high energy UV-photo-oxidation of several Australian soils, that removes most of the organic material that is not protected physically by association within aggregates (Knicker and Skjem-stad, 2000).13C NMR spectroscopic comparison of the residues after photo-oxidation and the untreated bulk soils, revealed a considerable increase in aromaticity in the residues for four of the ®ve soils. This suggested the presence of charred material, derived from former vegetation ®res. Pyrrolic-N was found to comprise a major fraction of the residues after photo-oxidation of the <53mm fractions of the char-containing soils with 15N NMR spectroscopy (Fig. 6). The authors concluded
that char largely comprises the inert or passive organic matter pool of many Australian soils. The nitrogen immobilized in heterocyclic aromatic forms as charred organic matter is supposed to be resistant against microbial attack.
input of fossil fuel combustion products and/or old vegetation ®res. They could thus show that the PAHs did not originate from recent synthesis by plants or the soil microbial biomass. This conclusion was achieved by a combination of IR-MS for the determination of speci®c
13C/12C ratios and AMS to determine the 14C age of
individual PAHs.
Major contributions to the organic matter in a Podzol investigated by Schmidt et al. (2000a) derive from industrial processes (char, coke, bituminous coal and metallic particles) and are highly aromatic in nature. Disperse atmospheric input of organic particles from coal industries may be common in soils of industrialized regions, altering both content and structure of organic matter in these soils. As a consequence, the organic matter in the surface horizons of these soils is a mixture of natural humic substances and organic particles from coal-processing industries. Presumably, anthropogenic organic particles accumulate also in other soils neigh-bouring industrialized areas, but remain undetected due to low levels of deposition.
7. Research perspectives
For the analysis of SOM structure a number of tech-niques have been developed. Very often a combination of techniques has been especially useful to elucidate SOM characteristics. Modern methods developed for the fractionation of soils and sediments in combination with chemolytic and spectroscopic techniques seem to be a promising approach for future work.13C and15N NMR spectroscopy can give constraints on the results from chemolytic or pyrolytic analyses. Although the presence in soils for dierent organic compounds has been proven, we do only partly know in what quantities they are present. Soil organic matter characterization could further bene®t from the application of non-destructive microscale techniques and microscopic techniques, that allow to have information on the chemical composition and physical allocation of organic and mineral materials in soils, such as X-ray spectroscopy (Thieme and Niemeyer, 1998; Yuan et al., 1998).
Future work should consider the composition and formation of organic matter in dierent soil types. Up to now, only a limited data set is available, which is limited mainly to the surface or A horizon of agri-cultural soils and to litter degradation in forest soils. Thus, the variability of SOM with respect to soil type and pedogenesis is not yet included in our data base. A broader data base has also to consider the site history and the climatic history at a speci®c site, which may have pronounced in¯uence on SOM sequestration (Schmidt et al., 1999a, 2000a; Amelung et al., 1999).
Organic matter in the subsoil has not been investi-gated in detail. The concentration of OM in the subsoil (B and C horizons) is low, although it has become clear that OM in these strata may represent a signi®cant pro-portion of the total amount of carbon sequestered in soils. Most probably, the organic matter in the subsoil is highly recalcitrant to biodegradation. To elucidate the
stabilization mechanisms in the sub-soil requires re®ned analytical techniques. It means analysis of OM in a matrix where quartz sand, clay minerals and iron oxides predominate and the concentrations of OC are lower than 0.05±0.1% OC. For such samples, the eect of mineral components on analytical performance may be crucial and has to be elucidated in detail. This also applies to the investigation of organic matter in organo-mineral associates in many soils.
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