Soil Biol. Biochem. Vol. 28, No. 1, pp.
33-37, 1996
Pergamabn 0038-0717(95)00101-8
THE F’UMIGATION-EXTRACTION METHOD TO ESTIMATE
SOIL MICROBIAL BIOMASS: CALIBRATION OF THE
km VALUE
RAINER GEORG JOERGENSEN*
Institut fiir Bodenwissenschaft, Von-Siebold-Str.
(Accepted
Summary--The HEN value (=extractable part of microbial biomass N after fumigation) of the
fumigation-extraction method was assessed using the C-to-N ratio of the organic matter which was
rendered extractable by CHCln fumigation. The data for this calibration approach was obtained from 51
arable and 23 grassland soils. The second calibration
N rendered extractable by CHCl, fumigation and
fumigation--incubation method by recalculating data
two approaches, we recommend using a /CEN value of
Biology & Biochemistry 17, 837-842, 1985).
INTRODUCTION
In grassland and <arable soils, the microbial biomass
contains on average 3.1% of total N in the O-30 cm
layer ranging from 0.5 to 6.6% (Joergensen, 1995).
When investigating and modelling immobilization
and mobilization processes of N in soil, it is
important to know as exactly as possible the real
amount of this nutrient stored in the soil microbial
biomass. For that purpose, the N fraction rendered
extractable by the fumigationextraction (FE)
method (Brookes et al., 1985) can be converted to
total biomass N according to the equation
biomass N = EN:REN,
where EN is (N extracted with 0.5 M KzS04 from
fumigated soil) minus (N extracted with 0.5 M KS04
from non-fumigated soil) and kENis the extractable
part of microbial biomass N after fumigation.
Brookes et al. (1985) found a kENvalue of 0.54 from
the linear regression between EN and biomass N
values of the fumigation-incubation (FI) method.
The determination of the kENvalue is more difficult
than that of the kEc value, because N content and,
thus, the C-to-N ratio of microorganisms grown
in vitro varies as a function of growth conditions and
growth phase within a large range in contrast to the
roughly constant C content of about 45% (Anderson
and Domsch, 1980; Jenkinson, 1988). However, the
C-to-N ratio of the soil microbial biomass seems to
*Author for correspondence.
tPresent address: Department of Agricultural Sciences,
Section of Soil, Water and Plant Nutrition, The Royal
Veterinary and Agricultural University, 40 Thor-
valdsensvej, 1871 Frederiksberg C (Copenhagen),
Denmark.
SBB 28,1--c
Copyright 0 1996 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0038-0717/96 $15.00 + 0.00
and TORSTEN MUELLER?
4, 37075 Giittingen, Germany
24 July 1995)
approach was to compare the relationship between
the C-to-N ratio measured in the flush of the
obtained from the literature. On the basis of these
0.54 as originally proposed by Brookes et al. (Soil
be less variable as suggested by relatively constant
C-to-N ratios found in the flush of the FI method
(Jenkinson, 1988) or in the extracts of the FE method
(Joergensen et al., 1992, 1993; Joergensen, 1995) after
analysing many soils for agricultural use. One
exception of significant larger biomass C-to-N ratios
are strongly acidified forest soils (Joergensen et al.,
1995).
Some of the experiments were carried out to
calibrate the & value directly (Bremer and van
Kessel, 1990, 1992). In most cases, however, the kEN
value was calibrated indirectly from the relationship
between EN and the biomass N values obtained with
the FI method (Brookes et al., 1985; Jenkinson,
1988). Consequently, the calibration of the kN value
of the FI method must be considered when
calibrating the kEN value. In contrast to the kc value,
the kN value cannot simply be calibrated by the
addition of cultivated microorganisms (Jenkinson,
1988). The kN value is affected by the N content of the
microorganisms; the larger the N content of the
microbial biomass, the smaller the k, value (Voroney
and Paul, 1984; Nicolardot et a/., 1986). For that
reason, the kN value was itself indirectly calibrated by
the C-to-N ratio measured in the flush (= Fc-to-&
ratio) and the assumption that the mean C-to-N ratio
of the microbial biomass is constant at 6.7 in most
soils (Anderson and Domsch, 1980; Shen et al.,
1984).
We have compared two different approaches for
indirect calibration, both based on the work of
Jenkinson (1988). Firstly, the kEN value was calibrated
on the basis of the C-to-N ratio found in the extracts
of the FE method (= EC-to-EN ratio) as proposed by
Joergensen et a/. (1992), in which a large group of
33
34 Rainer Georg Joergensen and Torsten Mueller
arable and grassland soils was analyzed. Secondly,
the kEN value was calibrated on the basis of the
relationship between the EN and the C-to-N ratio
measured in the hush of the FI method with data
obtained by Jenkinson (1988) from the literature.
MATERIALS AND METHODS
Soils
A group of 74 samples (5 1 arable and 23 grassland
soils) were collected in Lower Saxony, Germany. All
samples were taken from the A horizon, sieved
(< 2 mm), adjusted to a water holding capacity of
40%, conditioned for 10 days at 25°C and stored at
4°C until analysis. Soil conditions were analysed by
the standard methods described by Joergensen et al.
(1995). Precise details of all individual soils have been
described by Joergensen (1995). Some of the soils
were used in experiments by Kaiser et al. (1992) and
Joergensen et al. (1992, 1993). Soil properties
spanned a wide range. Soil pH(H20) ranged from 4.1
to 8.1. Soil organic C content ranged from 5.5 to
214.5 mg g-l soil and total N content from 0.53 to
19.3 mg g-’ soil.
Fumigation-extraction (FE)
The FE method was performed according to
Brookes et al. (1985) and Vance et al. (1987) using
25 g for the fumigated and 25 g for non-fumigated
treatments which were extracted with 100 ml 0.5 M
KS04. Organic C in the extracts was measured using
a Dohrman DC 80 automatic analyser (Wu et al.,
1990). The EC was (organic C extracted from
fumigated soil) minus (organic C extracted from
non-fumigated soil). NH:, NO; and organic N
were measured by Kjeldahl analysis according to
Joergensen and Meyer (1990). A 50-ml aliquot of the
KSO., extract was pipetted into a distillation flask
and 200 mg of MgO were added to volatize NH,
under alkaline conditions. When the distillate rose
back to the 30-ml mark on the receiver flask
(containing 5 ml 2% HjBOJ acid solution), the first
distillation was halted and 200 mg of Devarda alloy
were rapidly added to reduce NO; and NO; to NHX.
When the distillate again reached the 30-ml mark on
the receiver flask, the second distillation was halted.
Then, 10 ml cont. HSO, and 500 mg Se catalyst
mixture were added and the flask was heated gently
until all water had been removed. Thereafter, the
mixture was boiled for 3 h until the digest cleared.
After cooling, approximately 40 ml 10 M NaOH were
added for a third distillation. In each of the three
distillates, NH,-N was determined with standard
10 mM HCl by automatic titration to pH 4.8.
Calibration with data obtained from the literature
The relationship between C flush (Fc) and N flush
(FN) of the FI method was reanalysed for the data on
104 soils similarly obtained by Jenkinson (1988) from
the literature, where Fc is (COYC evolved from
fumigated soil during the O-10 d incubation period)
minus (COz-C evolved from non-fumigated soil
during the &lo d incubation period) and FN is
(NH,-N + NO,-N mineralized in fumigated soil
during the O-10 d incubation period) minus (NH.+-
N + NO,-N, mineralized in non-fumigated soil
during the cl0 d incubation period). We took the
results of: 20 soils from Ayanaba et al. (1976); 12 soils
from Powlson and Jenkinson (1976), excluding 1 soil
of pH 3.9; 6 soils from Ross et al. (1980), excluding
3 soils pH < 4.7; 30 soils from Carter and Rennie
(1982); 10 soils from Adams and Adams (1983),
excluding 6 soils of pH < 4.7; 1 soil from Voroney
and Paul (1984); 17 soils from Carter (1986); and 8
soils from Nicolardot et al. (1986), excluding one soil
of pH 4.8.
Statistics
The X2-test was used to assess the distribution
fitting. The results presented are arithmetic means of
triplicate analysis (three extractions, one C and N
measurement in each extract) and expressed on an
oven dry basis (about 24 h at 105C).
RESULTS AND DISCUSSION
Indirect calibration by the C-to-N ratio in the extracts
of the FE method
The EC content ranged from 38.5 to 1336.5 pg g-l
soil and the EN content from 5.99 to 274.3 pg g-i soil.
Both sets of values were significantly correlated
(r = 0.98; P < 0.0001) and formed the regression
equation EC = 5.28EN (Fig. 1). The mean coefficients
of variation between the 3 replicates of each soil were
+3.0% for EC, *5.9% for EN and f6.3% for the
EC-to-EN ratio. The data used for calibration must fit
a normal distribution or must be transformed into
one (Wardle and Parkinson, 1991). If the data fits a
log-normal distribution, the conversion values cannot
be taken from the regression coefficient of the
log-transformed data, but from the mean which is the
geometric mean. The EC-to-EN ratio fitted to a
log-normal distribution in the 74 soils (Fig. 2) and
ranged from 3.7 to 8.7 with a geometric mean of 5.55
(Table 1). Joergensen et al. (1992) proposed the
following equation to calculate the kENvalue based on
the work of Jenkinson (1988):
kEN= [B:C(l*kEc,
where /3 = biomass C-to-N ratio = 6.7 (Anderson
and Domsch, 1980; Shen et al., 1984)), LY= &-to-&
ratio = 5.55 and kcEc= 0.45 (Jenkinson, 1988;
Joergensen, 1995, 1996). From that equation, the
kEN value can be calculated as follows: kEN =
[6.7:5.55].0.45 = 0.54. This value is identical to the
kEN value of 0.54 proposed by Brookes et al. (1985).
 The FE method: calibration of the /CEN
value 35

EN [vgg-’ soil]
Fig. I. The linear relationship between EN and EC in 74 grassland and arable soils measured by the FE
method: EC = 5.28,&.

Indirect calibration by the relationship between EN and
the C-to-N ratio in thefrush of the FI method
Jenkinson (1988) analysed the relationship between
the C flush (Fc) and N flush (FN) of the FJ method
in 104 soils obtained from the literature. A regression
coefficient of 5.3 I was calculated from the linear
relationship between FC and FN after the regression
line was passed through zero. The kN value was
calculated from the following equation (Jenkinson,
1988):
kN = [P:cl]&,
where /I = bioma,ss C-to-N ratio = 6.7 (Anderson
and Domsch, 1980; Shen et al., 1984), CI= Fc-to-FN
ratio = 5.31, kc = 0.45 (Jenkinson, 1988), kc = the
fraction of microbial biomass C evolved as CO*-C
during the incubation period and kN = the fraction of
microbial biomass N mineralized to NHcN during
the incubation period. From that equation, a kN value
of 0.57 can be calculated for the FI method
(Jenkinson, 1988). This new kN value was 16.2%
smaller than the older kN value of 0.68 (Shen et al.,
1984). Brookes et al. (1985) found by means of
regression analysis the following linear relationship
between EN and FN:
Ei., = 0.79.K.
As consequence, kEN = 0.79.kN and thus a kEN value
of 0.45 was calculated for the FE method by
Jenkinson (1988). The kEC and kEN values have been
identical since Wu et al. (1990) established a kECvalue
of 0.45, i.e. the biomass C-to-N ratio does not differ
from the EC-to-EN ratio. This has the advantage of
affording an immediate access to the data measured

25

20
‘-:
0

z 15
6
s
‘: 10
Cz
IL
5

0
3.0 3.6 4.6 5.4 6.2 7.0 7.6 6.6 9.4
E~:EN
Fig. 2. Frequency distribution of the &-to-& ratio in 74 grassland and arable soils measured by the FE
method.
36 Rainer Georg Joergensen and Torsten Mueller

Table I. Direct and indirect calibration of the &ENvalue method. A kEN value of 0.55 can be obtained from the
Author km Range No. of soils kN value of 0.70 according to the equation
Direct kEN = 0.79.kN. For that reason, the data on the 104
hr situ/ahe//ingby theaddition of “N-labelled substrate
Azam rt al. (1989)* 0.26 0.17-0.42 I, = I
soils obtained from the literature do not demand the
Bremer and van Kessel (1990) 0.24 fl=l change of the kEN value proposed by Brookes et al.
Bremer and van Kessel (1992) 0.21 II = I (1985).
Indirect
CN Direct calibration by in situ labelling
Brookes et al. (1985) 0.54 n = 37
Jenkinson (1988) 0.45 n= 104
The direct calibration of the kEN value with in situ
Joergensen and Muellert 0.55 0.32-0.88 ?I=104 labelling of the microbial biomass was carried out by
G-to-Eb adding of 15N-labelled (NH&SO, and glucose
Joergensen and Mueller: 0.54 0.35-0.81 n = 74
followed by a short period of incubation (Bremer and
*Five varying concentrations of (‘INH&S04 (67-333 pg g-’ soil)
and glucose (C-to-N ratio of 30 at each addition rate).
van Kessel, 1990). The total biomass “N was (amount
iRecalculation of the data obtained by Jenkinson (1988) from the of 15N added) minus (15N0,-N + “NH4-N extracted
literature, see Fig. 3. with 0.5 M K2S04 from non-fumigated soil), ElsN was
:This work, see Fig. 1 and Fig. 2.
(“N extracted with 0.5 M KS04 from fumigated soil)
minus (15N extracted with 0.5 M K1S04 from
non-fumigated soil) and kEN = EISN-to-total biomass
in the extracts. However, the C-to-N ratio of the 15N ratio. This procedure is based on the validity of
complete microbial cell is usually larger than that of the following two assumptions analogous to the
the extractable part, which consists mainly of N-rich direct calibration by in situ ‘%-labelling: (1) the part
cytoplasm. A C-to-N ratio of 7.6 was found by of 15N non-extractable as NO; or NH: after
Nicolardot (1986) in an intact cell of Aspergillus incubation is completely incorporated into the
~‘Iuvus, but a C-to-N ratio of only 5.6 in the microbial biomass and only a negligible part is
cytoplasm. Consequently, the kEC and kEN value must transformed to non-biomass microbial metabolites;
be different. and (2) the extractability and the C-to-N ratio of the
The data from the literature sampled by Jenkinson young ‘5N-labelled biomass must be representative of
(1988) were recalculated to prove his kN value of 0.57 total microbial biomass N.
obtained by linear regression analysis of non-trans- Both assumptions are very likely wrong. Voroney
formed data. The recalculation showed that not only and Paul (1984) have shown that the C-to-N ratio of
FC and FN, but also the F&to-F, ratio were microflora grown on large amounts of glucose can be
log-normally distributed (Fig. 3). The soils with large doubled in comparison to the natural microbial
FC contents often had a relatively large Fc-to-FN ratio population. The assumption that the extractability of
and, thus, overproportionately affected the regression the 15N-labelled biomass is not representative of the
coefficient of the linear relationship between Fc and total microbial biomass N is supported by the work
FN. of Azam et al. (1989). They found an average kEN
The geometric mean of the Fc-to-FN ratio was 4.32 value of 0.26 after adding five varying concentrations
in the 104 soils obtained from the literature, whereby of (‘5NH&S04 and glucose at a C-to-N ratio of 30
the equation kN = [b:cx].kc yields a kN value of 0.70, (Table 1). Bremer and van Kessel (1990, 1992)
very similar to that of Shen et al. (1984) for the FI obtained k,, values approximately 30 and 13%

35r

28
_ I-

1.8 2.5 3.2 3.9 4.8 5.3 8.0 6.7 7.4 9.1
Fc:FN
Fig. 3. Frequency distribution of the &to-& ratio measured in 104 soils with the FI method (Jenkinson,
1988; Joergensen, 1995).
 The FE method: calibration of the DENvalue 37

smaller than the respective kEc values by using the Carter M. R. and Rennie D. A. (1982) Changes in soil
direct calibration approach. quality under zero tillage farming systems: distribution of
microbial biomass and mineralizable C and N potentials.
This means that the C-to-N ratio of the microbial Canadian Journal of Soil Science 62, 587-597..
biomass portion that is non-extractable by KZS04 Jenkinson D. S. (1988) The determination of microbial
must be considerably smaller than that of the biomass carbon‘ and’ nitrogen in soil. In Advances in
extractable, which is extremely unlikely as shown by Nitrogen Cycling in Agric&ral Ecosystems (J. R. Wilson,
Ed.). vv. 368-386. CAB International. Wallinaford.
Nicolardot (1986) The kEN values found by in situ .I

Joergensen R. G. (1995) Die quantitative Beitimmung

labelling with 15N are only half of the indirectly der mikrobiellen Biomasse in Bidden mit der Chloroform-
estimated values (Table 1). This points to the fact that Fumigations-Extraktions-Methode. Gdttinger Boden-
after a short time significant parts of N are kundliche Berichte. In press.
Joergensen R. G. (1996) The fumigation+xtraction method
transferred to the microbial residual mass which is
to estimate soil microbial biomass: calibration of the REC
composed of exoenzymes, exudates and necromass. value. Soil Biology &IBiochemistry 28, 25-31.
These observations are supported by considerably Joergensen R. G. and Meyer B. (1990) Nutrient changes in
larger REPvalues measured by Bremer and van Kessel decomposing beech leaf litter assessed using a solution
(1990) 6-12 h after substrate addition, which reached flux approach. Journal of Soil Science 41, 279-293.
Joeraensen R. G.. Mever B. and Mueller T. (1992) Zeiteane
a maximum value of 0.54. The kEN value was de; mikrobiellen i(iomasse in der Acierkrime hner
apparently affected by the incubation time after mitteleuroplischen L&s-Parabraunerde. G&ringer Bo-
substrate addition, by the amount of “N and glucose denkundliche Berichre 100, l-140.
added and also by the C-to-N ratio of the added Joergensen R. G., Kiibler H., Meyer B. and Welters V.
(1993) Die Beziehungen von mikrobiell gebundenem C, N
substrate. However, maximum kENvalues were found
und P in Acker- und GraslandbGden. VDLUFA-
at the lowest (Bremer and van Kessel, 1992) and the Schriftenreihe 37, 189-192.
largest (Azam et al., 1989) concentration of added 15N Joergensen R .G., Anderson T.-H. and Walters V. (1995) C
and glucose. We conclude from the results of these and N relationships of the soil microbial biomass in soils
direct calibration experiments that in situ labelling is of beech (Fagus syhatica L.) forests. Biology and Fertility
of Soils 19, 141-147.
unsuitable for obt,aining a reliable kENvalue for the Kaiser E.-A., Mueller T., Joergensen R. G., Insam H. and
FE method (see Joergensen, 1996). Heinemeyer 0. (I 992) Evaluation of methods to estimate
the soil microbial biomass and the relationship with soil
texture and organic matter. Soil Biology & Biochemistry
Acknowledgements-We would like to thank Dr Helga
24, 675-683.
Kiibler, Ingrid Ostermeyer and Karin Schmidt for their help Nicolardot B. (1986) &de comparte de la mineralisation
with the experiments,. de diffkrentes fractions cellulaires d’dspergillusflarus dans
un sol fumigi ou non au chloroforme. C.r. Academic
Science de Paris 303(111), 489494.
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