Soil Bid. Biocfrem.

Vol.

17, No.

6, pp. 837-842, 1985

0038-0717/85$3.00+ 0.00

Printed in Great Britain

Pergamon Press Ltd

CHLOROFORM
FUMIGATION
AND THE RELEASE OF
SOIL NITROGEN: A RAPID DIRECT EXTRACTION
METHOD TO MEASURE MICROBIAL BIOMASS
NITROGEN IN SOIL
P. C. BROOKES, ANDREA LANDMAN,* G. PRUDEN* and D. S. JENKINSON
Soils and Plant Nutrition Department, Rothamsted Experimental Station, Harpenden,
Her&. AL5 214, U.K.
(Accegted 14 MqJ 1985)
Summary-A new direct extraction method for measuring soil microbial biomass nitrogen (biomass N)
is described. The new method (fumigation-extraction)
is based on CHCI, fumigation, followed by
immediate extraction with 0.5 M &SO, and measurement of total N released by CHCl, in the soil extracts.
The amounts of NH,-N and total N extracted by K,SO, immediately after fumigation increased with
fumigation time up to 5 days. Total N released by CHClr after 1 day fumigation (1 day CHCIr-N) and
after 5 days fumigation (5 day CHCl,-N) were positively correlated with the flush of mineral N (FN) in
37 soils that had been fumigated, the fumigant removed and the soils incubated for 10 days
(fumigation-incubation).
The regression equations were 1 day CHCI,-N = (0.79 & 0.022) F, and 5 day
CHCl,-N = (1.01 & 0.027) F,, both regressions accounting for 92% of the variance in the data.
In field soils previously treated with r5N-Iabelled fertilizer, the amounts of labelled N, measured after
fumigation-extraction,
were very similar to the amounts of labelled N mineralized during
fumigation-incubation; both were about 4 times as heavily labelled as the soil N as a whole. These results
suggest that fumigation~xtraction
and fumigation-incubation both measure the same fraction of the soil
organic N (probably the cytoplasmic component of the soil microbial biomass) and that measurement of
the total N released by CHCI, fumigation for 24 h provides a rapid method for measuring biomass N.

INTRODUCTION

that the increased biochemical activity in a moist soil following CHCl, furnigation, fumigant removal and aerobic incubation is
due to the decomposition of the cells killed by the
fumigant by the recolonizing microbial population.
Under most conditions, the decomposability
of other
soil organic fractions is little, if at all, affected by
CHCI, fumigation (Jenkinson and Powlson, 1976).
The Rush of N, (FN), defined as the N mineralized
by a previously fumigated soil incubated for 10 days
under standard conditions, less that mineralized by a
similarly incubated, but non-fumigated control soil,
can be used to estimate soil microbial biomass N on
the basis that 68% of the N in the original biomass
is mineralized (i.e. k, = 0.68) during this period (Shen
et al., 1984). Voroney and Paul (1984) used a comparable approach, differing however in two important aspects. Firstly, they considered that the total
It is now well established

amount of N mineralized during aerobic incubation

of a fumigated
soil was derived from the original

biomass, i.e. they did not subtract a control and

secondly, that k, was lower (~0.4) and variable,
depending on the amount of N immobilized by the
recolonizing population.
Chloroform fumigation of soil causes a small immediate increase in 0.5 M K2 SC&-extractable NH,-N
(Jenkinson and Powlson, 1976) and a much larger
*Present address: Department of Soil Science, Agricultural
University. De Dreijen 3, 6703 BC Wageningen, The
Netherlands.
* Deceased.

increase in total (i.e. inorganic

and organic)
K,SO,-extractable N (Brookes et al., 1985). Further
release of N&-N and total N occur up to 5 days
fumigation. Brookes et al. (1985) suggested that the
fumigant increased total extractable N by lysing
living soil organisms, and that the extractability of
other soil fractions to K,SO, was little, if at all,
affected by the fumigant. In this paper we examine
the relationships between the flush of N (FN) in a
range of previously fumigated and aerobically incubated soils (fumigation-incubation)
and the total N
released by CHCl, and extracted
by &SO,
fumigation
immediately
after
(CHCl,-N)
(fumigation-extraction).
CHCl,-N is defined as the
amount of total (i.e. inorganic + organic) N extracted
by 0.5 M &SO, from soil immediately after fumigation fess that extracted from a non-fumigated soil
at the time fumigation commenced. Soils were exposed to CHCl, for either 1 day or 5 days in the
fumigation-extraction
procedure, 5 days being chosen because previous work (Brookes et al., 1985)
showed that little extra N was released by fumigating
For longer than 5 days. The amounts of CHCI,-N
reieased after 1 and 5 days fumigation are termed
1 day CHCl,-N and 5 day CHC&-N respectivety.
Our aim was to develop a new direct extraction
method for measuring biomass N. It is based on the
amount of N extracted by 0.5 M K*SO., after a
24 h CHCI, fumigation, less that extracted by K,SO,
from unfumigated soils. Such a method would remove the need for aerobic incubation, sidestep the
control issue of the approaches of Shen et al.
(1984) and Voroney and Paul (1984) and avoid the
problems caused by denitrification
and immo-

&38

P. C.

BROOKES et

al.

bilization of N in both the control and fumigated

samples during aerobic incubation (Jenkinson and
Powlson, 1976).
MATERIALS

AND METHODS

The soils, selected to encompass

a wide range
of agricultural systems, were mainly from two soil
series, Batcombe and Cottenham (Table I) Each
sample, consisting of IO or more separate soil cores
(5 em dia) was taken from O-10, 10-23 or O-23 cm
depth. Replicate samples from the same plot are
given alphabetical
suffixes in Tables 1-3. The
5N-labelled soils used came from microplots (Jenkinson et al., 1983) on the Park Grass permanent
grassland experiment at Rothamsted (Warren and
Johnston, 1964). Six of the microplots were located
on plot 9-b of the main experiment, which is limed
and receives 96 kg N ha- as (NH,),%& annually:
three of these microplots received 93.7 kg labeiled N
ha- as (NH&SO, (4.90 atom% 15Nexcess) in April
1980, 2yr before sampling (soils 7A-C), the others
100.3 kg N ha- as (NH,),SO,
(4.81 atomo/:, rSN
excess) in April 1981, 1 yr before sampling (soils
7D-F). The other six microplots were located on plot
14-d of the main experiment, which is unlimed and
receives 96 kg N ha- annually as NaNO,; three of
these microplots received 93.7 kg N ha- as KNO?
(4.85 atom% 15N excess) in April 1980 (soils 8A-C),
2 yr before sampling, the other three 97.0 kg N ha-
as KNO, (4.82 atom% $N excess) in April 1981, 1 yr
before sampling (soils X-F). These Park Grass soils,
stored at - 15X, were thawed before use, then
treated as described below. All results are given on an
oven-dry soil basis (lOSC, 24 h) and, unless otherwise stated, are the mean of triplicate determinations.

Soil freafmenfs

All soils were sieved (< 2 mm) and given a preliminary incubation in bulk at 40% water holding
capacity (WHC) for 7 days before use (Brookes et al.,
1985). Soil samples used for 1 day CHCl,-N and for
F, determinations were fumigated simultaneously at
40% WHC with alcohol-free CHCI, for 24 h (or for
5 days for 5 day CHCl,-N) before extraction or incubation. Extraction for 1 and 5 day CHCl,-N was
done with K,SO, immediately after fumigation and
without fumigant removal; samples used to determine
initial total and inorganic (i.e. NH, and NO,-N) N
were extracted at the time fumigation commenced.
For F, measurement, the fumigant was removed, the
soil moisture adjusted to 50% WHC and the soils
then incubated for 10 days at 25C before extraction
with 0.5 M K,SO, for measurement of extractable
NH,-N and NO,-N (Jenkinson and Powlson, 1976;
Shen et al., 1984). Methods for measuring CHCI,-N,
NH,-N, N03-N, total N and total C in soil are fully
described by Brookes et al. (1985).
RESULTS

AND DISCUSSION

The re~afionship between the flush of N (FN) and the

q~nf~fy of N released by CHCI, and extracted by
K2S0, (Cycle-~)

Table 2 gives the inorganic N extracted by

0.5 M K2S0, from the 37 soils before and after

by 0.5 M K,SO,

8.2
8.0
22.5
13.2
7.5
17.5
22.8
8.4
5.8
26.6
38.0
30.5
39.3
38.3
31.6
34.2
41.3
38.5
37.3
33.3
39.8
16.7
69.0
18.1
14.9
3.7
4.2
2.6
2.8
7.8
9.7
7.9
5.7
8.8
8.5
7.3
9.3

7.6
8.0
18.1
8.4
0.5
9.6
9.1
0.9
1.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
14.5
32.3
4.2
13.6
3.7
4.2
2.6
2.4
6.9
8.0
7.1
4.8
5.8
7.6
6.5
7.6

N extracted at
time 0
NH,-N + NO,-N
NO,-N

Mineral

N extracted

13.6
13.5
30.0
14.9
8.9
34.9
34.2
10.1
10.9
33.3
44.0
36.9
42.0
42.1
39.1
43.6
49. I
48.5
49.1
40.2
52.5
25.5
97.7
40.6
21.2
8.5
9.4
8.2
6.5
14.5
24.1
13.6
13.4
13.7
14.7
15.6
15.1

NH,-N + NO,-N
13.1
13.1
26.4
10.4
0.3
16.6
10.5
0
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
23.3
76.9
6.4
19.9
8.5
9.0
6.1
6.1
14.1
21.2
12.7
12.1
13.3
14.7
15.2
14.7
48.7
46.4
166.1
175.3
100.8
154.9
150.9
28.8
31.6
79. I
93.5
81.8
91.7
93.2
86.6
89.9
96.8
97.5
95.1
91.1
100.4
82.2
131.6
108.1
65.3
20.3
20.9
16.9
14.0
34.3
45.7
37.4
35.6
32.3
37.1
41.1
42.2

NH,-N + NO,-N

Fumigated

after 10 day incubation

NO,-N

soil

N extracted

Non-fumigated

Mineral

from soils before and after fumigation-incubation

soil

6.7
5.1
11.5
0.8
0.5
2.0
1.4
0
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8.4
35.8
3.4
7.1
3.6
4.6
4.0
3.1
5.9
7.8
7.1
7.9
5.0
6.5
5.7
6.1

NO,-N

by 0.5 M K,SO,

9.7
6.8
17.8
15.7
21.9
35.8
38.1
25.7
20.6
36.0
37.5
39.7
43.1
42.2
41.7
36.9
40.7
29.7
35.4
36.8
41.6
9.7
45.2
22.5
7.3
4.5
7.2
6.6
4.9
7.0
8.3
10.0
7.8
8.4
7.0
5.7
6.6

Total N extracted from

non-fumigated
soil at
time 0

and total N extracted

Total amounts of (NH, + NO,)-N extracted. corrected for denitrification

where possible by adding (difference between actual NO,-N
after fumigation incubation) to actual amount of NH,-N and NO,-N extracted from fumigated soil after fumigation-incubation.
ND = Not determined.

IA
1B
2
3
4
5A
5B
6A
6B
7A
7B
7c
7D
7E
IF
8A
88
SC
SD
SE
SF
9
10
II
12
13A
13B
14A
14B
l5A
15B
16A
168
17A
17B
18A
18B

Soil
sample
no.

Table 2. Mineral

initially

and actual NO,-N

extracted

from fumigated

37.3
36.9
139.9
165.6
149.5
141.6
141.6
50.9
47.3
96.9
97.7
89.9
95.5
91.9
89.4
91.6
90.7
88.1
87.9
95.2
91.8
70.0
93.1
83.9
53.7
16.4
13.8
12.0
10.0
29.9
35.5
31.3
29.9
37.1
34.4
40.4
38.5

40.0
37.3
110.9
126.3
123.7
136.7
127. I
43.3
42.5
81.3
82.5
78.4
78.9
78.3
80.6
76.7
80.5
69.5
78.5
81.3
82.7
55.4
84.2
66.0
42.2
13.1
11.9
13.7
12.0
25.7
37.9
19.3
27.5
18.1
22.9
31.5
25.0
extracted

Total N extracted after

5 day CHCI, fumigation

soil

(pg N g- soil)

Total N extracted after

I day CHCI, fumigation

from soils before and after I or 5 day fumigation

840

P. C.

BROOKESet

fumigation-incubation.
It also gives total N extracted
by I&SO, from the same soils, again immediately
before fumigation, or after a 1 or a 5 day fumigation.
From these data, 1 day CHCl,-N, 5 day CHCI,-N
and F,, defined as [(NHI-N f NO,-N extracted
from fumigated soil after 10 days aerobic incubation) - (NH4-N + N03-N extracted from similarly
incubated non-fumigated soil)], were calculated. If
denitrification occurred during the incubation of the
fumigated soil (and if the necessary NO3-N measurements were available) FN was corrected by adding (dzfirence between NO,-N extracted initially
and NO,-N extracted from fumigated soil after
fumigation-incubation)
to (actual amount of NH,-N
and NO,-N extracted from fumigated soil after
fumigation-incubation).
Both 1 day CHCl,-N and 5 day CHCl,-N were
closely correlated with the N mineralized from the
biomass (FN) in 37 soils during fumigationincubation (Fig. la and b). Both regressions accounted for 92% of the variance in the data and the
intercepts were not significantly different from zero.
The regression equations (calculated with the intercepts fitted through zero) were:
1 day CHCl,-N = (0.79 k 0.022) FN.

(11

5 day CHCl,-N = (1 .Ol _t 0,027) FN.

(2)

al,

non-unifo~ly
iabelled. About 0.25% of total soil N
was SN-Iabelled in plot 9-b and about 0.35% in plot
14-d, irrespective of whether the labelled fertilizer had
been applied 1 or 2 yr previously.
For a given soil (with one exception, see later) the
ratio of (labelled N)-to-(unlabelled N) was similar in
F,, 1 day CHCl,-N and in 5 day CHCl,-N. However
(again with the same exception) this ratio was considerably greater in plots that had received N-1abelled
fertilizer 1 yr before than in plots that had received it
2yr before. These results again suggest that
fumigation-incubation
and fumigation-extraction
both attack the same small fraction of soil N, a
fraction much more N-enriched than the bulk soil
N. Futhermore, this labelled fraction is much more
sensitive to when the labelled fertilizer was added
than is the labelled fraction of the total soil N. These
observations are thus consistent with the hypothesis
that F,, 1 and 5 day CHCl,-N all come from the
same fraction of the soil N, probably the cytoplasmic
(McGill et al., 1981) part of the soil microbial
biomass.
The labelled N content of F, was anomalously low
in soil SD-F (0.46 1_1g
N g- soil). The reasons for this
150

la)

These results show that the amount of CHCl,-N

extracted from soil after .5days fumigation is virtually
the same as the amount of N mineralized from the
biomass (FN) during fumigation-incubation
and that
1 day CHCl,-N accounts for about 79% of F,. This
suggests that both methods are measuring the same
pool of soil-N. Further support for this suggestion is
given in the next section.
Relationship
between
iv
mineralized
during
fumigation-incubation and CHCl, - 15N
When N-1abelled fertilizer N is applied to a crop,
some of it will be rapidly incorporated into the soil
biomass, which itself becomes N-enriched. However
a single pulse of labelled N will not unf~ormly label
the whole of the soil N and different fractions will be
Iabelled to different extents. A non-uniformly labelled
soil can therefore be used to see if two different
isolation procedures are in fact attacking the same
fraction. If both remove the same quantity of N,
containing the same proportion of label, there is a
strong presumption that both are attacking the same
fraction (Brookes et al., 1985).
Grassland soils that had received N-1abelled fertilizer 1 or 2 yr before sampling were used. Total and
labelled N were measured in the F, and CHCl,-N (1
and 5 day) fractions (Table 3). As in the preceding
section, unlabelled F, was a little greater than unlabelled 1 day CHCl,-N and a little less than unlabelled S-day CHCI,-N. The results show that the
amounts
of labelkd
N mineralized
during
fumigation-incubation
or
released
during
fumigationextraction
were comparable, although,
generally, labelled 5 day CHCl,-N tended to be
slightly greater than labelled F,. The ratio labelled
N-to-unlabelled
N was consistently and markedly
lower in the whole soil N than in F,, 1 day CHCl,-N
or 5 day CHCl,-N, showing that the soil was indeed

100

50

FNipgN

200

150

g-soil)

Fig. l(a). Relationship between the

ing fumigation-incubation
and N
fumigation. 1 day CHCl,-N = (0.79
bols see Table

flush of N (FN) followreleased after a 1 day

+ 0.022) F,. For sym1.
Y

150

(b)
=
.03

.
/

P
0

50

100

150

200

Fn @gN g-soil)
Fig. l(b). Relationship between the flush of N (FN) following fumigation-incubation
and N released after a 5 day
fumigation. 5 day CHCl,-N = (1 .OI + 0.027) F,. For symbols see Table 1.

Measurement
Table 3. Labelled and u&belled

Soil
sample
IIO.

Years between
addition of
labelled
fertilizer and
sampling

of microbial

N in total soil N, F,,

Fraction

biomass

841

1 day CHCI,-N and 5 day CHCI,-N)

Labelled N
(pg N g- soil)

Unlabelled N
(pegN gg soil)

Labelled N
unlabelled N

IA-C

Total N in soil
F,
I day CHCl,-N
5 day CHCl,-N

6.71
0.44
0.53
0.62

2810
46.8
37.9
50.8

0.0024
0.010
0.014
0.012

7&F

Total N in soil
F,
1 day CHCl,-N
5 day CHCl,-N

7.39
0.73
0.70
0.89

2830
48.2
37.6
50.4

0.0026
0.015
0.019
0.018

8A-C

Total N in soil
F,
1 day CHCI,-N
5 day CHCl,-N

7.27
0.62
0.48
0.53

2070
48.1
39.2
54.4

0.0035
0.013
0.012
0.010

SD-F

Total N in soil
F,
1 day CHCI,-N
5 day CHCI,-N

6.13
0.46
0.78
0.79

2000
46.7
38.9
48.1

0.0034
0.010
0.020
0.016

0.14

5.1

0.0038

LSD between
F, and CHCI,-N,
within soil. within
year (P = 6.05)

All measurements in this Table are means of single determinations on soils from each of the three replicate plots
((A-C) or (D-F)) that received a particular treatment with sN-labelled fertilizer.

are not known with certainty although it could be

related to the fact that sample 8D-F, unlike all the
others, had been thawed and refrozen once previously
before use.
Relationship
content

between

Measurement
of
fumigation-incubation

CHCI,-N

and soil biomass

soil biomass
N (BN) by
is based on the relationship
B, = P,ik,,

(3)

where F, is the N mineralized from the biomass and

k, is the fraction of biomass N mineralized, both
measured under standard conditions. We currently
use a k, of 0.68 and subtract a control for biomass
N measurements by fumigation-incubation
(Shen et
al., 1984). Biomass N can then be calculated from
5 day CHCl,-N measurements by combining equations (1) and (3).
B, = F,/0.68 = [(5 day CHCl,-N)/l.01]/0.68
= (5 day CHCl,-N)/0.69.

(4)

However it is more convenient to measure biomass

N by fumigationextraction
after a 24 h fumigation,
even though 1 day CHCl,-N is only 79% of the N
mineralized during fumigation-incubation.
B, is then
calculated from equation (2) and (3).
B, = F,/0.68 = [( 1 day CHCl,-N)/0.79]/0.68
= (1 day CHCl,-N)/0.54.

(5)

The one to one relationship between F, and 5 day

CHC&-N in equation (2) strongly suggests that k,
was much the same in the various soils examined. The
actual value for k, (0.68) used in deriving equations
(4) and (5) is perhaps controversial. It is based on
consideration of the measured relationship between
Fc and F, and the likely C-to-N ratio of the soil

microbial biomass (Shen et al., 1984) and is greater

than values obtained by allowing organisms grown in
vitro to decompose in soil. Thus, Jenkinson and
Powlson (1976) found kN for six bacteria, two yeasts,
one actinomycete and one earthworm species to
range from 0.39-0.59; there was no net mineralization of added fungal-N. Adams and Laughlin
(1981) also reported no net mineralization of fungal
N, and calculated an average k, of 0.32 (range
0.30-0.58) for yeasts and bacteria. These values were
all based on a lo-day incubation, whereas Marumoto
et al. (1982) suggested a value of 0.37 for a 28-day
incubation. The difference between our value of 0.68
and these much lower values measured in vitro suggests that the N in soil organisms may be more
completely mineralized during a IO-day incubation
than the N in organisms grown in vitro.
Low (and variable) values of kN were also obtained
by Voroney and Paul (1984) who measured k, in a
soil incubated with glucose and NO,-N. Taking F, to
be the N mineralized from the fumigated soil alone,
they found that kN varied from 0.2 (1 day of incubation) to 0.3 (day 42), based on the assumption of
of N immobilized
during
variable
amounts
fumigation-incubation.
It is likely that the relationship between F, and CHCl,-N in equations (1)
and (2) breaks down in soils that have recently
received large quantities of substrate.
The fumigation-extraction
microbial biomass N

method for measuring soil

The fumigation-extraction
method has certain adover biomass
N measurements
by
vantages
fumigation-incubation.
The problems associated
with the incubation stage-the
need for complete
fumigant removal and for a prolonged incubation
under carefully controlled conditions do not arise. It
also sidesteps the issue of whether or not to use a

842

P. C. BROOK3s et al.

control incubation (Voroney and Paul, 1984). No

correction
is necessary for N losses due to
denitrification because there is no incubation. Similarly, where immobilization of N is a problem in
or
fumigated
soils
during
either
control
fumigation-incubation
measurements (e.g. soils that
have recently received fresh substrate of wide C-to-N
ratio), fumigationextraction
measurement
could
well be a more reliable approach for biomass N
measurement.
Other advantages are that biomass N can be
measured immediately after a 24 h fumigation, compared to the 11 days (24 h fumigation and 10 days
aerobic
incubation)
required
for fumigationincubation. Thus it is easy to follow daily changes if
required, a somewhat cumbersome procedure with
fumigation-incubation.
It also seems probable that
fumigation-extraction
will give trustworthy biomass
N measurements in situations where fumigationincubation generally does not give reliable results, for
example in freshly sampled soils, in acid soils or in
soils that have recently received fresh substrates such
as straw. However, these possibilities should be
treated with caution until thoroughly tested, as such
soils were deliberately excluded in establishing the
close relationship between F, and CHCl,-N (Figs la
and b).
Relationship
nitrogen

between

biomass

nitrogen

and total soil

Soil biomass N (calculated from 1 day CHCl,-N

and k, = 0.54) was closely correlated with total soil
N (Table 1) for the 20 soils (treating soils 7 and 8 each
as two soils-see Table 1). The regression accounted
for 80% of the variance in the data and the intercept
was not significantly different from zero. The regression, with the intercept fitted through zero, was
Biomass N @g gg) = (399.1 + 24.5)% total soil N.
These results show that biomass N is a very
constant percentage of total soil N in these soils
(ranging from about 2-6x with a mean of just under
4x), despite differences in their previous agricultural
history.
Summary

of proposed

method

Triplicate portions of moist, unfumigated soil are

extracted
for 30 min with 0.5 M K,SO,
(1:4
soil : solution ratio). Simultaneously, further triplicate
portions of moist soil are fumigated with alcohol-free
CHCI, for 24 h at 25C then extracted with K,SO.,
similarly. The filtered extracts are then analysed for
total N after Kjeldahl digestion, or stored at 2C until
analysed. The amount of N released by CHCI, after
24 h fumigation (1 day CHCl,-N) is calculated from
[(total N in K,S04 extracts of fumigated soil after
24 h fumigation) minus (total N in K,S04 extracts

of non-fumigated
soil at start of fumigation)]. S$iI
calculated
from
microbial
biomass
N
is
(1 day CHCl,-N)/0.54. All results are expressed as
pg N gg O.D. soil.
Acknowledgements-We
thank M. L. Fearnhead for Technicon analyses, J. E. D. Brown, D. S. McCann and N. D.
Sills for technical
assistance,
D. S. Powlson and R. P.
Voroney (University of Saskatchewan)
for useful discussion
and T. J. Dixon and Lynn C. Parry for statistical analyses.

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