Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue7-8.2000:

Organic Geochemistry 31 (2000) 609±625
www.elsevier.nl/locate/orggeochem

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 degradation or pyrolysis and subsequent analysis of the fragments by GC MS. Alternative techniques for the examination 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 di€erentiate between distinct SOM pools that can be further 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 structural characterization of soil organic matter (SOM) or
SOM components. From these results it has become
clear that C cycling and stabilization in soils is intimately 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 techniques 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.

* Fax: +49-8161-714466.
E-mail address: koegel@weihenstephan.de

Examples are given for tracing the fate of individual
plant or microbial components in soils, and for analyzing the structural composition of organic nitrogen-containing compounds in soils. The presence of charred
organic matter from burning events or atmospheric

organic contamination may drastically a€ect 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 presented.

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

0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00042-5

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I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

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 (rearrangement, 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-Knabner, 1995). Hydrolysis is used to obtain hydrolyzable
amino acids from proteins. Carbohydrates are released
from polysaccharides after di€erent 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 components 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 hydrolyzable, 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 of O-alkyl C in the
CPMAS 13C 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 the O-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 promise 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 predominantly 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, pyrolysis 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 e€ect 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 macromolecular structures and the diculties 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
To overcome some of the problems envisaged in pyrolysis, an alternative technique has been introduced
which is based on simultaneous pyrolysis and methylation of the produced monomers with gaseous tetramethylammonium hydroxide (TMAH) (Challinor, 1995;
Cli€ord et al., 1995; MartõÂn et al., 1995; Saiz-Jimenez,
1996; del Rio and Hatcher, 1998). This procedure yields
methyl esters of carboxylic acids and methyl ethers of
hydroxyl groups, rendering many of the polar products
volatile for gas chromatographic analysis. The technique
of pyrolysis/methylation has already been successfully
applied for the characterization of lignins, cutins and
cutans, and proteins (McKinney et al., 1996; del Rio
et al., 1998).

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

611

Fig. 1. TICs of the pyrolysates of the solvent insoluble residues of three horizons from a Rothamsted soil. P, phenol; 3+4P, co-eluting
3- and 4-methylphenol; C16FA, hexadecanoic acid; 1-Pr:1, Prist-1-ene; HE, hemicellulose marker (4-hydroxy-5,6-dihydro-(2H)-pyran2-one); LG, Levoglucosane; C16Ni, hexadecanenitrile; x n-alk-1-enes; *= n-alkanes; * contaminants. Numbers in bold refer to
compounds in Table 1. Side chains (attached at positions 4) of phenol-(P), 2-methoxyphenol-(guaiacyl; G) and 2,6-dimethoxyphenol(syringyl-; S) components are indicated (from van Bergen et al., 1998).

612

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

Table 1
Nitrogen-containing pyrolysis products detected in the pyrolysates of the three di€erent soil horizons (from van Bergen et al., 1998)
Pyrolysis products

1
2
3
4
5
6

7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26

27
28
29
30

Pyrrole
N-methylpyrrole
2-Methylpyrrole
3-Methylpyrrole
C2-pyrroles
Pyridine
C1-pyridines
Indole
3-Methylindole
C1-Indole
Quinoline?
Isoquinoline?
Benzenamine
Benzonitrile
C1-Benzonitriles

Benzenacetonitrile
Benzenepropanenitrile
Acetamide
Acetylpyrrolidone
3-Acetamido-5-methylfuran
3-Acetamido-4-pyrone
or 3-Acetamido-2-pyrone
Oxazoline structures
Diketodipyrrole
2,5-Diketopiperazines der.
2,5-Diketopiperazines der
2,5-Diketopiperazine
Tetradecanenitrile
Hexadecanenitrile
Octadecanenitrile
Eicosanenitrile

Origina

Mw

Leaf litterb

Humic layerb

Mineral soilb

1

2

3

1

2

3

1

2

3

Pro, Hyp, Glu
AA/unknown
Pro, Hyp
Pro, Hyp
Hyp
AS/Ala
AS/Ala
Trp
Trp
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Phe
Phe
AS
AS
AS
AS

67
81
81
81
95
79
93
117
131
131
129
129
93
103
117
117
131
59
127
139
153

+
+
ÿ
ÿ
ÿ
+
ÿ
+
+
ÿ
ÿ
ÿ
ÿ
+
ÿ
+
+
+
ÿ
ÿ
ÿ

+
+
+
+
ÿ
+
ÿ
+
+
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
+
ÿ
+
ÿ
ÿ
ÿ

+
+
ÿ
ÿ
ÿ
+
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ

++
+
+
+
ÿ
+
++
+
+
ÿ
ÿ
ÿ
ÿ
+
ÿ
+
+
+
ÿ
+
ÿ

++
+
+
+
+
+
+
++
+
ÿ
+
+
ÿ
+
ÿ
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
ÿ
+
+
+
+
ÿ
ÿ
ÿ
ÿ

+++
+
+
+
+
++
+
++
+
+
+
+
?
++
+
+
+
+
ÿ
ÿ
ÿ

+++
+
+
+
+
++
+
++
+
+
+
+
ÿ
++
+
+
+
+
ÿ
ÿ
ÿ

++
+++
+
+
+
+
+
+
ÿ
ÿ
ÿ
ÿ
ÿ
++
+
+
ÿ
ÿ
ÿ
ÿ
ÿ

AS
Hyp-Hyp
Pro-Val, Pro-Arg
Pro-Ala
Pro-Pro
Unknown
Unknown
Unknown
Unknown

185
186
154
168
194
209
237
265
293

ÿ
+
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ

?
+
+
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ

ÿ
+
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ

+
++
+
+
+
ÿ
ÿ
ÿ
ÿ

+
++
+
+
+
ÿ
ÿ
ÿ
ÿ

ÿ
+
+
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ

ÿ
++
ÿ
ÿ
+
+
+
+
+

ÿ
++
+
ÿ
+
+
+
ÿ
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
ÿNot detected, + present, ++ abundant, +++ very abundant.

Sub-pyrolysis temperatures of 300 C in the presence
of TMAH was found to produce a suite of products
similar to that observed at higher pyrolysis temperatures
(Hatcher and Cli€ord, 1994; Cli€ord 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 oxidation 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 sucient 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 using 13C-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 sedimentary 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

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

transferred to a C4 plant (maize) with pyrolysis-gas
chromatography and isotope ratio mass spectrometry (PyGC/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 movement and selective sorption of dissolved organic matter
and bioturbation e€ects 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 and 15N NMR spectroscopy
13
C and 15N 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 of 13C and 15N 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
13
C NMR spectra are generally recorded as free induction

613

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 signals 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 horizons. This was attributed to the fact that spectra from
solid samples low in organic carbon are more dicult to
obtain because of sensitivity problems (Mahieu et al.,
1999). The review of Mahieu et al. (1999) shows that 13C
NMR spectra from bulk soils are remarkably similar,
dominated by signals from O-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 combination with 13C- and 15N-labeled parent materials
added to the soil, the evolution of these C and N labels
can be followed in di€erent C and N pools. Dipolar
dephasing or interrupted decoupling is a pulse sequence
that can be used to di€erentiate carbons at the same
chemical shift but with di€erences of molecular motions
or di€erences in substitution level (quaternary vs. nonquaternary 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 a€ected by
strong proton dipolar coupling are preferentially lost.
The loss of 13C signal intensity with increasing Tdd is
more rapid for protonated carbons that have strong
dipolar interactions with hydrogens. Carbons without
directly attached hydrogens and protonated carbons
with high molecular motion, e.g. methoxyl and methyl
groups, experience weaker dipolar interactions with
hydrogens and thus show a slow decay of signal intensity. The rate of loss of signal intensity can be described
by exponential decay functions, from which the relative
contribution of each of these fractions can be calculated.
Thus the fraction experiencing strong dipolar interactions can be di€erentiated from the fraction with weak
dipolar interactions. Webster et al. (1997) have used this

614

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

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
di€erent delay times. The subspectra thus generated
correspond to signals from domains with di€erent proton
spin-lattice relaxation time constants. By doing so it is
possible to ``fractionate'' OM in di€erent structural
components. These di€erences in proton spin relaxation
time constants have been applied to edit CPMAS NMR
spectra of di€erent types of soil organic matter, by generating subspectra associated with the fast and slow
relaxing pools of protons. The PSRE technique was
used recently to di€erentiate between di€erent SOM
pools in soils. Golchin et al. (1997b) were able to di€erentiate between partly decomposed plant residues and
charcoal. Clinton et al. (1996) identi®ed di€erent SOM
pools in forest litter using PSRE. Fig. 2 shows the differentiation between the slow and fast relaxing subcomponents 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 dominated 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 condensed aromatic structures in soils, such as charred
organic matter, bituminous coal residues or soot
(KoÈgel-Knabner and Knicker, 2000). The CPMAS 13C
NMR spectrum of the Oh horizon of a mine soil developed from forest-remediated Tertiary sandy overburden
material a€ected by lignite dust from nearby briquetteproducing 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
of O-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 CPMAS 13C NMR spectrum
of the same soil material.

Fig. 2. Proton spin relaxation edited subspectra A±C from the
13
C NMR spectrum of a grassland (top) and a forest soil (bottom) (from Condron and Newman, 1998). Top: grassland soil;
subspectrum A 24% (of total signal intensity), T1p(H) 6.3 ms,
T1(H) 26 ms; subspectrum B 65% (of total signal intensity), T1p
(H) 3.3 ms, T1(H) 16 ms; subspectrum C 11% (of total signal
intensity), T1(H) 2.8 ms, T1(H) 28 ms. Bottom: forest soil; subspectrum A 17% (of total signal intensity), T1p (H) 6.4 ms,
T1(H) 31 ms; sub-spectrum B 57% (of total signal intensity),
T1p (H) 3.2 ms, T1(H) 13 ms; subspectrum C 26% (of total
signal intensity), T1p(H) 2.8 ms, T1(H) 22 ms.

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

615

Fig. 3. 13C CPMAS NMR spectra of the forest ¯oor and Ai horizon of a mine soil pro®le, reference lignite-derived material, and
Bloch decay spectrum of the Ai horizon. (from KoÈgel-Knabner and Knicker, 2000). Horizon designations are according to the German soil classi®cation system (AG Boden, 1994).

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 di€erent 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 settings 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 transformation products (= humic substances?) form the
organic matter in soils. Thus, in contrast to sediments,

each soil horizon represents a mixture of these materials
in di€erent stages of degradation. Therefore, analysis of
SOM has bene®ted tremendously from physical fractionation according to size and/or density (Baldock and
Skjemstad, 2000). These methods are designed to fractionate SOM into pools of di€erent turnover times as
they di€erentiate 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 di€erent 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 di€erent
types of substructures, that have been designated
primary (organo-mineral associates) and secondary
organo-mineral compounds, i.e. soil aggregates (Christensen, 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.

616

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

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 di€erent
types of ultrasonic treatments for dispersion. A number
of publications have been produced without giving
details of the isolation procedure for primary organomineral 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 di€erent ultrasonic isolation procedures. The amount of ultrasonic energy input applied
to soils has to be calibrated and cannot be compared
between di€erent 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 mlÿ1 or 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 composition of the organic matter in these fractions often
remains limited. Such an approach should especially
include a comparison of soils developed under di€erent
pedogenetic environments, with special emphasis on
di€erent mineral composition.

occluded in aggregates, and OM associated in organomineral associations (microaggregates). As shown by
solid-state 13C 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 stabilized 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 di€erent 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 by 13C 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 di€erences between soils
are found, if the clay fractions from di€erent 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, Oalkyl, aromatic and carboxyl/amide carbon. The Luvisol
and Alisol have Lessive dynamics (migration of clay and
associated OM) and both show high proportions of Oalkyl 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 di€erent pedogenetic environments have

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 of 13C 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 compounds (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

Fig. 4. Solid-state 13C NMR spectra of the clay fraction isolated from soils of di€ering pedogenesis.

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I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

a strong e€ect 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 e€ect of di€erent pedogenetic environments in the
bulk soils.

4. Individual plant or microbial components
4.1. Polysaccharides
Polysaccharides enter the soil from both plant and microbial residues. Polysaccharides are commonly analyzed after
hydrolysis, which also allows to di€erentiate between
crystalline (cellulose) and non-cellulosic polysaccharides
(hemicelluloses from plants and microbial polysaccharides). 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 di€erent 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 structures and the polysaccharides showed more diverse
polymeric structures, more modi®cation, as indicated
from the greater abundance of furan structures in pyrolysis products, and lower molecular weight than the
parent soil material (Table 2).
4.2. Lipids
Free and bound lipids of plant origin are partly preserved in soils. The total lipid extracts of a soil from
Rothamsted under di€erent 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 contribution 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 suberan components could not be detected by Curie-point
pyrolysis (KoÈgel-Knabner et al., 1992).
Lipids may be trapped in the macromolecular network 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 and n-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 nalkane distribution.
These results on the free and bound lipid component
in soils show that a number of di€erent 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 quantitative 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/z 182, 162)

Anhydropentose
(m/z 150, 132)

Anhydrodeoxyhexose
(m/z 146, 164)

Furans
(m/z 114, 116, 128, 144)

Soil Lf
Soil Oh
Mineral soil
DOM A
DOM B

69.2
62.7
55
20.8
17.0

15.9
13.3
9.9
12.9
15.3

4.7
7.3
8.8
16.1
13.9

10.3
16.9
26.4
50.2
53.8

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I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

4.3. Lignin
CuO oxidation is now extensively used to characterize
the lignin component of SOM and SOM fractions. Oxidation with CuO transforms the lignin network to phenol units with aldehyde, carboxylic acid and ketone
functionality. Depending on the type of lignin present in
di€erent tissues of gymnosperms, angiosperms or
grasses, monomers with a vanillyl or syringyl substitution pattern are released. After extraction, puri®cation
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-called l parameter) have been used to
estimate the relative amount of lignin in soils (KoÈgelKnabner, 1993; Shevchenko and Bailey, 1996). The state of
lignin degradation can be recognized by the ratios of the
oxidized derivatives (carboxylic acids) versus the corresponding 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 shortening 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 biopolymer composition during degradation in soils have
been observed also by Kuder and Kruge (1998) with PyGC/MS. Major changes observed in a peat bog pro®le
include loss of ester-bond ferulic and coumaric acids,
increased oxidation at the Ca, shortening of alkyl sidechains, and demethylation.
The lignin signature of forest soils can be di€erentiated 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 ligninderived 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 di€erent 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 agricultural soils (Schmidt, 1998).

The lignin signatures obtained from CuO oxidation
are now also used to di€erentiate between organic matter
derived from di€erent 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 e€ects of vegetation and land management 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 di€erent soils.
The relevance of a number of other plant and microbial 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 (Lorenz 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 biodegradation may make them unavailable for the colorimetric 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 composition of fungal melanins by 13C and 15N NMR spectroscopy in comparison to SOM from representative soils
and to composts. They found large di€erences within
the structural composition of the melanins, but also in
comparison to the soils and composts. Gomes et al.
(1996) investigated the melanins from di€erent actinomycetes 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
Di€erent approaches have been used to quantify biomarkers for microbial residues in soils. Fungal and
bacterial hexosamines (glucosamine, galactosamine,
mannosamine) and bacterial muramic acid can be used

619

I. KoÈgel-Knabner / Organic Geochemistry 31 (2000) 609±625

Fig. 5. Yield of CuO lignin oxidation products from the A horizons of agricultural and forest soils (from Schmidt, 1998).

as markers after hydrolysis and gas chromatographic
determination (KoÈgel-Knabner et al., 1990; KoÈgelKnabner, 1995). Chantigny et al. (1997) used glucosamine
and muramic acid to di€erentiate between bacterial and
fungal contributions to soil aggregation and concluded
that fungi play a predominant role in soil macroaggregate formation. Zhang et al. (1998) found that the
major part of the hexosamines (glucosamine, galactosamine, 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 a€ect their degradative behavior, as the dynamics
of the amino sugars were di€erent to that of muramic
acid. Guggenberger et al. (1999) examined the e€ect 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

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 di€erent 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 coecients between soil properties of four soil
pairs (conventional tillage±no tillage) and parameters for
microbial cell wall residues (from G. Guggenberger, unpublished)a
Soil property

Glucosamine mg kgÿ1

gluN/murb

pH
clay content (g kgÿ1)
water content (g gÿ1)
fungal biomass (mg gÿ1)
POMc-C (g kgÿ1)
MWD (mm)d

NS
NS
0.59*
0.52*
0.80***
0.64**

NS
NS
0.68**
0.76**
0.73***
0.77***

a

Plant proteins undergo rapid biodegradation when
entering the soil, and at the same time ecient 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 KoÈgelKnabner, 1998). The solid-state 15N NMR spectroscopic

n=24.
Ratio glucosamine/muramic acid.
c
Particulate organic matter.
d
Mean weight diameter of water stable aggregates, logtransformed.
*, **, *** statistically signi®cant at the P