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

Organic Geochemistry 31 (2000) 655±668
www.elsevier.nl/locate/orggeochem

Refractory organic carbon in C-depleted arable soils, as
studied by 13C NMR spectroscopy and carbohydrate analysis
Rita Kiem a,*, Heike Knicker a, Martin KoÈrschens b, Ingrid KoÈgel-Knabner a
a
Chair of Soil Science, Technische UniversitaÈt MuÈnchen, 85350 Freising-Weihenstephan, Germany
Department of Soil Research, UFZ-Umweltforschungszentrum Leipzig-Halle GmbH, 062460 Bad LauchstaÈdt, Germany

b

Abstract
Soil organic matter (SOM) comprises refractory compounds, to which a turnover time of more than 1000 years has
been attributed in SOM models. The goal of this study is to characterize the chemical structure of refractory compounds of organic carbon in arable soils by means of 13C NMR spectroscopy and analysis of carbohydrates. C-depleted soils that are expected to be enriched in refractory compounds are compared with fertilized soils from long-term
agroecosystem experiments. In the C-depleted soils, lower proportions of O/N-alkyl C and higher proportions of aromatic and carboxyl C compared with the fertilized counterparts are observed. Ratios of alkyl to O-alkyl C are higher in
the depleted soils than in the fertilized ones. Along with the overall C-depletion, the absolute amount of all carbon
species was reduced. This net decrease is highest for the O/N-alkyl C and smallest for the aromatic C. Yields of wet
chemically determined carbohydrates positively correlate with the relative intensities of O-alkyl C in the NMR spectra,
and con®rm the net decrease of O-alkyl C compounds along with C-depletion. The refractory organic carbon pool in
arable soils appears to have a lower contribution of O/N-alkyl C, and a higher proportion of recalcitrant aromatic

structures compared with more labile fractions of organic carbon. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Soil organic carbon; Refractory/passive pool; Long-term agroecosystem experiments;
Carbohydrates; Hydrolysis residue

1. Introduction
SOM is a heterogeneous mixture of compounds, which
represent a continuum from fresh plant residues to
strongly humi®ed material (KoÈgel-Knabner, 1993). The
di€erent components in this continuum di€er with
respect to their turnover time in soil. On the basis of these
distinct turnover times, in SOM-models, the total SOM
pool is divided into conceptual fractions (Van Veen and
Paul, 1981; Parton et al., 1987; Cambardella, 1998).
Essentially, SOM in these models has been considered to
consist of a compartment of active components with a
residence time (or turnover time) of years to decades, and
a passive (or refractory) fraction remaining in soil for
hundreds to thousands of years. In some model approaches, ``active'' components are further distinguished into

* Corresponding author: Tel. +49-8161-713147; fax +498161-714466.

E-mail address: kiem@weihenstephan.de (R. Kiem).

13

C CPMAS NMR spectroscopy;

a labile fraction of plant litter and microbial biomass,
and an intermediate fraction of particulate organic matter
which is stabilized for a few decades. Factors that determine stability of organic carbon in soil have been summarized as chemical recalcitrance of organic molecules
against microbial attack, interactions between organic and
mineral compounds, and accessibility of organics to
microbes and enzymes (Sollins et al., 1996). Among the
chemically most resistant compounds are aromatic and
paranic structures (Oades, 1995). With regard to interactions and accessibility, the stabilization of organic carbon in soils was suggested to be in¯uenced by the clay
content, and those factors that control the aggregation
status (Oades, 1995).
Long-term agroecosystem studies provide a means to
investigate the e€ect of management practices on C
sequestration in soils. In this study we investigated the
chemical structure of at least two contrasting treatments

from long-term agroecosystem experiments, comparing
the structure of soil organic carbon (SOC) of fertilized
plots with that of treatments depleted in organic carbon,

0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00047-4

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R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

i.e. unmanured plots and bare fallows (Fig. 1). In agroecosystem experiments usually each treatment is established on several replicate plots, which are randomly
arranged within the experimental ®eld. Due to this
design, environmental factors such as changes in geological substrate, soil texture, or atmospheric input of e.g.
mineral matter should randomly a€ect the various
treatments. Mean di€erences in SOM observable
between the treatments should, therefore, be attributable
to the type of soil management.
Fertilized plots receive organic and mineral fertilizers
in order to guarantee high crop yields. Thus, high

amounts of organic materials enter the soil, as crop
residues and farmyard manure. In the plots where no
inorganic or organic nutrients are added, crop production is reduced compared with the fertilized plots. Consequently, in the unmanured plots the total input of
organic matter into the soil is lower than in the fertilized
counterparts. Bare fallows are kept free of any vegetation cover. Thus, these soils do not receive any (or a
negligible quantity of) organic matter input. In the fallows investigated in this study, weed plants are removed
manually avoiding the use of herbicides. The pronounced di€erences in organic input Ð at the long
term Ð result in a di€erentiation in the total SOC level of
the plots (Fig. 1). Furthermore, the labile and intermediate fractions of SOM are expected to be strongly
a€ected by the type of soil management and the amounts
of organic input, respectively, whereas the passive/
refractory pool should remain una€ected at a time scale
of decades (Elliott et al., 1996). Compounds with a
residence time of years to decades would be depleted in
the plots with low/missing organic input, and as a consequence the passive pool should make up a higher
proportion of total SOC compared with the fertilized
treatments (Fig. 1). The relative accumulation of

refractory compounds in the C-depleted soils is the basic
assumption of our experimental approach.

The objective of this study is to assess the chemical
composition of refractory organic carbon in arable soils,
by comparing C-depleted treatments with fertilized soils,
which di€er in the relative proportion of refractory compounds. This includes the study of the gross chemical
composition by means of 13C nuclear magnetic resonance
(NMR) spectroscopy, and the analysis of carbohydrates as
a speci®c compound class, which is quantitatively of great
importance for SOM (Lowe, 1978).

2. Materials and methods
2.1. Study sites and soil sampling
We have selected soils from eight European long-term
agroecosystem experiments (Table 1). The study sites
are located in di€erent regions of Central and Eastern
Europe, covering a range of climatic features, of geological substrates and of soil types. At each experimental
site, samples were obtained from di€erent treatments:
plots with the combined addition of mineral and organic
fertilization, and unmanured plots with the same crop
rotation as in the fertilized treatments but without fertilization. In the experiments of Bad LauchstaÈdt and
Prague bare fallows were established. The two contrasting treatments are denoted as fertilized plots and Cdepleted plots (unmanured soils and bare fallows) (see

Fig. 1).
Except for the bare fallows in Prague and Bad
LauchstaÈdt, all types of treatments are replicated at least
three-fold on separate plots arranged in a randomized
way within the experimental ®elds (3±5 replications
depending on the experiment). The bare fallow plots of
Prague and Experiment A (Bad LauchstaÈdt) are 2±3 m2
in size and have no replications. The bare fallow treatment
of Experiment B (Bad LauchstaÈdt) is replicated twice.
Soil samples were collected from a depth 0±20 cm.
Soil material collected at 10 sampling points on each of
the replication plots of a certain treatment was mixed.
Subsequently, samples were air-dried and components
>2 mm were removed by dry sieving. For elemental
analysis and hydrolysis of carbohydrates, aliquots of the
samples were ground using a ball mill.
2.2. Elemental analysis and pH

Fig. 1. Gradients in organic input, organic carbon levels and
SOM composition with respect to plot treatment in the longterm agroecosystem experiments.

C and N contents were determined by dry combustion
using a Elementar Vario EL Analyzer. Inorganic and
organic carbon was di€erentiated by determining the
amount of carbon before (total carbon) and after ignition of the samples at 550 C for 3 h (inorganic carbon).
Inorganic carbon was not detected in any sample; thus,
the total carbon amount is referred to as organic carbon.

Table 1
Site characteristics of the long-term agroecosystem experiments and treatments considered in the present study
Start of
experiment

Soil classi®cation
(FAO)

Clay
(%)

Mean

annual
temperature
( C)

Mean
annual
precipitation
(mm)

Treatments

Thyrow
(Germany)

1937

Albic Luvisol

2.7

8.6

520

Fertilized

Groû Kreutz
(Germany)

1967

Albic Luvisol

4.0

8.9

537

Unmanured

Fertilized

Skierniewice
(Poland)

1923

Luvisol

6.0

7.9

527

Unmanured
Fertilized

Puch
(Germany)

1983a

Orthic Luvisol

18

7.9

927

Unmanured
Fertilized

1953b
1966

Dystric Cambisol

18

6.3

900

Haplic Chernozem

23

8.6

490

Lauterbach
(Germany)
Bad LauchstaÈdt
(Germany)
Experiment A

1902c

Fertilized

1956d

Unmanured
Bare fallow
Fertilized

Experiment B

1984

Prague
(Cech Republic)

1958

a
b
c
d

Bare fallow
Fertilized
Unmanured

Luvi-haplic
Chernozem

29

8.1

450

Bare fallow
Bare fallow+
fertilization
Bare fallow (1)
Bare fallow with soil
tillage (0-20 cm) (2)

Literature

NPK+lime+farmyard manure
(15 t haÿ1 year ÿ1)
No fertilization
N+farmyard manure
(3 t haÿ1 year ÿ1)
No fertilization
NPK+lime+farmyard manure
(6 t haÿ1 year ÿ1)
No fertilization
NPK+lime+farmyard manure
(10 t haÿ1 year ÿ1)
Without crops and fertilization
NPK+lime+farmyard manure
No fertilization (but liming)

NPK+farmyard manure
(15 t haÿ1 year ÿ1)
No fertilization
Without crops and fertilization
Farmyard manure
(200 t haÿ1 year ÿ1)
Without crops and fertilization
Farmyard manure
(80 t haÿ1 year ÿ1)
Without crops and fertilization

Schnieder (1990)

Asmus (1990)

Mercik et al. (1997)

Krauss et al. (1997)
Diez et al. (1997)
Reichelt (1990)

KoÈrschens and Eich
(1990)

KoÈrschens et al.
(1998)
KubaÂt and NovaÂk
(1992)

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

Location

Fertilized treatment since 1983.
Bare fallow since 1953.
Fertilized and unmanured plots since 1902.
Bare fallow since 1956.

657

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R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

The pH values were measured in the supernatant of a soil
suspension in 0.01 M CaCl2 by using a glass electrode.
The ratio of soil material (w) to CaCl2 solution (v) was
1:2.5 (Schlichting et al., 1995).
2.3.

13

C CPMAS NMR spectroscopy

Sieved samples were treated with 10% hydro¯uoric
acid (HF) to remove paramagnetic species and mineral
matter, resulting in a concentration of organic material
(Schmidt et al., 1997). Fifty millilitres of 10% (v/v) HF
were added to 10 g of soil in a polyethylene bottle. The
suspension was shaken horizontally for approximately 12
h. After centrifugation, the supernatant was removed. The
HF treatment was repeated four times. Finally, the residue
was washed with deionzed water and freeze dried.
The solid-state 13C nuclear magnetic resonance
(NMR) spectra were obtained on a Bruker DSX 200
spectrometer operating at a 13C resonance frequency of
50.3 MHz by using the cross polarization magic-angle
spinning (CP-MAS) technique (Schaefer and Stejskal,
1976). Samples were packed into a rotor of zirconium
dioxide with a diameter of 7 mm, and spun at a frequency
of 6.8 kHz. A pulse delay of 400 ms and a contact time
of 1 ms was used. Due to low sensitivity of the sandy
soil samples from Thyrow, Groû Kreutz and Skierniewice, the spectra of these samples were obtained by
using a pulse delay of 250 ms. A ramped 1H-pulse was
used during contact time in order to circumvent inexact
Hartmann-Hahn conditions (Peersen et al., 1993). After
accumulation of 23,000±350,000 scans and prior to
Fourier transformation, a line broadening of 100±150 Hz
was applied. The chemical shift scale is referenced to tetramethylsilane (= 0 ppm). The spectra are divided into
four major chemical shift regions, assignable to alkyl C
(0±45 ppm), O/N-alkyl C (45±110 ppm), aromatic C
(110±160 ppm) and carboxyl/carbonyl C (160±220
ppm). A detailed scheme of most tentative assignment
of chemical shift regions to di€erent carbon types is
given in Table 2. Signal intensities for aromatic and
carboxyl carbon were corrected for spinning side bands,
adding the intensities of the ranges 276±220 ppm and 0
Table 2
Tentative assignment of signals in the

toÿ50 ppm to those of the aromatic carbon region. One
side band of the carboxyl carbon is found in the range 323±
276 ppm. Assuming that the second side band for carboxyl
C, between 0 and 45 ppm, is of equal size, the integral of the
®rst side band was doubled and added to the signal 160±
220 ppm; then, the intensity of the ®rst side band (326±276
ppm) was subtracted from the alkyl C region.
In the depleted soils, to quantify the decrease of C in the
various shift ranges, the proportions of the individual shift
ranges were normalized to the OC of the fertilized soils:
ÿ

OCDepleted
Carbon species % of OCDepleted

OCFertilized
ˆ % of OCFertilized
OCDepleted
OCFertilized

…1†

Organic Carbon of the depleted plot
(g kgÿ1)
Organic Carbon of the fertilized plot
(g kgÿ1)

2.4. Carbohydrate analysis
Analysis of carbohydrates was carried out according
to KoÈgel-Knabner (1995). The method includes acid
hydrolysis of carbohydrates followed by a colorimetric
determination of sugar monomers by the MBTH (3methyl-2-benzothiazolinone hydrazone hydrochloride)
procedure. In this procedure, monosaccharides are
reduced to alditols, followed by an oxidation of the
terminal glycol (±CH2OH) groups of the alditols yielding two moles of formaldehyde per mole of original
monosaccharide. The formaldehyde concentration is
determined photometrically at 635 nm after reaction
with MBTH (Pakulski and Benner, 1992).
For the hydrolysis of non-cellulosic carbohydrates,
soil samples were incubated with 1 M HCl at 105 C for
5 h. For the hydrolysis of total carbohydrates (cellulosic
and non-cellulosic), the samples were incubated with 12
M H2SO4 at room temperature for 16 h, and subsequently with 1 M H2SO4 at 105 C for 5 h. The cellulosic
fraction of carbohydrates was determined by calculating
the di€erence of monosaccharides released by H2SO4

13

C NMR spectra (from Almendros et al., 1992; Knicker and LuÈdemann, 1995)

Chemical shift
range (ppm)

Assignment

0±45
45±60

Terminal CH3 groups (0±25); CH2 groups in chains (30) (lipids, proteins)
OCH3 in aromatic structures (lignin) and in polysaccharides (hemicelluloses); a-amino C (amino acids)
C-6 of some polysaccharides
Higher alcohols, C-2 to C-5 of hexoses; a-, b-, g-C in b-O-4 linked units (lignin)
(103±105) C-1 in polymeric carbohydrates (anomeric C) and C-2 and C-6 in syringyl units (lignin)
Protonated and C-substituted aromatics; ole®nic carbons
Aromatic COR and CNR groups
Carboxyl-C in aliphatic and aromatic acids, in esters, in amides (lipids, proteins); Carbonyl-C (aldehydes, ketones)

60±90
90±110
110±140
140±160
160±220

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R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

(total carbohydrates) and by HCl hydrolysis (non-cellulosic carbohydrates). Yields of monosaccharides were
expressed relative to a calibration curve of glucose. The
amount of glucose-C was calculated by multiplying the
total glucose mass with 0.4. For correction of mass difference between glucose monomers and polysaccharide
structures, dominating in SOM, the yields were multiplied with a factor of 0.9.
All analyses were run in triplicate. The standard deviation
relative to the mean value ranged between 2 and 25% for
the non-cellulosic carbohydrates, and between 3 and 23%
for the measurement of total carbohydrates, respectively.
To assess the net decrease of carbohydrate C in the
depleted plots, the content of total carbohydrate C was
normalized to the OC of the fertilized plots:
ÿ

OCDepleted
Carbohydrate C % of OCDepleted

OCFertilized
ˆ % of OCFertilized
OCDepleted
OCFertilized

…2†

Organic Carbon of the depleted plot
(g kgÿ1)
Organic Carbon of the fertilized plot
(g kgÿ1)

3. Results and discussion
3.1. Carbon and nitrogen content, pH values
The contrasting management practices lead to a considerable di€erence in the content of organic carbon and
nitrogen (Table 3). Unmanured plots and bare fallows
contain between 40 and 67% of the organic carbon and

between 41 and 67% of the nitrogen of the respective fertilized plots. Lacking any input of organic material, the bare
fallow of Experiment A (Bad LauchstaÈdt) is more depleted
in C and N than the corresponding unmanured soil. As the
relative decrease is similar for organic carbon and total
nitrogen, in most of the experiments the C/N-ratio of the
depleted soils is not subject to change compared with the
fertilized plots. Only in 4 experiments, 3 of which are bare
fallows, C-depleted soils have slightly higher C/N ratios
than the fertilized counterparts (Table 3).
In Prague, soil tillage of the bare fallow results in a further loss of organic matter compared with the untilled
fallow. This may be explained by the e€ect of tillage on
soil structure. Tillage leads to the breakdown of aggregates of various size. Upon disintegration of aggregates,
OM previously protected within the aggregate (``physical
protection'') is assumed to become exposed to microorganisms and degradative enzymes (Christensen, 1996).
In Thyrow, Skierniewice and Puch the lack of lime
application in the depleted plots Ð in contrast to the
fertilized ones Ð resulted in lower pH values (Table 3).
The chernozemic soil of Bad LauchstaÈdt is not given
any lime at all, and a similar pH is maintained in the
di€erently managed plots of this soil. In Groû Kreutz
and Lauterbach the fertilized plots show a lower pH
value than the depleted, unfertilized soils. One possible
explanation of this decrease in pH may lie in the nitri®cation of ammonium-N present in the organic and
inorganic fertilizers, respectively (Paul and Clark, 1989).
3.2. Chemical structure of SOC (13C NMR)
13
C nuclear magnetic resonance (NMR) spectra of the
soils are given in Figs. 2 and 3, relative signal intensities

Table 3
Content of organic carbon and total nitrogen, C/N ratios and pH of di€erent treatments of long-term agroecosystem experiments
Organic carbon (g kgÿ1)

Total nitrogen (g kgÿ1)

C/N ratio

Experimental site

Fertilized
plot

Depleted
plot

OC Depleted
(% of
OCFertilized)

Fertilized
plot

Depleted
plot

N Depleted
(% of
NFertilized)

Fertilized
plot

Depleted
plot

Fertilized
plot

Depleted
plot

Thyrow
Groû Kreutz
Skierniewice
Puch
Lauterbach
Bad LauchstaÈdt
Experiment A
Unmanured plot
Bare fallow
Experiment B
Prague
Bare fallow
Bare fallow+tillage

6.8
10.3
8.8
12.0
48.3

3.2
4.1
4.4
7.0
30.1

47
40
51
58
62

0.61
1.00
0.76
1.44
4.39

0.27
0.43
0.39
0.91
2.74

44
43
51
63
62

11
10
11
8
11

12
10
11
8
11

6.1
5.8
6.1
6.9
6.0

4.1
6.6
4.5
5.2
6.6

24.0

16.0
14.9
19.7
14.5
12.8

67
62
48
50
44

1.97

1.32
1.12
1.64
1.22
1.07

67
57
44
47
41

12

12
13
12
12
12

7.2

7.5
n.d.a
7.1
6.4
6.9

a

41.3
29.1

n.d., not determined.

3.68
2.62

11
11

pH

7.1
7.1

660

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

Fig. 3. 13C CPMAS NMR spectra of soils from two experimental sites di€ering in treatment.
Fig. 2. 13C CPMAS NMR spectra of soils from three experimental sites di€ering in treatment.

are presented in Table 4. In the spectra, the resonance
line discernable around 30 ppm, in the region of alkyl
carbon, can probably be assigned to methylene structures.
The peak around 56 ppm (in the region of O- and N-substituted alkyl carbon, 45±110 ppm) may originate from
methoxyl groups and N-substituted alkyl carbon. The
peaks around 72 and 104 ppm are most probably assigned
to carbohydrates. The signal between 110 and 140 ppm
peaking at 130 ppm may originate from protonated and
C-substituted aryl carbon, as well as from unsaturated
alkyl structures. The signal around 175 ppm is attributable to carboxyl/amide functional groups.
Di€erences in the relative intensity distribution
between the spectra of the depleted and the fertilized
plots are most apparent for the O/N-alkyl C and the
aromatic C, respectively (Table 4). Compared with the
fertilized plots, low organic input lead to a relative

decrease of the proportion of O/N-alkyl C, and to a
relative enrichment of aromatic C. Except for Puch and
Lauterbach, the percentage of aromaticity Ð given as
the ratio of aromatic C to the sum of aromatic and total
aliphatic C in Table 4 Ð increases by a factor of 1.2 on
an average. Aromaticity is exceptionally high in the bare
fallow (A) of Bad LauchstaÈdt and the unmanured plot
of Thyrow. By comparison, in the majority of the soils
of this study, the O/N-alkyl C region is the quantitatively most important one, as it is reported for soils
from di€erent regions of the world (e.g. Baldock et al.,
1992; Haider, 1992; Guggenberger et al., 1995).
As seen in this study, in long-term experiments at
Rothamsted Experimental Station the relative contribution of O-alkyl C diminished along with reduced organic
input (Kinchesh et al., 1995). In a wide range of soils,
the O-alkyl C range has been found to be negatively
correlated with the aromatic C region (Mahieu et al.,
1999). The changes in relative signal distribution found

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R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668
Table 4a
Relative contributions of carbon species to the total signal intensity in
ments in long-term agroecosystem experiments
Experimental site

Thyrow
Groû Kreutz
Skierniewice
Puch
Lauterbach
Bad LauchstaÈdt
Experiment A
Unmanured plot
Bare fallow
Experiment B
Prague Bare fallow
Bare fallow+tillage
a
b
c

13

C NMR spectroscopy of soil samples under di€erent treat-

% Alkyl C
(0±45 ppm)

% O/N-Alkyl C
(45±110 ppm)

% Aromatic C
(110±160 ppm)

% Carboxyl C
(160±220 ppm)

Alkyl C
O/NÿAlkyl C

% Aromaticityc

Fert.a

0b

Fert.

0

Fert.

0

Fert.

0

Fert.

0

Fert.

0

19
22
21
19
22

17
20
22
22
25

35
41
38
43
43

28
38
36
41
43

30
25
26
27
22

36
28
30
25
20

15
11
15
11
13

17
14
11
12
12

0.55
0.55
0.55
0.43
0.51

0.62
0.53
0.66
0.54
0.57

36
28
31
30
25

44
33
34
28
22

27

20
20
19
19
18

33

33
28
35
35
35

26

33
34
32
30
34

14

13
17
14
15
12

0.81

0.60
0.72
0.55
0.54
0.51

30

39
41
37
36
39

21
20

39
41

28
26

13
13

0.53
0.50

32
30

Fertilized plots.
Depleted plots.
Aromatic C …110ÿ160†
Aromatic C …110ÿ160† ‡ Aliphatic C …0ÿ110†


100 (FruÈnd et al., 1994).

Table 4b
Absolute amounts of the carbon species (13C NMR spectroscopy) under di€erent treatments in long-term agroecosystem experiments

Experimental site

Thyrow
Groû Kreutz
Skierniewice
Puch
Lauterbach
Bad LauchstaÈdt
Experiment A
Unmanured plot
Bare fallow
Experiment B
Prague
Bare fallow
Bare fallow+tillage
a
b

Alkyl C
(0-45 ppm)
(g C kgÿ1)

O/N-Alkyl C
(45±110 ppm)
(g C kgÿ1)

Aromatic C
(110±160 ppm)
(g C kgÿ1)

Carboxyl C
(160±220 ppm)
(g C kgÿ1)

Fert.a

0b

Fert.

0

Fert.

0

Fert.

0

1.33
2.31
1.82
2.24
10.52

0.57
0.83
0.97
1.58
7.39

2.42
4.24
3.33
5.16
20.76

0.92
1.57
1.62
2.91
12.96

2.09
2.59
2.32
3.22
10.57

1.19
1.16
1.35
1.74
5.86

1.00
1.18
1.28
3.22
6.32

0.57
0.59
0.49
1.38
3.49

6.42

3.18
3.04
3.79
2.77
2.29

7.93

5.28
4.25
6.84
5.14
4.54

6.28

5.36
5.00
6.31
4.41
4.37

3.31

2.14
2.56
2.73
2.19
1.59

8.50
5.94

16.01
11.90

11.51
7.48

5.20
3.81

Fertilized plots.
Depleted plots.

in our experiments, the concomitant decrease of O-alkyl
C and increase of aromatic C, are in agreement with
these correlations.
The total range of aromatic carbon can be further
divided into a region of O-aryl C (140±160 ppm) and of
aryl C (110±140 ppm). A prominent signal in the O-aryl
C region with a peak around 150 ppm, mainly assignable

to lignin units (LuÈdemann and Nimz, 1974), is usually
found in plant materials in ®rst stages of decomposition
(KoÈgel et al., 1988). In our soils the contribution of Oaryl C (140±160 ppm) is generally low, ranging from 6 to
10% of total signal intensity (Fig. 4), indicating that
lignin does not constitute a major component of SOC.
The aromatic region of most of our soils is characterized

662

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

by a broad signal in the aryl C range (110±140 ppm) with
a peak around 130 ppm (Figs. 2 and 3). The increase of
aromaticity found in the C-depleted soils (except for
Puch and Lauterbach) is mainly attributable to an
increase of this aryl C signal (Fig. 4). Sp2-hybridized
carbon of various origin contribute to the signal around
130 ppm, for example carbon in condensed aromatic
rings (Skjemstad and Dalal, 1987). Possible sources of
condensed ring structures in soil are charred plant residues (charred organic carbon) and coal material, both
giving a pronounced signal around 130 ppm in 13C NMR
spectra (Haumaier and Zech, 1995; Skjemstad et al.,
1996; Rumpel et al., 1998; Schmidt et al., 1999). Charred
organic carbon was identi®ed in chernozemic soils of
Australia and Germany (Skjemstad et al., 1996; Schmidt
et al., 1999). In these studies it was suggested that the
charred material may originate from natural vegetation
®res or from human use of the ®re, e.g. for clearing of
forests in Central Europe. There may possibly be a
contribution of charred organic carbon in some of the
soils investigated in this study. The evalution of this
hypothesis by direct measurement of charred material
should be a subject of future research. A further source
of condensed aromatic rings present in SOM is given
by the atmospheric deposition of dust and coal-like
particles, emitted by the coal processing industry

(Schmidt et al., 1996). As some of the soils are located in the surroundings of industrialized areas, the
possibility of dust emissions into the soils should also
be considered.
Except for Puch and Lauterbach, in the depleted soils
a slightly lower intensity of alkyl C is observed compared with the fertilized plots (Table 4). In the soils of
Puch and Lauterbach, the signal intensity of alkyl C is
higher in the depleted than in the fertilized soils. The proportion of carboxyl C either increases slightly or remains
unchanged in the depleted soils (Table 4). Knicker (1993)
showed that the variation associated with phase- and
baseline correction of Fourier-transformed spectra is
highest for the shift ranges 160±220, 45±60 and 0±45
ppm. The relative standard deviation of the signal
intensity attributed to these regions was up to 13%. The
relative standard deviation for intensities in the ranges
60±110 and 110±160 ppm was found to be up to 6.5%.
Consequently, the di€erences in relative signal intensity
observed between the treatments of this study can be
considered to be more sound for the O/N-alkyl C and
aromatic C region than for alkyl and carboxyl C.
According to Baldock et al. (1997), the ratio of alkyl
to O-alkyl C can be taken as an indicator for assessing
the degree of decomposition of organic materials. In
six of the experiments, the ratio of alkyl to O/N-alkyl

Fig. 4. Relative distribution of signal intensity of the aryl C region (110±140 ppm) and the O-aryl C region (140±160 ppm) in fertilized
and depleted plots.

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

carbon is higher in the depleted than in the fertilized
soils (Table 4). A higher proportion of alkyl C relative
to O-alkyl C would be related to a higher degree of
decomposition of the residual organic matter in the
depleted plots compared with the fertilized ones. An
increase of alkyl C relative to O-alkyl C was also noted
in cultivated soils in comparison with the corresponding
native sites (Oades et al., 1988; Preston et al., 1994).
3.3. Depletion of C associated with di€erent C-species
Table 4b shows that all carbon forms are diminished
in terms of absolute carbon amount, indicating that all
carbon species were a€ected by degradation processes.

663

Applying Eq. (1), the carbon amounts of the di€erent
species are normalized to the OC content of the fertilized plots. In Fig. 5, the percentages of carbon normalized to the fertilized plots are given for the di€erent
C-species. Ratios between the percentages of depleted
and fertilized treatments are calculated (mean values in
Fig. 5). These ratios demonstrate that on an average the
extent of carbon decrease follows the order O/N-alkyl C
> alkyl C > carboxyl C > aromatic C. The higher
decrease associated with O/N-alkyl C than with aromatic C compounds is in line with the higher biodegradability of the ®rst and the higher recalcitrance of the
latter, respectively. Setting the total decrease of OC to
100%, the major part of this decrease is accounted for

Fig. 5. Contribution of the four carbon species expressed as percentage of organic carbon of the fertilized plots. The ratio given for
each carbon species is the mean value of the 8 experiments.

664

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

OC content of the fertilized plot. The decrease of carbon
in the 2 depleted plots is smallest for the aromatic C.
When comparing the mostly depleted bare fallow with
the unmanured plot, the carbon present in alkyl, aromatic and carboxyl structures is more resistant against
further degradation than O/N-alkyl carbon compounds.
This ®nding again points out the labile nature of O/Nalkyl C.

by O/N-alkyl C (42% of the C loss). Alkyl and aromatic
C account for 22% each, and carboxyl C for 14%.
In the bare fallows of Prague, soil tillage lead to a
further decrease of all carbon species except the aromatic
C, corroborating the refractory nature of aromatic C
(Fig. 5). In Experiment A of Bad LauchstaÈdt a gradient
in the OC content exists among the 3 plots with di€erent
management regime, i. e. the fertilized plot, the unmanured plot, and the bare fallow (see Table 3). In Fig. 6,
proportions of the carbon species are normalized to the

3.4. Carbohydrates
Total carbohydrate C contents, expressed as a percentage of OC, are similar in the fertilized and depleted
plots of the experiments (Table 5), except for the bare
fallow of Experiment A, Bad LauchstaÈdt, which is
depleted in carbohydrates compared with the other soils.
In both treatments, the carbohydrate pool is dominated
by the non-cellulosic fraction (Table 5). But the cellulose
proportion of total carbohydrates is slightly smaller in
the depleted than in the fertilized soils. This loss of cellulose re¯ects the reduced input of plant materials, as
cellulose is turned over rapidly in soils (Haider, 1992).
Analogous to the carbon species, the carbohydrate
content of the depleted soils was normalized to the OC
of the fertilized soils [see Eq. (2)] (Table 5). The depleted
plots contain between 39 and 72% of the carbohydrate
C present in the fertilized plots (mean value 52%).
Comparing the ratios determined for carbohydrate C
and O/N-alkyl C (52% or 0.52, and 0.48, respectively),
the relative extent of decline in carbohydrate C and O/Nalkyl C are of similar magnitude. The data of Experiment

Fig. 6. Contribution of the four carbon species (13C NMR)
and total carbohydrate C (MBTH method) in Experiment A
(Bad LauchstaÈdt), expressed as percentage of organic carbon of
the fertilized plot.

Table 5
Amounts of total carbohydrates, proportions of cellulose, and total carbohydrates normalized to OC of the fertilized plots in longterm agroecosystem experiments
Total
carbohydrate C
(% of OC)

Cellulosic C
(% of total
carbohydrate C )

Total carbohydrate C
(% of OC of fertilized plot)

Experimental site

Fertilized
plot

Depleted
plot

Fertilized
plot

Depleted
plot

Fertilized
plot

Depleted
plot

Carbo: ÿ CDepleted
(%)
Carbo: ÿ CFertilized

Thyrow
Groû Kreutz
Skierniewice
Puch
Lauterbach
Bad LauchstaÈdt
Experiment A
Unmanured plot
Bare fallow
Experiment B
Prague Bare fallow
Bare fallow+tillage

13.9
14.6
15.5
15.7
14.8

15.4
15.7
13.5
18.5
13.6

11
13
14
13
12

3
n.d.
n.d.
7
n.d.

13.9
14.6
15.5
15.7
14.8

7.3
6.3
6.8
10.8
8.5

52
43
44
69
57

11.2

12.0
8.2
10.2
11.5
10.2

9

4
n.d.a
13
n.d
n.d.

11.2

8.0
5.1
4.9
5.8
4.5

72
46
39
55
43

a

n.d., not detectable.

12.5
10.5

17
n.d.

12.5
10.5

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

A (Bad LauchstaÈdt) show that there is an analogous
trend for the changes in O/N-alkyl C on the one hand
and carbohydrate C on the other hand, with progressive
decrease of organic carbon (Fig. 6). In summary, the
carbohydrate results con®rm those of 13C NMR
regarding the decline of O/N-alkyl C compounds in the
depleted plots.
The contents of carbohydrate C signi®cantly correlate
with the signal intensities of O-alkyl C (60±110 ppm) in
the soils (Fig. 7). On an average, carbohydrate C (normalized to OC) accounts for 47% of the relative signal
intensity in the range 60±110 ppm (O-alkyl C). In the
work of KoÈgel-Knabner et al. (1988) a comparable
relationship between hydrolyzable carbohydrates and
intensity in the shift range of O-alkyl C (50±110 ppm)
was found for forest ¯oors. The discrepancy between
yields of carbohydrates obtained by hydrolysis and
intensities of O-alkyl C in NMR spectra may be
explained by several reasons (KoÈgel-Knabner, 1997).
First, it may be due to methodological problems associated with the hydrolysis procedure. As the digestion of
polymeric carbohydrates to monosaccharides is a prerequisite for their determination by the MBTH procedure, incomplete breakdown of polysaccharides during
hydrolysis leads to an underestimation of colorimetrically determined carbohydrates. As shown by
Allard et al. (1997), acid hydrolysis of cell material at
high temperatures for several hours lead to the precipitation of a dark-colored residue, which was identi®ed as melanoidin-like polymer, formed in the presence
of both sugars and amino acids. Second, the discrepancy
may result from the fact that other alcohol and ether
groups present for example in lignin side chains contribute to the O-alkyl C signal in 13C NMR spectra.
To assess the contribution of both hydrolyzable carbohydrates and non-hydrolyzable structures to the O-alkyl C

Fig. 7. Correlation between O-alkyl C signal intensities and
yields of total carbohydrate C in various plots of long-term
agroecosystem experiments. The solid line is given by the
regression equation.

665

Fig. 8. 13C CPMAS NMR spectra of soils before and after
hydrolysis with H2SO4.

region, 13C NMR spectra were obtained from the residues after H2SO4 hydrolysis of four soils (Fig. 8). The
spectra show that most of the carbohydrates (with
resonances around 72 and 105 ppm) were hydrolyzed.
Between 21 and 31% of the signal intensity of the Oalkyl C region of the untreated soils remain in the spectra of the hydrolysis residues (Table 6). Apart from the
contribution of a small portion of non-hydrolyzable
carbohydrates, this residual signal intensity may be
derived from lignin side chains, other non-speci®ed
alcohol and ether structures, and from overlapping signals of alkyl or aromatic carbon. According to Table 6,
between 39 and 51% of the O-alkyl C intensity are
identi®ed as carbohydrates by means of hydrolysis and
MBTH reaction, leaving between 25 and 40% of the
signal intensity as hydrolyzable, but non-identi®ed carbon. This proportion of hydrolyzable compounds which
are not identi®ed by the MBTH method was calculated
by di€erence, taking the two other ``fractions'' into
account. As stated above, the gap between the sum of
carbohydrates (MBTH method) plus non-hydrolyzable
compounds and the total O-alkyl C signal may be due
to: (i) di- or oligomeric carbohydrates in the hydrolyzate
which do not form a colored complex during the MBTH
procedure, (ii) carbohydrates which are involved in the
formation of melanoidin-like polymers, and (iii) monosaccharides which are lost during the hydrolysis procedure (Beudert, 1988).

666

R. Kiem et al. / Organic Geochemistry 31 (2000) 655±668

Table 6
Distribution of the signal in the chemical shift range 60±110 ppm (13C NMR) in soils from long-term experiments
O-Alkyl C (60±110 ppm)=100%
Soils

Hydrolyzable C,
identi®ed as carbohydrate Ca (%)

Hydrolyzable C,
non identi®edb (%)

Non hydrolyzable Cc
(%)

Thyrow
fertilized plot
Bad LauchstaÈdt, Experiment A
fertilized plot
Bad LauchstaÈdt, Experiment B
bare fallow
Bad LauchstaÈdt, Experiment B
fertilized plot

51

25

24

47

25

28

39

40

21

44

25

31

a
b
c

Total carbohydrate C determined by H2SO4 hydrolysis+MBTH method.
Calculated values by taking into account the carbohydrate C and the non hydrolyzable O-alkyl C.
Determined by 13C NMR analysis of the hydrolysis residue.

4. Summary and conclusions

Acknowledgements

In the experimental approach of this study it was
supposed that exhaustive depletion of OC in arable soils
leads to a relative accumulation of refractory compounds in the total SOM. Comparing C-depleted plots,
i. e. unmanured plots and bare fallows, with fertilized
plots from long-term agroecosystem experiments, the
following trends have been observed.
In comparison with the fertilized plots, the residual
SOM in the depleted plots is characterized by: (i) a
relative decrease in O/N-alkyl C compounds, (ii) a relative accumulation of aromatic C, especially in aryl C
compounds, and (iii) in most cases a slightly higher
degree of oxidation as indicated by the relative proportion of carboxyl functional groups. The proportion of
alkyl C either increases or is diminished in the depleted
plots. However, alkyl C is found to be enriched relative
to O-alkyl C in most of the depleted soils.
Regarding the absolute decline of OC, the amount of
all carbon species is diminished in the depleted plots
compared with the level present in the fertilized ones.
However, the various carbon species di€er with respect
to the extent of this carbon decrease. The decrease was
shown to follow the order O/N-alkyl C>alkyl C>carboxyl C> aromatic C. The di€erence in the behaviour
of O/N-alkyl C and aromatic C is in line with the degree
of biodegradability of these structures. The extent of
decrease of O/N-alkyl carbon is con®rmed by the results
obtained from wet chemical analysis of carbohydrates.
Our data demonstrate the value of long-term agroecosystem experiments for studying the composition of
the slowly turned over SOC pool. According to our
results, this pool of organic carbon is relatively depleted
in O/N-alkyl C compounds, whereas it is relatively
enriched in aromatic carbon in comparison with active/
labile fractions of OC.

The work was ®nancially supported by the Deutsche
Forschungsgemeinschaft. The authors would like to
thank Michael Baumecker from the Experimental Station at Thyrow (Germany), Dr. Pommer from the
Bayerische Landesanstalt fuÈr Bodenkultur und P¯anzenbau at Freising (Germany), and Prof. Stanislaw
Mercik from Warsaw Agricultural University (Poland)
for the help in obtaining soil samples from the various
experimental sites. We are grateful to Dr. Jaromir
KubaÂt from the Research Institute of Crop Production
at Prague (Cech Republic) for providing soil samples of
the bare fallow experiment.

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