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The determination of δ13C in soil microbial

biomass using fumigation-extraction

ARTICLE in SOIL BIOLOGY AND BIOCHEMISTRY · JULY 2003

Impact Factor: 3.93 · DOI: 10.1016/S0038-0717(03)00151-2

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 Soil Biology & Biochemistry 35 (2003) 947–954
www.elsevier.com/locate/soilbio

The determination of d13C in soil microbial biomass

using fumigation-extraction
Martin Potthoff a,*, Norman Loftfielda, Franz Bueggerb, Barbara Wicka, Bettina Johna,
Rainer Georg Joergensenc, Heiner Flessaa
a
Institute of Soil Science and Forest Nutrition, University of Göttingen, Büsgenweg 2, 37077 Göttingen, Germany
b
Institute of Soil Ecology, GSF-National Research Center for Environment and Health, 85764 Neuherberg, Germany
c
Department of Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany
Received 22 August 2002; received in revised form 28 February 2003; accepted 14 March 2003

Abstract
The determination of the isotopic composition of the microbial biomass C in soil is an important tool to study soil microbial ecology and
the decomposition and microbial immobilization of soil organic C. We discuss advantages and disadvantages of different methods to
determine 13C/12C in soil microbial biomass and propose a new procedure that is based on the UV-catalyzed liquid oxidation of fumigated
and non-fumigated soil extracts combined with trapping of the released CO2 in liquid nitrogen and subsequent determination of d13 CO2 -C by
a gas chromatograph connected with an isotope ratio mass spectrometer (IRMS). This method was evaluated using test solutions with known
isotopic composition and soil extracts. Additionally, the method was compared with an off-line sample preparation technique combined with
isotope analysis by a dual-inlet IRMS and an on-line analysis using an elemental analyser connected with an IRMS. All methods applied
obtained comparable results and there were no significant differences between the d13 C values measured. The off-line preparation procedure
had the highest precision but it was also the most labour-intensive. The choice of the most suitable method depends mainly on the number of
samples that have to be analysed, the salt concentration of the extracts and the differences of d13 C that have to be detected.
The application of this method with liquid oxidation and subsequent GC-IRMS analysis showed that microbial biomass C of a grassland
soil was 13C-enriched by 2‰ d13 CPDB compared with the total soil organic C. The addition of maize straw resulted in a rapid immobilization
of maize C in the microbial biomass.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Soil microbial biomass; d13 C; Fumigation-extraction; Mass spectrometry

1. Introduction (van Veen et al., 1985). Both, the fumigation-incubation

Studies on the decomposition of 14C labelled rye grass
showed that a significant fraction with strong isotope
enrichment was extractable after chloroform fumigation of
soil (Jenkinson, 1966). The use of the isotope technique
supported the hypothesis that CHCl3 labile material in soil is
of microbial origin. This observation led to the development
of the fumigation methods for measuring the soil microbial
biomass (Jenkinson and Powlson, 1976) followed by an
increase in knowledge about the contribution of soil
microorganisms to the turnover of plant nutrients
* Corresponding author. Present Address: Department of Vegetable
Crops, University of California, 1 Shields Avenue, Davis, CA 95616, USA.
Tel.: þ 1-530-752-9164; fax: þ1-530-752-9659.
E-mail address: mpottho1@gwdg.de (M. Potthoff).
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0038-0717(03)00151-2
(FI) and the fumigation-extraction (FE) method allow direct
estimates of C and N in the soil microbial tissue (Jenkinson,
1988). Therefore, they are suitable for experiments looking
at the turnover of isotope labelled material. However, the
use of the FI method is restricted in terms of 14C, 13C, and
15
N (van Veen et al., 1987; Nicolardot et al., 1986, 1994;
Qian and Doran, 1996; Qian et al., 1997), and the FI method
is limited to aerobic, neutral soils without actively
decomposing substrates (Jenkinson, 1988; Ocio and
Brookes, 1990). In contrast, the FE method can be used
with a larger range of isotopes, i.e. 14C, 13C, 15N, 32P and 35S
(Sparling and West, 1988; Ryan and Aravena, 1994; Hedley
and Stewart, 1982; Wu et al., 1994), in all types of soil,
especially in the presence of added actively decomposing
substrates (Ocio and Brookes, 1990; Joergensen, 1996).
948 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954
Recently, the use of 13C has increased in soil microbial
ecology, because of an improved sensitivity in detecting
13
C. In contrast to 14C, 13C-labelled material can be used in
field experiments without any further safety precautions.
Also important is the fact that the difference in d13 C values
between C3 (e.g. wheat) and C4 plants (e.g. maize and sugar
cane) can be used as natural label for the differentiation
between different C pools in decomposition experiments.
For these reasons, the determination of the natural
abundance of 13C in faunal tissue (Schmidt et al., 1997)
and soil organic matter (Boutton, 1996) has been widely
applied. Bulk samples from these materials can be directly
analysed by an elemental analyser (EA) connected to an
isotope ratio mass spectrometer (EA-IRMS) after drying
and grinding. This is not possible for the soil microbial
biomass, because it can be released from bulk soil with
actively decomposing substrate only after fumigation and
extraction or incubation. However, the fumigation extrac-
tion method has gained wider acceptance due to the
advantages described above. Several attempts have been
made to determine d13 C values of the soil microbial biomass
by the FE method after addition of 13C-labelled C-substrates
(Gaillard et al., 1999; Trinsoutrot et al., 2000), but also in
the range of natural 13C abundance (Ryan et al., 1995). At
present, there is no standard procedure of extracting soil
samples and preparing the extracts for 13C/12C analysis, and
a vast variety of methods has been described (Table 1). The
FE approaches employed differ mainly by the type and
concentration of the solution used for extraction and by the
following on-line or off-line preparation procedure for the
oxidation of the extracted organic C and the purification of
the CO2 produced.
In this study, we discuss the advantages and disadvan-
tages of published methods of 13C/12C determination in soil
microbial biomass and we describe a new procedure to
determine d13 C in FE extracts after UV activated persul-
phate oxidation of organic C to CO2. This method requires
only one C digestion for the quantification of the released
CO2 by infra-red detection and the following mass spectro-
metric determination of the isotopic composition (13C/12C).
The aim of the present work was (i) to evaluate this
procedure using cane sugar and beet sugar in K2SO4
solutions, K2SO4 extracts of fumigated and non-fumigated
grassland soil, and dissolved organic C leached from a
sandy arable soil and (ii) to compare the proposed method
with an off-line sample preparation technique combined
with isotope analysis by a dual-inlet IRMS and an on-line
analysis using an EA-IRMS system.
2. Material and methods
2.1. Test solutions
We used three different solutions to evaluate the methods
to analyse the d13 C of soil microbial C.
(1) 0.5 M K2SO4 stock solutions were spiked with organic
C (10, 20, 40, 60 mg C ml21) derived from cane sugar
(d13 C ¼ 210:50‰) or beet sugar (d13 C ¼ 224:82‰)
with known d13 C values.
(2) Grassland soil samples incubated with and without
maize straw (10 g air dried maize straw mixed with
1 kg soil; incubation time 28 days) were extracted (FE)
with 0.5 M K2SO4 according to Vance et al. (1987).
The organic C content of the soil was 12 mg g21, the
C/N ratio was 10, the soil microbial biomass C was
300 mg kg21, and the d13 C value of the total soil
organic C was 2 26.93‰. The maize straw had a d13 C
value of 2 12.04‰.
(3) Dissolved organic C solutions were derived by
leaching a sandy arable soil with 0.01 M CaCl2 (Flessa
et al., 2000).
2.2. d13 C of test solutions and FE extracts
The following three methods were used to oxidise the
organic C in test solutions and FE extracts, to purify the CO2
produced, and to analyse the d13 C of the CO2-C.
Drying and grinding. The solutions were acidified with
HCl to pH 3.5 to eliminate inorganic C. Subsamples of 10 ml
were dried at 70 8C to a constant dry weight. The grinding of
the dried samples was done using an agate-mortar. The
ground material was analysed for d13 C by EA-IRMS
(Finnigan Mat delta plus, Fissions EA, Bremen, Germany).
Liquid oxidation with freeze-trapping of CO2. The
solutions were acidified with sodiumhexametaphosphate
and H3PO4 to pH 2. Aliquots of 1 ml were digested with UV
and potassium peroxodisulphate þ H3PO4 (pH 3) in an
automatic C analyser (Dohrman DC 80) (Wu et al., 1990).
The released CO2 passed the IR detector of the C analyser
using synthetic air as carrier gas and was trapped in 1 ml glass
vials at the gas outlet of the analyser by liquid nitrogen. Vials
were kept gas tight using rubber lids. They were placed in the
autosampler of a GC-IRMS system (Finnigan, Delta C,
Bremen, Germany) for on-line 13C/12C analysis (Fig. 1).
Liquid oxidation with off-line cryogenic purification of
CO2. FE extracts were digested in evacuated glass flasks at
130 8C using Na2S2O8 as oxidant of organic C. The released
CO2 was purified in a manually operated vacuum-prep-
aration line with cryo-traps (Fig. 2). In a first step, CO2 was
purified by drawing the gas through a water trap (2 70 8C,
dry ice with acetone) into a cryogenic trap (2 196 8C, liquid
N2). Then, the trapped CO2 was frozen in a standard volume
dipped into a second cryogenic trap (2 196 8C, liquid N2).
This volume was connected to a pressure sensor and was
used to determine the amount of CO2 (released from the FE
extracts) after removing the cryo-trap and shortly heating
the standard volume with the trapped gas to room
temperature. In a final step, the CO2 was trapped in a
glass vial (again by cryo-trapping with liquid N2) that could
be connected to the dual-inlet system of an IRMS (Finnigan,
Delta S, Bremen, Germany).
 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954 949

Table 1
Different methods of d13 C analysis in soil microbial biomass

Reference Method of 13C/12C analysis in soil Subject

microbial biomass

Ryan and Aravena, 1994; Fumigation-extraction (0.5 M K2SO4) Immobilization of maize C

Ryan et al., 1995 Freeze-drying of extracts followed in microbial biomass, determination
by off-line preparation: of available root C
Thermal oxidation (550 8C) and cryogenic
purification of CO2
Determination by IRMS with a dual-inlet
system
Angers et al., 1995 Fumigation-extraction (0.05 M K2SO4) Immobilization of maize C
Drying of extracts followed by off-line in microbial biomass
preparation:
Thermal oxidation (800 8C) and cryogenic
purification of CO2
Determination by IRMS with a dual-inlet
system
Bruulsema and Duxbury, 1996 Fumigation-extraction (0.25 M K2SO4) Method evaluation
Drying of extracts followed
by on line analysis:
Determination by EA-IRMS
Qian and Doran, 1996; FI, trapping of CO2 in NaOH Immobilization of maize root
Qian and Doran, 1997 Precipitation with CaCl2 in excess as CaCO3 C in microbial biomass;
Determination by EA-IRMS Quantification of maize root- derived
available C
Gaillard et al., 1999 Fumigation-extraction (0.03 M K2SO4) Immobilization of labelled (13C, 15N)
Freeze-drying of extracts followed wheat straw C and N in microbial
by on line analysis: biomass
Determination by EA-IRMS
Rochette et al., 1999 Fumigation-extraction (0.25 M K2SO4) Immobilisation of maize C
Extracts were dialyzed to in microbial biomass
remove salt and then
freeze-dried
Determination by EA-IRMS
Gregorich et al., 2000 Fumigation-extraction (ultra pure water) Immobilization of maize C in
Freeze-drying of extracts followed total SOC, water extractable
by on line analysis: SOC, and microbial biomass
Determination by EA-IRMS
13
Šantrůčková et al., 2000 Fumigation-extraction (0.5 M K2SO4) C discrimination by microbial
Freeze-drying of extracts followed biomass
by off-line preparation:
Thermal oxidation (900 8C) and cryogenic
purification of CO2
Determination by IRMS with
a dual-inlet system
Trinsoutrot et al., 2000 Fumigation-extraction (0.25 M K2SO4) Immobilization of labelled (13C, 15N)
No further details residue C and N in microbial biomass
Liang et al., 2002 Fumigation-extraction (pure water) Immobilization of maize root
Freeze-drying of extracts followed C in microbial biomass
by on line analysis:
Determination by EA-IRMS

2.3. Calculations where CSA is the C of the sample and CPDB is the C of the
PDB standard.
The isotopic composition of a sample was calculated as The C content of the fumigated extracts ðCf Þ is the sum of
follows: the C content of the non-fumigated (control) extracts ðCc Þ
and the additional extracted C from cell lysis by chloroform
d13 CSA ð‰Þ ¼ð13 CSA =12 CSA 2 13 CPDB =12 CPDB Þ= fumigation (chloroform-labile C; Cb ):
ð13 CPDB =12 CPDB Þ £ 1000 Cf ¼ Cc þ Cb
950 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954

Fig. 1. Scheme of the method to determine d13 C in soil microbial C by liquid oxidation of organic C in extracts derived from the FE-method. Oxidation and
infra-red CO2 detection is combined with freeze-trapping of the CO2 released and subsequent determination of d13 C by GC-IRMS.

Accordingly, the d13 C of FE extracts is determination of d13 C by EA-IRMS (method 1) resulted in

d13 Cf £ Cf ¼ ðd13 Cc £ Cc Þ þ ðd13 Cb £ Cb Þ
ðRyan et al:; 1995Þ
The d13 C values were expressed as means ðn ¼ 5Þ with
standard deviation. The t-test was applied to determine
significant differences between the methods and the test
solutions used.
3. Results
Drying and grinding of 0.5 M K2SO4 solutions spiked
with sugar to different C concentrations and subsequent
inconsistent d13 C values (data not shown). The large
quantities of salt hampered the mass spectrometric analysis
by affecting the oxidation process. The combustion was
often incomplete and the combustion time not reproducible,
especially for samples which contained less than 60 mg
C ml21. In order to ensure a proper flash combustion of
dried 0.5 M K2SO4 extracts in the EA-IRMS equipment, the
amount of salt entering the combustion chamber should not
exceed 80 mg K2SO4 and the amount of C should be at least
20 mg.
The d13 C-values of the sugar solutions obtained by liquid
oxidation with freeze-trapping of CO2 and following
13 12
C/ C analysis by GC-IRMS (method 2) were reproduci-
ble, not affected by different C to salt ratios of the sugar

Fig. 2. Scheme of the method to determine d13 C in soil microbial carbon by liquid oxidation of organic C in extracts derived from the FE-method and off-line
purification of the CO2 released using a vacuum-preparation line with cryo-traps and subsequent determination of d13 C by a dual-inlet IRMS.
 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954
Fig. 3. d13 C values of (a) cane sugar solutions and (b) beet sugar solutions
made of 0.5 M K2SO4 as analysed by liquid oxidation with freeze-trapping
of CO2 and GC-IRMS analysis (n ¼ 4; standard deviation) and the d13 C
value of the beet sugar and the cane sugar ðn ¼ 5Þ:
solutions, and they were close to the expected values of
2 10.5‰ (cane sugar) and 2 24.5‰ (beet sugar) (Fig. 3).
The sugar – K2SO4 solutions obtained from cane sugar had a
mean d13 C value of 2 10.5‰ (^ 0.5‰; n ¼ 16) and the
mean d13 C value of the solutions containing beet sugar was
2 24.8‰ (^ 0.3‰; n ¼ 16). No significant differences
between the expected and the determined values were
obtained for all C concentrations. The three methods of
sample preparation and d13 C analysis were compared by
analysing 0.5 M K2SO4 solutions spiked with 60 mg C ml21
from cane sugar or beet sugar. The three methods resulted in
very similar d13 C values and there were no significant
differences either between the methods applied or between
the measured and the expected d13 C values (Fig. 4).
However, the off-line preparation and subsequent analysis
by the dual-inlet system of the IRMS resulted in a
significantly reduced standard deviation (^ 0.05‰) of the
d13 C value as compared with the other methods (approxi-
mately ^ 0.3‰).
Samples of dissolved organic C leached from a
sandy arable soil were analysed to test the reliability of
the described methods when determining the isotopic
composition of a more complex mixture of organic
compounds. Again, there was no significant difference
between the d13 C value determined by the method based on
drying and grinding of the sample with on-line analysis by
EA-IRMS (method 1, 2 27.2 ^ 0.4‰, n ¼ 5) and the d13 C
value obtained by liquid oxidation with freeze-trapping of
CO2 and following 13C/ 12C analysis by GC-IRMS
(method 2, 2 27.5 ^ 0.2‰, n ¼ 5) (data not shown).
951
Fig. 4. d13 C values of (a) cane sugar solutions and (b) beet sugar solutions
(0.5 M K2SO4 with 60 mg C ml21) as analysed by three different methods:
(1) drying and grinding of FE-extracts, 13C/12C analysis by EA-IRMS; (2)
liquid oxidation of FE-extracts with cryo-trapping of the CO2 released and
13 12
C/ C analysis by GC-IRMS; and (3) liquid oxidation of FE-extracts with
off-line purification of the CO2 released and 13C/12C analysis by dual-inlet
IRMS (n ¼ 4; standard deviation).
The two liquid oxidation methods resulted in a complete
oxidation of the sugars added to the K2SO4 stock solution.
The CO2-C yield achieved in the vacuum-preparation line
(method 3) was on an average 102 ^ 2% (mean ^ standard
deviation) of the sugar C added. A similar CO2-C recovery
rate was found for sugar oxidation by method 2. Oxidation
of more complex organic compounds of soil extracts was
highly reproducible (Table 2) and there was no significant
difference between the DOC concentrations measured with
different methods (data not shown).
Method 2 (liquid oxidation with freeze-trapping of CO2;
GC-IRMS) was applied to FE extracts from grassland soil
samples incubated with and without maize straw. The 13C
content was enriched in the extractable part of the soil
Table 2
Total organic carbon and maize derived carbon in 0.5 M K2SO4 soil
extracts of fumigated and non-fumigated grassland soil samples, in the
microbial biomass (chloroform-labile C) (mean ^ standard deviation,
n ¼ 12), and in the soil organic matter (mean ^ standard deviation,
n ¼ 5) measured 28 days after the addition of maize straw
SOC fraction TOC Maize derived TOC
(mg g21) (mg g21)
Extract without fumigation 144 (^5) 21 (^7)
Fumigated extract 306 (^13) 119 (^17)
Chloroform-labile microbial C 161 (^8) 97 (^11)
Organic soil C 11,800 (^100) 380 (^30)
952 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954
Fig. 5. d13 C values of 0.5 M K2SO4 soil extracts of fumigated and non fumigated grassland soil samples with or without addition of maize straw as analysed by
liquid oxidation and cryo-trapping of the CO2 released with subsequent analysis by GC-IRMS (n ¼ 12; standard deviation), calculated d13 C values of
microbial biomass C (chloroform-labile C), and d13 C value of the total soil organic C (n ¼ 5; standard deviation).
organic C compared to the total soil organic C (þ 3.5‰ for
the non fumigated extract and þ 2.7‰ for the fumigated
extract, control soil without maize). The d13 C value of the
soil microbial (chloroform-labile) biomass C was about 2‰
higher than the d13 C of the total organic C. The soil extracts
without maize contained 54 mg C ml21 (fumigated) and
30 mg C ml21 (non-fumigated). The incorporation of maize
residues resulted in increased C concentrations in the
fumigated (þ 25 mg C ml21) and the non-fumigated extract
(þ 6 mg C ml21). The content of microbial biomass was
about 30% greater in the maize-treated soil than in the control
soil. The uptake of maize C by the microbial biomass was
reflected by the significantly increased d13 C value of
the chloroform-labile C (Fig. 5). Approximately 60% of the
microbial biomass C were derived from maize C after the
incubation period of 28 days.
4. Discussion
The 0.5 M concentration of K2SO4 commonly used to
extract microbial biomass C (Beck et al., 1997) hampered
accurate mass spectrometric analysis of dried extracts by
EA-IRMS. The large quantities of salt (with a relatively
small amount of organic C) impaired the flash combustion
in the EA. This problem was also described by Gregorich
et al. (2000). We did not obtain reliable d13 C values for
dried samples containing more than 80 mg K2SO4 in the
combustion chamber.
For this reason, the K2SO4 concentration in the extractant
was reduced in a number of studies to minimize the amounts
of salt entering the combustion chamber of the EA-IRMS
(Table 1). These reductions range from 0.25 M K2SO4
solutions to pure water solutions. A reduction in concen-
tration is possible without creating differences in CHCl3
labile organic C as long as flocculation of the soil colloids is
ensured. Joergensen (1995) showed that the amount of
CHCl3 labile organic C extracted with a 0.01 M CaCl2
solution did not differ from a 0.5 M K2SO4 solution.
However, the organic C content extracted from non-
fumigated soil was strongly affected by the composition
and ionic strength of the extraction solution. Differences in
the extraction efficiency of CHCl3 labile organic C only
occurred when the flocculation of soil particles was
incomplete, as for example with 0.001 M K2SO4 or water
as extractant (Haney et al., 2001). Nevertheless, the use of
0.5 M K2SO4 is inevitably necessary if microbial biomass N
has to be determined simultaneously. Additionally, the high
salt concentration prevents microbial decomposition of very
easily decomposable CHCl3 labile organic material.
Another possibility to reduce the high salt concen-
trations of the FE extracts was proposed by Wanniarachchi
(unpublished data) and later applied by Rochette et al.
(1999). They used a dialysis membrane to remove the salt.
However, we found that a considerable part of the
extracted C was lost by this procedure. About 25% of the
C from the extract was lost after 1 h and about 70% after
5 h although we used dialysis membranes with a lower cut-
off point (molecular weight of 1000) than the membrane
proposed by Wanniarachchi (unpublished) (molecular
weight of 3500). Similar results were reported by
Balesdent and Mariotti (1996) who lost about 20 –30% of
fulvic acid C by applying dialysis to Na-hexametapho-
sphate extracts.
Ryan et al. (1995); Ryan and Aravena (1994) proposed
an off-line sample preparation technique to purify CO2
evolved during the sample oxidation. The oxidation was
performed by combustion of freeze-dried FE extracts (0.5 M
K 2 SO 4) at 550 8C, the produced CO 2 was purified
cryogenically, and the isotopic composition (13C/12C) was
determined with a dual-inlet IRMS (Table 1). They obtained
standard deviations of the d13 C values between 0.1 and
 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954
0.9‰ for the FE extracts and between 0.9 and 1.9‰ for the
calculated microbial C.
The off-line preparation technique used in our study
(liquid oxidation and cryogenic purification of CO2 with
following analysis by dual-inlet IRMS, method 3, Fig. 2)
resulted in significantly smaller standard deviations of the
d13 C values as compared with the other methods tested (EA-
IRMS or liquid oxidation with freeze-trapping of CO2 and
GC-IRMS analysis). It was the method with the highest
precision, however, the manual preparation of samples with
off-line cryogenic purification of CO2 requires technical
experience and is laborious (only about 10 samples per day
could be prepared and analysed). This method has a high
precision due to repeated measurements of sample and
reference gases during a single isotope ratio determination
and the large amount of C and homogeneity of the samples
(no pulverisation is necessary). There is the risk of
contamination with ambient CO2 in both methods analysed
(i.e. by a leak in the vacuum-preparation line or by leaky
rubber lids of sample vials). However, the risk that a
contamination is not realized is greater for our method 2
(liquid oxidation with freeze-trapping of CO2 and on-line
GC-IRMS analysis) than for method 3 (off-line cryogenic
purification of CO2 and analysis with dual-inlet IRMS)
because in the latter method, the operational sequence can
be controlled by the vacuum in the preparation line and
vacuum tight vials were used to collect and process the CO2.
This method seems to be superior especially if small
differences in d13 C have to be detected. We like to point out
that a specific problem arises when CaCl2 extracts are
processed. Chloric gas (Cl2) is produced which cannot be
separated from CO2 by cryo-trapping. In this case, we
recommend to precipitate Cl by adding a 0.2 M AgNO3
solution before processing the extracts in the vacuum-
preparation line.
The proposed method 2 (UV-catalyzed liquid oxidation
of the extracts with following detection of 13C/12C by GC-
IRMS) has the advantage that the amount of work involved
is reduced considerably since drying and grinding is omitted
and purification of the trapped CO2 is achieved on-line by
the gas chromatograph connected with the IRMS. This
method is recommendable if a large number of samples has
to be analysed and if the extraction was done with 0.5 M
K2SO4. The method 1 (drying and grinding of samples with
following 13C/12C analysis by EA-IRMS) can be achieved
with standard equipment available in most stable isotope
laboratories. It is suitable to analyse a large number of
samples and might be superior when extraction can be done
with reduced K2SO4 concentrations or pure water. The
choice of the best method depends mainly on the number of
samples that have to be analysed, the salt concentration of
the extracts and the difference of d13 C that has to be
detected.
Our results of grassland soil samples confirmed that
extractable soil organic C is enriched in 13C compared to
the bulk soil C as reported previously by Flessa et al.
953
(2000). We found that microbial biomass C (chloroform-
labile C) was 13C-enriched by ca. 2‰ compared with the
total soil organic C. The same 13C enrichment in the soil
microbial biomass was described by Šantrůčková et al.
(2000) who analysed 21 tropical and temperate grassland
soils. Ryan et al. (1995) found a difference in d13 C of
1.8‰ between soil microbial biomass C and total soil
organic matter for an arable soil in Canada. Gleixner et al.
(1993) detected d13 C values for basidiomycetes (fungi)
being 1.5 – 6.0‰ higher than their substrate (wood tissue).
Comparable values (3.5‰ enrichment of 13C) were also
obtained for heterotrophic bacteria (Vibrio harveyi) by
Macko and Estep (1984). The 13C enrichment in microbial
biomass C is a result of the isotope discrimination during
biosynthesis of new biomass and isotopic composition of
organic compounds preferentially used by soil microor-
ganisms. The depletion of 13C in the respired CO2
compared with biomass C indicates that catabolic reactions
prefer light isotopes (Šantrůčková et al., 2000). The
increasing enrichment of 13C with soil depth is a result
of this isotope fractionation by soil microflora (Boutton,
1996).
We found a considerable immobilization of maize C in
the microbial biomass 28 days after the incorporation of
maize leaves (60% of microbial biomass C derived from
maize C, Table 2). This observation is in good agreement
with other studies showing that the decomposition of plant
residues is accompanied by a fast C assimilation in the
microbial biomass (Ladd et al., 1995; Trinsoutrot et al.,
2000). However, the immobilization of maize C in soil
microbial biomass represented less than 3.5% of the
added maize C. The incorporation of residue C in the
soil microbial biomass may be useful as an indicator for
the active or zymogenous part of the soil microbial
population (Potthoff et al., 2001).
With reference to a kEC value of 0.45 (Wu et al.,
1990; Joergensen, 1996) d13 C values received by the FE
method are valid for the chloroform-labile C, which
represents 45% of the soil microbial biomass C
(Aoyama et al., 2000). At the moment, it is completely
unknown to what extent d13 C values differ between the
chloroform-labile and the non-labile part of microbial
biomass C, although such differences are likely.
However, d13 C values obtained by FE still give a
characteristic feature of the soil microbial biomass,
since the fumigation methods provide the only direct
access to soil microbial C.
Acknowledgements
We acknowledge Annette Muhs, Anja Becker and Lars
Szwec for technical assistance, Jens Dyckmans and
Bernard Ludwig for several helpful suggestions and the
Deutsche Forschungsgemeinschaft (DFG) for financial
support.
954 M. Potthoff et al. / Soil Biology & Biochemistry 35 (2003) 947–954
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