Soil Bml. Bmhem. Vol. 20. So 5. pp 63-630.

1988 0038-0717
88 S3.M)t0.00
Printed m Great Britam Pergamon Pressplc

TEMPORAL VARIATIONS IN SOIL MICROBIAL BIOMASS

C AND N UNDER A SPRING BARLEY CROP
K. RITZ and D. ROBINSON’*
Department of Microbiology and ‘Soil Fertility. Macaulay Institute for Soil Research, Craigiebuckler.
Aberdeen AB9 ZQJ, U.K.

(kcepted 8 March 1988)

Summary-Concentrations of microbial biomass C and N were estimated using the chloroform

fumigation-incubation technique periodically throughout the life of a spring barley crop grown in NE
Scotland. The crop was fertilized at rates equivalent to 25. 75 or I25 kg N ha-‘. Biomass C showed a
sharp increase up to approx. 50 days after sowing and declined thereafter, showing no relationship to crop
growth. Biomass N was somewhat variable over time. There was no consistent effect of fertilizer rate on
either biomass C or N concentrations. The soil-water content was unusually stable throughout the growing
season. There was no correlation between C-flush and N-flush values at any time.

INTRODUCTIO’ populations under a barley crop during I yr. Bac-

terial and fungal populations were estimated by direct
There is an increasing awareness of the importance of microscopy whilst protozoa were estimated by the
the soil microbial biomass in relation to soil fertility most probable number technique. The time courses,
and crop nutrition. The biomass is a key feature of based on I7 samples over the year, were somewhat
nutrient conservation and cycling processes in soil variable for all 3 groups. There was some evidence
and as such represents an important reservoir of that bacterial biomass was reduced by dry weather
potentially-available plant nutrients. Very few studies conditions. Protozoan populations showed a ten-
have been concerned with temporal changes in the dency to peak in late spring and early autumn.
biomass in relation to the growth of annual crops. We report a field experiment where amounts of C
Lynch and Panting (1980a) followed changes in and N in the soil microbial biomass were estimated
biomass C over time under winter wheat using periodically by the fumigation technique throughout
a modification of the chloroform fumigation-incu- the life of a spring barley crop.
bation technique originally proposed by Jenkinson
and Powlson (1976). The time course, based on 6 >l,\TERIALS AND METHODS
sampling times, showed approximately constant
amounts of biomass C from autumn to early spring. Soil
a peak in late spring. and a continual decline to the Soil samples were taken from a field site at the
following autumn. The peak coincided with heading Cross of Jackston, Aberdeenshire (OS. reference NJ
of the crop. In a second study, based on 7 sampling 748 331; altitude I83 m). The soil is a freely-drained
times in the period November to July, they found a loam (Foundland association) derived from slates
similar springtime increase in biomass C. followed by and schists, with a pH (CaCI,) of 6.4 (Glentworth and
a decline (Lynch and Panting, 1982). Carter and Muir, 1963).
Rennie (1984) also applied the fumigation technique
to obtain estimates of biomass in soils supporting Experimental design
wheat crops, at 4 successive growth stages of the Plots (9 x 3.2 m). ploughed out of grass the pre-
plants. The crops were grown at several sites and vious autumn, were sown with spring barley
fertilized with 15N labelled urea. They regarded the (Hordeurn cctlgare L. cv. Golden Promise) on I5 April
NH,-N flush following fumigation and incubation as 1985. The plots were fertilized 29 days after sowing,
an indicator of biomass C. The NH, flush was found at the 2-3 leaf growth stage of the plants, with
to generally increase from plant emergence up ammonium nitrate at a rate equivalent to 25, 75 or
to booting, and declined thereafter. Recovery of I25 kg N ha-‘, hereafter referred to as 25N. 75N or
fertiliser N in the ammonium Rush was often higher l25N plots respectively. Fertilizer levels were applied
during the early growth stages of the plants and according to a randomized block design with 3
declined at crop maturity. In a detailed study where replicate blocks per treatment. Basal P was combine
the biomass was partitioned into bacteria, fungi and drilled at a rate equivalent to 22 kg P ha-‘, as
protozoa, Schniirer et al. (1936) found no pro- superphosphate. Soil samples were taken from the
nounced seasonal trends in the sizes of microbial O-l 5 cm horizon directly beneath the crop row, using
a screw auger. For each plot, several cores were taken
*Present addresses: Department of Mycology and Bac- along a 50cm run of crop, bulked and sieved
tcriology. and ‘Physiology and Crop Produciion. (~2 mm). Samples were taken immediately before
Scottish Crop Research Institute. Inbergowrie. Dundre sowing, here following a completely randomized
DD2 5DA. U.K. design (IO samples). since the plot structure had not
626 K. RITZ and D. ROBNON

0 50 100 150 200

Time since sowing (days)

Fig. I. Calendar. soil water content (line graph) and rainfall (histogram) for field site in this study

been laid out at this time. Samples were also taken RESULTS
immediately before fertilizer application, and at
approximately regular intervals thereafter until mid- Soil-water content
November. Rainfall on this site in 1985 was low in spring but
frequent and regular throughout the summer (Fig. I).
Biomass determinations Consequently, the soil water content was stable
The chloroform fumigation-incubation technique throughout the growing season from mid-May on-
was used to estimate both biomass C (Jenkinson and wards (Fig. I). The soil was driest in early May, with
Powlson, 1976) and N (Shen er al., 1984). Duplicate a water content of 15%. Thus, only small additions
subsamples (30 g) of soil were fumigated, inoculated of water were required to bring the soils to 55% of
with 0.4% (w/w) unfumigated soil and held for IO their water-holding capacity for all but the samples
days at 25’C. Incubation was carried out at 55% of taken in early May.
the soil water-holding capacity (sieved soil), corre-
sponding to 30% water content. For biomass C Soil respiration
estimation, duplicate control samples were incubated Fumigated soils always evolved more CO, over IO
for IO days corresponding to days IO-20 relative to days incubation than their respective controls (Fig.
the fumigated samples. Total CO& evolved from the 2). The C-flush, indicating biomass C, showed a
soils was measured by gas chromatography (Sparling, sharp increase up to approx. 50 days after sowing,
1981). The C-flush was taken as the difference and declined thereafter at a decreasing rate (Fig. 3).
between CO& evolved by fumigated vs control There was no significant effect (P > 0.05) of fertilizer
samples. Biomass C was estimated using a kc value rates upon the C-flush except at the 52-day sampling;
of 0.45 (Jenkinson and Ladd. 1981). For biomass N here 25N plots apparently supported a significantly
estimation, duplicate control samples were incubated smaller biomass than 75 N or l25N plots (P < 0.05;
for IO days in parallel with fumigated samples. Fig. 3). Between approx. 50 and 140 days, the C-flush
Mineral N was extracted into 2bt KCI (soil:extractant from soils samples from 25N plots was consistently
ratio I : 5) and determined calorimetrically. Am- lower than from 75N or l25N plots. Assuming a kc
monium-N was determined using the salicylate- of 0.45 is valid (see below), biomass C therefore
dichloriosocyanurate reaction (Fraser and Russell, ranged from 20655Opg C g-’ soil (Fig. 3).
1969) and NO,-N was measured as NO,-N following corresponding to 250-685 kg C ha-’ in the &I 5 cm
reduction by copperized cadmium (Henriksen and horizon.
Selmer-Olsen. 1970). The N-flush was calculated as
Nitrogen minerali:ation
the difference between mineral N extracted from
fumigated vs control soils. Biomass N was estimated Amounts of mineral-N in the fumigated soils were
using a k, value of 0.68 (Shen er al., 1984). always higher than in control soils after IO days
incubation even when background concentrations of
Construction o/‘ time courses mineral-N were high due to fertilizer application (Fig.
Since the randomized block design was not estab- 4). This was primarily due to considerably higher
lished at sowing, samples taken at this time are not amounts of NH,-N in the fumigated vs the control
strictly comparable with subsequent samples. Pre- soils. Concentrations of NH,-N in fumigated soils
sowing points (time 0 on graphs) were therefore were on average 44 times greater than those in
joined to the rest of the time-courses by broken lines. control soils (range 16-172 x ). Nitrification always
All other points are joined assuming that they are occurred in the control soils. Amounts of NO,-N in
directly comparable. This is likely to be a valid control soils exceeded those in fumigated soils by
assumption since the incubation conditions and lO.5-25.5pg N g-’ dry soil.
analytical methods were kept as consistent as was Amounts of mineral-N in both fumigated and
practically possible at all sampling times. control soils showed a sharp increase following fertil-
 Variations in biomass C and N 621

I I I I
50 100 150 200
Time since sowing (days)

Fig. 2. Soil respiration by fumigated (closed symbols) and control (open symbols) soils. 0 25N plots;
q 75N plots; A 125N plots.

izer application and generally declined thereafter
(Fig. 4). In 125N plots, elevated concentrations
mineral-N remained in the soil for longer periods
than in 25N or 75N plots (Fig. 4). This pattern is
consistent with mineral-N content of soils immedi-
ately following sampling (data not presented). The
mineral-N flush, indicating biomass N, was some-
what variable and did not show simple trends in
relation to either time or fertilizer rate (Fig. 5).
Analysis of variance of the combined data (all sam-
ples excluding time 0) indicated significant variation
in the N-flush over time (P < 0.001) but no signifi-
cant effect of fertilizer level (P > 0.05). Generally,
values for N-flush increased up to 60 days after
sowing, declined until approx. 150 days, and there-
of after recovered to concentrations prevailing at 60
days (Fig. 5). The main exception to this trend was
in l25N plots, where the post 60-day decline was less
marked (Fig. 5). The N-flush was significantly greater
in mid-November than in early April (P < 0.001;
November data pooled).
Anomalous samples
On two sampling occasions the soil-water content
was greater than 30% (122 and I64 days after sowing;
Fig. 1). In these cases the samples proved extremely
difficult to sieve, requiring great force to push the soil
through the mesh. This treatment resulted in an

XI i I I I x I I I I
I I I I
SO 100 150 200

Time since sowing (days)

Fig. 3. C-flush due to fumigation and biomass C estimates for 25N plots (0). 75N plots (0) and l25N
plots (A). Bars show standard errors. $& Values significantly different within time (P c 0.01).
628 K. RITZand D. Roerssos

50 100 150 200

Time since sowing (days.1

Fig. 4. Extractable mineral N from fumigated (closed symbols) and control (open symbols) soils. 0 ZSN
plots: 0 75N plots: A l2SN plots. Arrow shows time of fertilizer application.

extruded rather than sieved soil, with a highly with the plateaux forming at approx. I20 days after
modified structure. This problem has been reported sowing for 2SN plots. and 100 days for 75N and
previously for grassland soil (Ross et al., 1985). The l25N plots (Fig. 6). Plant yields from 2SN plots were
biomass estimates were clearly influenced in such a always lower than from 75N or l25N plots, particu-
way that they were not comparable with the estimates larly from 100 days after sowing (Fig. 6). There was
obtained for other “sievable” samples, as also re- no significant difference between plant yields from
ported by Ross (1987). In addition, these samples 75N or l25N plots at any time (P > 0.05).
were incubated at a higher soil-water content than
55%. since no attempt was made to dry them.
Consequently, biomass data from these sampling DISCLSSION
times have been omitted from the time courses.
Biomass C
Crop growfh It is unlikely that the observed
peak in biomass C
Crop growth followed typical sigmoidal patterns, in the spring was due solely to a crop-related factor.

Time since sowing (days1

Fig. 5. hlineral-S Rush due to fumigation and biomass N estimates for 25N plots (0). 7SN (G) and 125X
plots (A). Bars show standard errors. * Values significantly different Nithin time (P < 0.05).
 Variations in biomass C and N
I I
50 100 150
Time since sowing (days1
Fig. 6. Dry wt of above-ground crop mass for 25N plots
(0). 7SN plots (0) and 12SN plots (A).
At the time of the peak, the above-ground crop mass
was only 62gm-- ’ in all treatments (Fig. 6); the
amount of root material would have been similarly
low and unlikely to have stimulated the biomass to
such an extent as was observed. Several factors could
account for the additional stimulation, including soil
disturbance during sowing, fertilizer application, the
increase in soil-water content in early May and
springtime rises in soil temperature. Disturbance of
the soil during sowing would disrupt soil aggregates
and render previously inaccessible soil organic matter
available for microbial assimilation (Powlson, 1980).
Fertilizer application would stimulate the biomass if
it were N-limited, but there was no effect of fertilizer
rate upon biomass levels except during the post
50-day decline. A number of studies have shown how
soil microbial biomass is stimulated by rewetting fol-
lowing partial drying (e.g. Bottner, 1985) and how soil
microbial populations and rainfall events are often
correlated (e.g. Campbell and Biderbeck, 1976;
Schniirer er nl., 1986). At this site, soil-water content
was (unusually) stable after mid-May. which may
account for the apparent lack of variation in biomass
C after this time. Soil temperature data specific to
this site are not available. Meterological Office data
(Meteorological Office, 1985) for soil temperature
(&30cm). averaged for this region of NE Scotland.
shows an approximately constant rate of increase of
3’C month-’ from March (3.7’C) to May. Lynch and
Panting (1980a, 1982) also found springtime increases
in biomass which they attributed to a combination of
temperature increases and root growth.
The apparent decline in biomass from 50 to 80 days
after sowing is difficult to account for. This period
was one of maximal crop growth rate (Fig. 6) and it
would be expected that root development and exu-
dation would be sufficiently high to at least partially
support the elevated amounts of biomass. Previous
studies suggest that crop growth often stimulates an
629
increase in the size of the biomass (Lynch and
Panting, 1980a; Carter and Rennie, 1982). and a
number of authors have related the size of the
biomass to (often postulated) amounts of root ma-
terial prevailing in the soil (e.g. Adams and Laughlin,
1981; Carter, 1986; Lynch and Panting, 1980b). The
results from this experiment do not follow this pat-
tern. Some of the increase may be methodological,
reflecting the availability of substrates in the soil
sampled in late spring. Van der Werf and Verstraete
(1987) also found no stimulation of total biomass
by growth of a winter wheat crop, but that the
ache component of the biomass was stimulated
considerably.
The general decline in N-flush from 60 to 100 days
after sowing (Fig. 5) also coincides with the period of
rapid crop growth (Fig. 6). This decline is presumably
related to the decline in biomass C. The plants would
have been taking N up actively until about I20 days
after sowing, when dry matter accumulation ceased
(Fig. 6). The implication is that N lost from the
biomass may have been transferred to the crop. When
the crop ceased acting as a N sink. biomass N levels
(but not C) recovered. Between approx. 100 and 150
days after sowing, N-flush data ranks in accord with
fertilizer rates, suggesting that at this stage microbial
N-pool sizes were influenced by fertilizer rate. That
there was no significant effect of fertilizer rate upon
the size of the biomass (as C-flush or N-flush) sug-
gests that the biomass was generally not N-limited.
These results support those of Van der Werf and
Verstraete (1987). who also found that total biomass
was not influenced by fertilizer rate. Lynch and
Panting (1982) found that N fertilization significantly
increased biomass C contents in soil supporting
winter wheat, but only when the crop was direct-
drilled.
In many studies of the chloroform fumigation-
incubation method, the ratio of C-flush-to-N-Rush
has been shown to be remarkably constant between
different soil types (see Shen er al., 1984). In this
experiment, there was no significant correlation be-
tween C-flush and N-flush at any time (pooled data
for all harvests, r = 0.158; P > 0.05). This disparity in
results is difficult to account for, especially since the
soil in the study was from the same site, sampled at
different times.
Sign@ance of conrersion factors
To convert flush data to biomass data, a con-
version factor (k). based on the proportion of killed
biomass which is converted to CO2 (kc) or mineral-N
(kN) is used. If time-courses based on the fumigation-
incubation method are to be considered as showing
true variations in biomass contents, then the assump-
tion must be made that k factors are constant over
time. This is likely to be the case for C; published
estimates of k, vary little between 0.41 (Anderson
and Domsch, 1980) and 0.45 (Jenkinson ef al., 1979).
However, k, values are more prone to variation and
have been reported to vary from 0.20 (Voroney and
Paul, 1984) to 0.68 (Shen ef al., 1984). Voroney and
Paul (1984) have suggested that k, may be adjusted
according to the ratio of the C- and N-Rushes
630 K. RITZ and D. Roe~ssos

(C,-to-N,). They reason that where C,-to-N, is high, method for the determination of cation-exchange capacity
the microbes involved in mineralizing N are likely to of clay minerals. Cfav Minerals 8. X9-230.
become somewhat N-limited and a greater pro- Glentworth R. and Muir J. (1963) The Solis of fhe Counrrj
portion of the mineralized N will be reimmobilized, round Aberdeen. Inoerurie and Fraserburgh. Memoirs of
the Soil Survey of Great Britain. HMSO, Edinburgh.
resulting in lower k, values. Conversely, lower
Henriksen A. and Selmer-Olsen A. R. (1970) Automated
C,-to-N,, values will result in higher k, values. methods of determining nitrite and nitrate in water and
They showed experimentally that k, = -0.014 soil extracts. Annl~sr !?$ 514-518.
C,-to-N, + 0.39 (Voroney and Paul. 1984). Here, C, Jenkinson D. S. and Ladd J. N. (1981) hlicrobial biomass
is the CO,-C evolved over 10 days by fumigated soil in soil: measurement and turnover. In Soil Biochemisrry,
(i.e. no subtraction of control) and N, is the net Vol. 5 (E. A. Paul and J. N. Ladd. Eds). pp. 415-471.
production of NH,-N in the soil (i.e. no account Dekker, New York.
taken of NOJ-N). We do not consider it valid to Jenkinson D. S. and Powlson D. S. (1976) The effects of
correct k, using this equation with our data since, biocidal treatments on metabolism in soil-V. A method
for measuring soil biomass. Soil Biology & Biochemistry
unlike those of Voroney and Paul, our soils were
8, 209-213.
reinoculated with unfumigated soil, and the level of Jenkinson D. S., Davidson S. A. and Powlson D. S. (1979)
reinoculation has a significant effect on the C-flush Adenosine triphosphate and microbial biomass in soil.
(Chapman, 1987). The C-to-N ratios of the Rushes Soil Biology & Biochemisq II, 521-527.
measured in this experiment varied significantly over Lynch J. M. and Panting L. M. (1980a) Cultivation and the
time (P < O.OS), ranging from 3.1 to 10.4, with no soil biomass. Soil Biology & Biochemistrv 12, 29-33.
consistent trends or significant effect of fertilizer rate Lynch J. M. and Panting L. M. (1980b) Variation in the size
(P > 0.05). Assuming that the C-to-N ratio of the -of the soil biomass.- Soil Biology’ & Biochemistry 12,
547-550.
flush, however it is estimated, does influence k,, there
Lynch J. M. and Panting L. M. (1982) Effects of season,
is therefore evidence that in this experiment k, varied cultivation and nitrogen fertiliser on the size of the soil
significantly over time. Consequently the time- microbial biomass. Journal of rhe Science of Food and
courses of N-flush are likely to be poor indicators of Agriculfure 33. 249-W.
temporal variations in biomass. This conclusion is Meteorological Office (1985) .Ilonrh!, tC.eafher Repporn.
supported by the relative stability of the time-courses HMSO. London.
for C-flush compared to those for N-flush. Powlson D. S. (1980) The effects of grmding on mrcrobial
and non-microbial organic matter in sorl. Journul of Soil
Science 31, 77-85.
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