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33-37, 1996
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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 approach was to compare the relationship between
N rendered extractable by CHCl, fumigation and the C-to-N ratio measured in the flush of the
fumigation--incubation method by recalculating data obtained from the literature. On the basis of these
two approaches, we recommend using a /CEN value of 0.54 as originally proposed by Brookes et al. (Soil
Biology & Biochemistry 17, 837-842, 1985).
arable and grassland soils was analyzed. Secondly, the literature, where Fc is (COYC evolved from
the kEN value was calibrated on the basis of the fumigated soil during the O-10 d incubation period)
relationship between the EN and the C-to-N ratio minus (COz-C evolved from non-fumigated soil
measured in the hush of the FI method with data during the &lo d incubation period) and FN is
obtained by Jenkinson (1988) from the literature. (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
MATERIALS AND METHODS
during the cl0 d incubation period). We took the
Soils results of: 20 soils from Ayanaba et al. (1976); 12 soils
from Powlson and Jenkinson (1976), excluding 1 soil
A group of 74 samples (5 1 arable and 23 grassland of pH 3.9; 6 soils from Ross et al. (1980), excluding
soils) were collected in Lower Saxony, Germany. All 3 soils pH < 4.7; 30 soils from Carter and Rennie
samples were taken from the A horizon, sieved (1982); 10 soils from Adams and Adams (1983),
(< 2 mm), adjusted to a water holding capacity of excluding 6 soils of pH < 4.7; 1 soil from Voroney
40%, conditioned for 10 days at 25°C and stored at and Paul (1984); 17 soils from Carter (1986); and 8
4°C until analysis. Soil conditions were analysed by soils from Nicolardot et al. (1986), excluding one soil
the standard methods described by Joergensen et al. of pH 4.8.
(1995). Precise details of all individual soils have been
described by Joergensen (1995). Some of the soils
Statistics
were used in experiments by Kaiser et al. (1992) and
Joergensen et al. (1992, 1993). Soil properties The X2-test was used to assess the distribution
spanned a wide range. Soil pH(H20) ranged from 4.1 fitting. The results presented are arithmetic means of
to 8.1. Soil organic C content ranged from 5.5 to triplicate analysis (three extractions, one C and N
214.5 mg g-l soil and total N content from 0.53 to measurement in each extract) and expressed on an
19.3 mg g-’ soil. oven dry basis (about 24 h at 105C).
Fumigation-extraction (FE)
RESULTS AND DISCUSSION
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 Indirect calibration by the C-to-N ratio in the extracts
treatments which were extracted with 100 ml 0.5 M of the FE method
KS04. Organic C in the extracts was measured using The EC content ranged from 38.5 to 1336.5 pg g-l
a Dohrman DC 80 automatic analyser (Wu et al., soil and the EN content from 5.99 to 274.3 pg g-i soil.
1990). The EC was (organic C extracted from Both sets of values were significantly correlated
fumigated soil) minus (organic C extracted from (r = 0.98; P < 0.0001) and formed the regression
non-fumigated soil). NH:, NO; and organic N equation EC = 5.28EN (Fig. 1). The mean coefficients
were measured by Kjeldahl analysis according to of variation between the 3 replicates of each soil were
Joergensen and Meyer (1990). A 50-ml aliquot of the +3.0% for EC, *5.9% for EN and f6.3% for the
KSO., extract was pipetted into a distillation flask EC-to-EN ratio. The data used for calibration must fit
and 200 mg of MgO were added to volatize NH, a normal distribution or must be transformed into
under alkaline conditions. When the distillate rose one (Wardle and Parkinson, 1991). If the data fits a
back to the 30-ml mark on the receiver flask log-normal distribution, the conversion values cannot
(containing 5 ml 2% HjBOJ acid solution), the first be taken from the regression coefficient of the
distillation was halted and 200 mg of Devarda alloy log-transformed data, but from the mean which is the
were rapidly added to reduce NO; and NO; to NHX. geometric mean. The EC-to-EN ratio fitted to a
When the distillate again reached the 30-ml mark on log-normal distribution in the 74 soils (Fig. 2) and
the receiver flask, the second distillation was halted. ranged from 3.7 to 8.7 with a geometric mean of 5.55
Then, 10 ml cont. HSO, and 500 mg Se catalyst (Table 1). Joergensen et al. (1992) proposed the
mixture were added and the flask was heated gently following equation to calculate the kENvalue based on
until all water had been removed. Thereafter, the the work of Jenkinson (1988):
mixture was boiled for 3 h until the digest cleared.
After cooling, approximately 40 ml 10 M NaOH were kEN= [B:C(l*kEc,
added for a third distillation. In each of the three
distillates, NH,-N was determined with standard where /3 = biomass C-to-N ratio = 6.7 (Anderson
10 mM HCl by automatic titration to pH 4.8. and Domsch, 1980; Shen et al., 1984)), LY= &-to-&
ratio = 5.55 and kcEc= 0.45 (Jenkinson, 1988;
Calibration with data obtained from the literature Joergensen, 1995, 1996). From that equation, the
The relationship between C flush (Fc) and N flush kEN value can be calculated as follows: kEN =
(FN) of the FI method was reanalysed for the data on [6.7:5.55].0.45 = 0.54. This value is identical to the
104 soils similarly obtained by Jenkinson (1988) from 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 microbial biomass N mineralized to NHcN during
the C-to-N ratio in thefrush of the FI method the incubation period. From that equation, a kN value
Jenkinson (1988) analysed the relationship between of 0.57 can be calculated for the FI method
the C flush (Fc) and N flush (FN) of the FJ method (Jenkinson, 1988). This new kN value was 16.2%
in 104 soils obtained from the literature. A regression smaller than the older kN value of 0.68 (Shen et al.,
coefficient of 5.3 I was calculated from the linear 1984). Brookes et al. (1985) found by means of
relationship between FC and FN after the regression regression analysis the following linear relationship
line was passed through zero. The kN value was between EN and FN:
calculated from the following equation (Jenkinson, Ei., = 0.79.K.
1988):
As consequence, kEN = 0.79.kN and thus a kEN value
kN = [P:cl]&,
of 0.45 was calculated for the FE method by
where /I = bioma,ss C-to-N ratio = 6.7 (Anderson Jenkinson (1988). The kEC and kEN values have been
and Domsch, 1980; Shen et al., 1984), CI= Fc-to-FN identical since Wu et al. (1990) established a kECvalue
ratio = 5.31, kc = 0.45 (Jenkinson, 1988), kc = the of 0.45, i.e. the biomass C-to-N ratio does not differ
fraction of microbial biomass C evolved as CO*-C from the EC-to-EN ratio. This has the advantage of
during the incubation period and kN = the fraction 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