Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol13.Issue3.Dec1999:
Applied Soil Ecology 13 (1999) 209±218
Temporal variations in microbial biomass C and cellulolytic enzyme
activity in arable soils: effects of organic matter input
Kasia Debosza,*, Peter H. Rasmussenb, Asger R. Pedersena
a
Department of Crop Physiology and Soil Science, Danish Institute of Agricultural Sciences,
Research Centre Foulum, PO Box 50, DK-8830, Tjele, Denmark
b
Plant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University,
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 8 December 1998; received in revised form 26 April 1999; accepted 17 May 1999
Abstract
Temporal variations in soil microbial biomass C concentration and in activity of extracellular enzymes of the cellulolytic
complex were investigated in a ®eld experiment after eight years of cultivation with either low organic matter input (low-OM)
or high organic matter input (high-OM). The cultivation systems differed in whether their source of fertiliser was mainly
mineral or organic, in whether a winter cover crop was grown, and whether straw was mulched or removed. Sampling
occurred at approximately monthly intervals, over a period of two years. Distinct temporal variations in microbial biomass C
concentration and activity of extracellular enzymes of the cellulolytic complex were observed. The temporal pattern was
generally similar in the low-OM and high-OM cultivation systems. Temporal variations may have been driven by
environmental factors (e.g., temperature and moisture) and crop growth, i.e. by factors common to both systems but not
differences in organic matter input. Pronounced and constant increases in b-glucosidase activity (40%) and endocellulase
activity (30%) in high-OM were detected across all sampling periods. The increases in microbial biomass C concentration and
cellobiohydrolase activity varied over the sampling periods (0±60% and 24±92%, respectively). Over the experimental period
a mean of 148 6.0 mg biomass C gÿ1, a b-glucosidase activity of 123 3.3 nmol gÿ1 hÿ1, a cellobiohydrolase activity of
122 2.4 nmol gÿ1 hÿ1 and an endocellulase activity of 33.8 0.9 nmol gÿ1 hÿ1 were recorded in the low-OM soil (0±
20 cm). The corresponding means in the high-OM treatment were 189 6.6 mg biomass C gÿ1, a b-glucosidase activity of
174 4.1 nmol gÿ1 hÿ1, a cellobiohydrolase activity of 173 3.4 nmol gÿ1 hÿ1 and an endocellulase activity of
44.2 1.1 nmol gÿ1 hÿ1. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Integrated arable farming system; Soil quality indicators; Microbial biomass; Extracellular cellulase; Fluorogenic compound assay
1. Introduction
Soils managed with organic inputs generally have
larger and more active microbial populations than
*Corresponding author. Fax: +45-89-99-1619
E-mail adress: kasia.debosz@agrsci.dk (K. Debosz)
those managed with mineral fertilisers (Dick, 1984;
Doran et al., 1987; Powlson et al., 1987; Werner and
Dindal, 1990; Fauci and Dick, 1994). Soil microbial
biomass and activity are greatly stimulated by the
addition of manure through modi®cation of soil physical characteristics and addition of readily available C
and N sources (Reganold, 1988; Fraser et al., 1988).
0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 3 4 - 7
210
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
Since microorganisms are more responsive to changes
in the soil environment than is the soil organic matter
(Powlson et al., 1987), effects of experimental treatments are more readily detectable in the microbial
fractions (Dick, 1994). Previous studies have compared temporal responses of microbial biomass and
soil enzymes under different management or different
temperature and moisture regimes (Martyniuk and
Wagner, 1978; Lynch and Panting, 1982 Kaiser and
Heinemeyer, 1993; Joergensen et al., 1994; Ritz et al.,
1997; Jensen et al., 1997). McGill et al. (1986)
proposed that seasonal temporal changes in soil
microbial biomass are directly linked to the turnover
of organic matter and the cycling of nutrients in soil.
Temporal changes in the size of microbial pool may
enhance the ef®ciency of N use in agricultural systems
(Granatstein et al., 1987; Bonde et al., 1988; Bremer
and van Kessel, 1992).
Doran and Parkin (1994) proposed a set of soil
quality indicators that are sensitive to changes in soil
management and integrate biological, physical, and
chemical properties. Biological methods used to
assess soil quality should re¯ect cropping and management histories in long-term studies. In discussing
soil quality indicators, Karlen et al. (1997) included
soil microbial biomass C and extracellular enzyme
activity as biological indicators.
There is considerable interest in adopting alternative agricultural practices such as organic or biodynamic and low-input systems in the belief that the
present conventional agricultural system using
mineral fertilisers has detrimental effects on physical,
chemical and biological properties of soils (Nguyen
et al., 1995). Long-term bene®ts from the use of crop
rotations and animal manure include better soil tilth,
improved water in®ltration, increased soil organic
matter content, and increased biological activity
(Wardle, 1992).
Here, we report the effects of up to eight years
of high (high-OM) and low (low-OM) organic matter
input in arable farming systems on microbial biomass
carbon concentrations and selected cellulolytic
enzyme activity in the soil. The systems being compared in a long-term ®eld trial are cereal rotations
that differ in their source of fertiliser (mineral or
organic), in whether a catch crop is grown, and
whether the straw is mulched or removed. In the
high-OM system only a small part of the nutrient
supply is provided by mineral fertilisation and the
nutrient supply to crops and soil microorganisms
will be mainly from organic inputs. The objective
of this research was to determine the extent of temporal variations in microbial biomass C concentration,
cellobiohydrolase, b-glucosidase and endocellulase
activity over two successive years. A second objective
was to evaluate the effects of high and low organic
matter input on soil quality indicators after eight years
of cultivation.
2. Materials and methods
2.1. Site and soil
The study was part of a ®eld experiment on the
integrated arable farming initiated in 1987, located
at Research Centre Foulum, Denmark. It is situated
at (568300 N and 98340 E, 50 m above mean sea
level). The soil is a loamy sand with 67% sand and
8% clay. Mean annual (1961±1990) temperature
is 7.38C and ranges from ÿ0.58C in January to
15.48C in July. Average mean annual (1961±1990)
rainfall is 626 mm, of which 50% falls in autumn and
winter.
Previously these ®elds were uniformly cultivated,
and had no organic manure additions for 14 years
before the start of the experiment. In the spring of
1988, randomized blocks were established, with three
replicate blocks per treatment. The treatments differed
mainly in the form of nutrients applied (Rasmussen
and Hansen, 1994). This study included a high-OM
treatment with high inputs of organic matter in the
form of pig slurry, a catch crop and incorporation of
straw, and a low-OM treatment with mineral fertilisers, no catch crop and straw removed (short stubble
remaining) at harvest (Table 1). Both high- and lowinput treatments received a reduced input of pesticides
(Secher et al., 1995). Each spring, 30 t pig slurry haÿ1
were applied to the high-OM treatment, supplemented
by ammonium nitrate (AN) evenly spread over the
surface (Table 1). The slurry was surface-applied,
then ploughed in. Straw left after harvest was cut
before mulching. The low-OM treatment received
mineral fertilisers (NPK and AN) in the spring. In
the high-OM treatment, a four-year crop rotation of
winter wheat, spring barley with a catch crop sown
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
211
Table 1
Soil properties, treatments, crop rotation and OM inputs
Year of initiation
Soil properties in 1995
Fertilizer practice
Ploughing
Mulching
Crop rotationb
High-OM
Low-OM
1988
Total C 1.7 (%), Total N 0.13 (%),
CEC 9 (meq 100 gÿ1), pH-CaCl2 5.9
Pig slurry (30 t) and AN a,
(80 kg mineral N and 30 kg organic N haÿ1)
Mouldboard ploughed, spring
straw
winter wheat, spring barley, fodder rapec,
1988
Total C 1.6 (%), Total N 0.13 (%), CEC 6
(meq 100 gÿ1), pH-CaCl2 5.7
NPK and AN (90 kg N haÿ1)
Mouldboard ploughed, spring
Straw removed
winter wheat, spring barley, spring barley, spring rape
spring barley=ryegrassd, spring rape
Annual input of OM
e
6477 kg haÿ1
0
a
Ammonium nitrate.
Crops at sampling time underlined.
c
Catch crop.
d
Undersown catch crop.
e
Average annual input of OM in the form of straw, ryegrass and pig slurry, stubble and roots not included.
b
after harvest, spring barley undersown with a catch
crop in the spring, and pea in the ®rst and spring rape
in the second crop rotation was practised. Fodder rape
(Brassica napus L. ssp. oleifera (Metzg.) Sinsk.), or
tansy-leaf phacelia (Phacelia tanacetifolia) was sown
after harvest of the ®rst spring barley crop in the
rotation was harvested. Perennial ryegrass (Lolium
perenne L.) was undersown in the second spring
barley crop in the crop rotation. In the low-OM
treatment a four-year crop rotation of winter wheat,
spring barley, spring barley and pea in the ®rst and
spring rape in the second crop rotation was practised.
No catch crop was sown in the low-OM treatment. In
1995 and 1996 both treatments were sown to a spring
barley, as the main crop. In the high-OM treatment
fodder rape was sown in autumn 1995 as a catch crop
and perennial ryegrass was the undersown catch crop
in 1996.
Soil was sampled (0±20 cm) on 20 occasions
between May 1995 and December 1996. On each
sampling date, four soil cores (2.5-cm diameter) were
collected at random from each block and pooled to
give one sample per replicate. Freshly sampled soil
was thoroughly mixed, and roots and visible plant
residues removed. Soil was frozen (approximately
ÿ188C) for later determination of microbial biomass
C and enzyme activity. Gravimetric water content was
determined by drying duplicate 10 g portions of soil at
1058C for 24 h.
2.2. Biomass C
Microbial biomass C was determined by the fumigation±extraction method Vance et al. (1987). Soluble
C and chloroform labile C (C solubilized by CHCl3
during an 18 h fumigation period) were extracted with
0.5 M K2SO4 in a soil : solution ratio of 1 : 3 (wt : v).
The extract was centrifuged and the supernatant ®ltered through a combusted (2508C, 5 h) Whatman GF/
C ®lter and a 0.22 mm Millipore Millex-GP ®lter unit.
Organic C in all 0.5 M K2SO4 extracts was determined
by an automated UV persulphate oxidation procedure
using a Dorhrman DC-180 Carbon Analyzer (Wu
et al., 1990). Biomass C was calculated as soluble
C in fumigated minus soluble C in non-fumigated soil
using 0.45 as the kc-factor (Kaiser et al., 1992) and
determined in duplicate.
2.3. Enzyme assays
The activity of b-glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91) and endocellulase (EC
3.2.1.4) were measured in ®eld moist samples by
the use of different ¯uorogenic substrates (see below).
During hydrolysis of the substrates, the aglycone 4methylumbelliferone (4-MU) is formed, which is
highly ¯uorescent at an alkaline pH. The assays were
performed according to the methods described by
Tilbeurgh and Clayssens (1985), with the following
212
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
modi®cations. The b-glucosidase activity was determined by the addition of 2.0 ml of 0.05 M acetate
buffer, (pH 5.0) to 20±80 mg soil. The mixture was left
for ®ve minutes in polyethylene tubes at 308C. The
enzymatic reaction was initiated by the addition of
0.5 ml substrate solution, and incubated for 20 min in
a water bath at 308C. The substrate concentration of 4methylumbelliferyl b-D-glycoside was 100 mM. The
reaction was stopped by the addition of 2.5 ml 96%
ethanol (v/v). The mixture was left for 20±30 min at
58C, then transferred into centrifuge tubes and centrifuged at 58C for 10 min at 1500 g. The supernatant
(4.5 ml) was transferred to a second polyethylene
tube, and 0.5 ml 2.5 M Tris buffer (pH 10) was added
to convert 4-MU into its ¯uorescent anionic form.
Fluorescence was determined with a Perkin±Elmer
¯uorometer (LS50) at 448 nm emission and 365 nm
excitation.
The activity of cellobiohydrolase was determined
using 4-methylumbelliferyl b-D-cellobioside as substrate. Soil samples of 50 mg were pre-incubated
with 2.0 ml 0.05 M acetate buffer, (pH 5.0) for ®ve
minutes, at 308C. The assay conditions and separation
of method for 4-MU were the same as described above
for b-glucosidase. The substrate concentration in the
assay was 100 mM. Endocellulase activity was determined using 4-methylumbelliferyl b-D-cellotrioside
as the substrate. The assay was performed in the same
way as described above, but the incubation period was
increased to 50 min. Soil samples of 50 mg were
used and the substrate concentration in the assay was
25 mM. All enzyme activity measurements were determined in six replicates.
2.4. Statistical models and analysis
A split-plot design with soil treatment as the main
plot treatment and sampling date as the sub-plot
treatment was used. Within each block the 20 measurements of each of the four variables, microbial
biomass C concentration, cellobiohydrolase, b-glucosidase and endocellulase activity, were analysed for
serial correlation by means of standard semi-variogram techniques (Cressie, 1991). Based on these
analyses it was concluded that the serial correlation
was negligible for all four variables, and so it was
ignored in subsequent analyses. A linear statistical
model with systematic interaction between treatment
and sampling date and with random block effect was
accepted. The correlation analysis did, however, indicate some variance heterogeneity between treatment
groups, and even between blocks. However, only
variance heterogeneity between treatment for the variables b-glucosidase and endocellulase activity was
signi®cant (p < 0.05). The analyses were performed
by means of the SAS-procedure PROC MIXED (SAS
Institute Inc., 1996a) using the REML-methodology
(Searle et al., 1992; Diggle et al., 1994).
In the subsequent statistical analyses the systematic
part of the models, i.e. treatment and sampling date
effect were analysed. For microbial biomass C concentration and cellobiohydrolase activity for which
variance homogeneity was applicable, the classical
analysis of variance was used (PROC GLM, SAS
Institute Inc., 1989). For the effects of b-glucosidase
and endocellulase, iterative REML-methods (PROC
MIXED, SAS Institute Inc., 1996b) were applied to
allow for treatment speci®c variances. In both models,
the sampling date speci®c treatment effect and the
difference between treatments of the average level per
year of the four variables were formulated as contrasts,
and hence analysed by standard techniques. The average level per year, for the four variables, was approximated by a trapeze-sum, in order to account for the
non-equidistant measurement of time points.
In order to test for a temporal trend over the twoyear observation period, a linear time trend was added
to the systematic part of the model. This linear time
trend had a treatment speci®c intercept for all variables, whereas the slope was treatment speci®c for
microbial biomass concentration and cellobiohydrolase activity, but the same across treatments for endocellulase and b-glucosidase activity in accordance
with the statistical model derived above. Furthermore,
either the sampling date factor or the sampling
date treatment interaction factor was treated as a
random effect in order to test the signi®cance of the
added linear term.
3. Results
Mean monthly rainfall and air temperature over the
two-year observation period are shown in Fig. 1. Total
precipitation for the two-year sampling period was
1010 mm, 242 mm below the long-term average.
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
213
Fig. 1. Top: Mean monthly air temperature (line) and mean monthly rainfall (bars) from January 1995 and to January 1997 at the sampling
site. Bottom: Annual management practices in low-OM and high-OM input systems. S. barley, spring barley; CC, catch crop; T, ploughing; O,
organic fertilizers; F, mineral fertilizers; H, harvest; S, straw mulching.
Autumn 1995 and 1996 were both relatively wet,
while the spring of 1996 as well as the summers of
1995 and 1996 were extremely dry (Fig. 1). The mean
temperature for the experimental period was 7.28C,
which is comparable with the long-term average. The
soil was frozen from December 1995 to April 1996,
with a mean temperature of ÿ38C, and a mean temperature of ÿ28C in November and December 1996
(Fig. 1). The prolonged periods of frost in 1995 caused
slow and weak development of the catch crop (fodder
rape) in the high-OM treatment.
With the exception of a higher CEC for high-OM
treatment, there was little or no difference in soil
properties between treatments (Table 1). Percentage
total carbon levels were similar for the soils of both
treatments.
Signi®cant effects of both treatment and sampling
date were found for all four variables studied
(p < 0.05). For microbial biomass C concentration
and cellobiohydrolase activity the treatment and the
sampling date effects interacted signi®cantly, which
resulted in the treatment effect varying over the sampling period. The interaction between sampling date
and treatment implied that the model-predicted level
for each sampling date and treatment was equal to the
average of the true measurements for sampling date
and treatment (Fig. 2(a) and (b)). For b-glucosidase
and endocellulase activity the treatment effect was
constant over the sampling period and as a result the
predicted levels differed from the measured averages
for b-glucosidase and endocellulase activity (Fig. 2(c)
and (d)). In the treatment with high-OM input the soil
had consistently higher biomass C levels than low-OM
soil when all sampling dates were considered together
(p < 0.01). We found no signi®cant differences
between the summer and autumn sampling dates of
the respective years (Fig. 2(a)). There was no signi®cant linear temporal trend in microbial biomass C
concentration over the two-year period. Mean annual
biomass C concentration, calculated by the trapezesum on the data in Fig. 2(a), differed signi®cantly with
organic matter input (Table 2).
The high-OM treatment had consistently higher
cellobiohydrolase activity (24±92%) than the lowOM treatment at all sampling dates (p < 0.001). The
difference between treatments was signi®cant when
214
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
Fig. 2. Measured and model-predicted average levels of microbial biomass C concentration and extracellular enzyme activity in soils with
cereal rotation, with low-OM and high-OM organic matter input, April 1995±December 1996: (a) Microbial biomass C concentration; (b)
Cellobiohydrolase activity; (c) b-glucosidase activity; (d) Endocellulase activity. (*) measured and (ÐÐÐ) predicted values in low-OM
treatment, (*) measured and (- - -) predicted values in high-OM treatment.
Table 2
Mean annual microbial biomass C concentration (MB-C), and extracellular enzymes activity in 1995 and 1996 in soils with cereal rotation, as
related to low (low-OM) and high (high-OM) organic matter input
1995
Microbial biomass C (mg C gÿ1 soil)
Cellobiohydrolase activity (n mol 4-MU gÿ1 hÿ1)
b-glucosidase activity (nmol 4-MU gÿ1 hÿ1)
Endocellulase activity (nmol 4-MU gÿ1 hÿ1)
1996
Low-OM
High-OM
Low-OM
High-OM
160.9
115.1
114.5
30.6
184.6xx a
162.1xxx b
165.7xxx b
41.1xxx b
140.4
130.5
133.5
38.5
182.8xxx b
185.3xxx b
184.5xxx b
49.0xxx b
Note: Means per treatment and experimental year were estimated by trapeze-sum method. The p-values were derived from statistical tests
comparing the predicted yearly average level in low and high-OM level.
a
p < 0.01.
b
p < 0.001.
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
considering the whole entire time-course (p < 0.001),
but not on all individual sampling dates (Fig. 2(b)).
Soil with high-OM input had the greatest mean cellobiohydrolase activity in both years (Table 2). The
overall temporal trend showed fairly uniform cellobiohydrolase activity for most of the sampling dates
for both treatments, with a slight increase throughout
the experimental period (p < 0.05), indicating increasing cellobiohydrolase activity between 1995 and 1996
in both treatments.
Temporal ¯uctuations in b-glucosidase activity
were distinct in both treatments (Fig. 2(c)). There
was a signi®cant effect (p < 0.0001) of sampling date
on b-glucosidase activity, demonstrated by low activity from August 1995 to April 1996 and relatively high
activities on the other sampling dates (Fig. 2(c)). The
effect of treatment was constant and signi®cant over
all sampling dates (p < 0.001). b-glucosidase activity
followed a similar temporal pattern in the two treatments and was ca. 40% higher in the high-OM treatment than in the low-OM treatment on all sampling
dates. There was no signi®cant linear temporal trend in
b-glucosidase activity. The average difference in bglucosidase activity between high-OM and low-OM
was highly signi®cant (Table 2).
Temporal variations in endocellulase activity
showed a different pattern from those for b-glucosidase activity, with the highest activity in the autumn/
winter and early summer sampling samplings
(Fig. 2(d)). Despite temporal ¯uctuations of endocellulase activity the effect of treatment was constant and
signi®cant over all sampling dates (p < 0.001). On all
sampling dates, the endocellulase activity in the highOM treatment was ca. 30% higher in the low-OM
treatment (Fig. 2(d)) and the average level over the
two years differed signi®cantly with organic matter
input (Table 2). There was a consistent and signi®cant
temporal trend in endocellulase activity in both treatments (p < 0.05), indicating that endocellulase activity
increased during the two years.
4. Discussion
4.1. Temporal trends
Microbial biomass C concentration and extracellular soil enzymes of the cellulolytic complex displayed
215
marked temporal variability, similar in both low- and
high-OM treatments (Fig. 2). Different amounts of
annual OM input (Table 1) did not have any large
in¯uence on temporal variations of microbial biomass
C concentration and enzyme activity. This suggests
that temporal variations may have been driven by
climatic (e.g. temperature and moisture) factors and
by crop growth, i.e. by factors common to both
systems. Temporal changes in soil moisture, soil
temperature, and C input from crop roots, rhizosphere
products (i.e. root exudates, mucilage, sloughed cells,
etc.), and crop residues can have a large effect on soil
microbial biomass and activity (Ross, 1987). Crop
growth often stimulates an increase in the size of
microbial biomass during the growing season (Lynch
and Panting, 1982; McGill et al., 1986; Fraser et al.,
1988; Kaiser and Heinemeyer, 1993). Granatstein et
al. (1987) observed a signi®cant increase in microbial
biomass after harvest. Patra et al. (1990) argued that
biomass C concentration remained fairly constant
during the year due to a relatively even distribution
of litter with time. In this study, however, the peak in
microbial biomass C concentration occurred in early
May 1995 when no crop growth had yet taken place
(Fig. 2(a)). The autumn (October 1995) increase in
biomass C concentrations coincided with the onset of
rain (Fig. 1) and with the addition of new crop residues. Moisture probably became a limiting factor
during the summer 1995 (Fig. 1), with the lowest
biomass levels occurring in July (Fig. 2(a)). In contrast, a signi®cant increase in microbial biomass was
observed in July 1996 during the period of lowest soil
moisture. In many studies, the temporal variations of
microbial biomass were attributed to variations in soil
water content. Wetting and drying of soil has both
been shown to increase (Ross, 1989; Joergensen et al.,
1994), lower (Kieft et al., 1987; van Gestel et al.,
1992) or to have no effect on soil microbial biomass
levels (Ross, 1989; van Gestel et al., 1992).
Enzyme activity displayed different temporal patterns of the cellulase complex of enzymes as revealed
in different assays (Fig. 2(b)±(d)). According to Bolton et al. (1985), Martens et al. (1992) and Dick (1994)
some cellulases are closely related to inputs of fresh
organic materials, plant growth and plant residues,
while others appear to be more sensitive to soil
temperature and moisture. Temporal variations in
community structure and activity, induced by manure
216
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
decomposition and crop residue decomposition, may
modulate microbial biomass and the extracellular
enzymes that are released into the soil by the microbial
biomass (Fauci and Dick, 1994; Ritz et al., 1997;
Bossio et al., 1998).
Temporal variations in the size of microbial biomass C concentration and extracellular enzyme activities can be differentiated from that representing longterm changes in the biomass and enzyme activity in
response to inorganic fertiliser, organic manure, straw
incorporation or crop rotation with the statistical
approaches used in this study. Cellobiohydrolase
and endocellulase activity over two years showed a
consistent trend, i.e. a steady increase in the enzyme
activity in both high and low-OM treatments, but such
an increase did not appear in biomass C and bglucosidase. An increase in cellobiohydrolase and
endocellulase suggested that there were fundamental
differences in C-supplying mechanisms to the microbial community in both treatments in addition to
current-season manure or crop-derived inputs (Ritz
et al., 1997). These results suggest that a new equilibrium had not yet been attained after the change in
management. Following a sustained management
change such as the addition of organic fertiliser, crop
residues and crop rotation, it may take many years for
the soil organic matter to reach a new equilibrium (van
der Linden et al., 1987).
4.2. Soil quality and fertility
With the increasing emphasis on sustainable agriculture and soil quality it is important to determine the
effect of different organic inputs on soil organic matter
(Christensen and Johnston, 1997). Changes in the
quality and quantity of soil organic matter occur
slowly. These changes are dif®cult to quantify in
short-term studies because they are small in relation
to the large background of organic matter and the
Ê gren, 1991). Due
natural soil variability (Bosatta and A
to their dynamic nature, soil microbial biomass and
soil enzymes respond quickly to changes in organic
matter input (Powlson and Jenkinson, 1981; Powlson
et al., 1987; Dick, 1994).
In this study, no changes in total carbon levels in
soils were observed after eight years (Table 1). The
increased levels of microbial biomass C concentration
and cellulolytic enzyme activity in the high-OM treat-
ment (Table 2) re¯ect high annual inputs of organic
matter (6477 kg haÿ1) in the form of pig slurry, straw
residue and winter cover crop. Several studies have
looked at the shifts in microbial numbers and activity
in soil as related to changes in C inputs. In some
studies they were attributed to amount and diversity of
organic inputs (Fraser et al., 1988; Knudsen et al.,
1999; Martyniuk and Wagner, 1978; Powlson et al.,
1987). The importance of microbial biomass and
extracellular enzyme activity in assessment of soil
quality is established by the essential role of soil
microorganisms in nutrient cycling within agricultural
ecosystems (Rice et al., 1986). Of particular importance are free enzymes in the extracellular space, for
example for the initial depolymerization steps of
cellulose (Burns, 1982).
5. Conclusions
Large temporal variation in microbial biomass C
concentration and extracellular enzyme activity were
observed, which could be related to environmental
factors (e.g., temperature and moisture) and crop
growth but not differences in organic matter input.
Microbial biomass C concentrations and the activity
of extracellular cellulases appeared to be very good
indicators of early changes in soil biological status.
Biomass C concentration and extracellular enzyme
activity increased (30%) as a consequence of changing the cultivation system from one of low to one of
high organic matter input after eight years. In addition,
our results demonstrated that due to temporal variability repeated sampling is required to calculate annual
estimates of the size of microbial biomass C concentration and cellulolytic enzyme activity for the purpose of comparative ecosystem study.
Acknowledgements
We thank Anette Clausen, Ewa Hùrsdal, Bodil
Mùllnitz and Erling Nielsen for their ®eld, laboratory
and technical assistance and Gunnar Mikkelsen for
fruitful discussion and help in choosing the treatments.
Funding was provided by the AIR-3 Programme of
European Union, the Danish Ministry of Food, Agriculture and Fisheries Programme `Research on
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
Organic Farming' and the Danish Environmental
Research Programme `Centre for Sustainable Land
Use and Management of Contaminants, Carbon and
Nitrogen'.
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Temporal variations in microbial biomass C and cellulolytic enzyme
activity in arable soils: effects of organic matter input
Kasia Debosza,*, Peter H. Rasmussenb, Asger R. Pedersena
a
Department of Crop Physiology and Soil Science, Danish Institute of Agricultural Sciences,
Research Centre Foulum, PO Box 50, DK-8830, Tjele, Denmark
b
Plant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University,
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 8 December 1998; received in revised form 26 April 1999; accepted 17 May 1999
Abstract
Temporal variations in soil microbial biomass C concentration and in activity of extracellular enzymes of the cellulolytic
complex were investigated in a ®eld experiment after eight years of cultivation with either low organic matter input (low-OM)
or high organic matter input (high-OM). The cultivation systems differed in whether their source of fertiliser was mainly
mineral or organic, in whether a winter cover crop was grown, and whether straw was mulched or removed. Sampling
occurred at approximately monthly intervals, over a period of two years. Distinct temporal variations in microbial biomass C
concentration and activity of extracellular enzymes of the cellulolytic complex were observed. The temporal pattern was
generally similar in the low-OM and high-OM cultivation systems. Temporal variations may have been driven by
environmental factors (e.g., temperature and moisture) and crop growth, i.e. by factors common to both systems but not
differences in organic matter input. Pronounced and constant increases in b-glucosidase activity (40%) and endocellulase
activity (30%) in high-OM were detected across all sampling periods. The increases in microbial biomass C concentration and
cellobiohydrolase activity varied over the sampling periods (0±60% and 24±92%, respectively). Over the experimental period
a mean of 148 6.0 mg biomass C gÿ1, a b-glucosidase activity of 123 3.3 nmol gÿ1 hÿ1, a cellobiohydrolase activity of
122 2.4 nmol gÿ1 hÿ1 and an endocellulase activity of 33.8 0.9 nmol gÿ1 hÿ1 were recorded in the low-OM soil (0±
20 cm). The corresponding means in the high-OM treatment were 189 6.6 mg biomass C gÿ1, a b-glucosidase activity of
174 4.1 nmol gÿ1 hÿ1, a cellobiohydrolase activity of 173 3.4 nmol gÿ1 hÿ1 and an endocellulase activity of
44.2 1.1 nmol gÿ1 hÿ1. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Integrated arable farming system; Soil quality indicators; Microbial biomass; Extracellular cellulase; Fluorogenic compound assay
1. Introduction
Soils managed with organic inputs generally have
larger and more active microbial populations than
*Corresponding author. Fax: +45-89-99-1619
E-mail adress: kasia.debosz@agrsci.dk (K. Debosz)
those managed with mineral fertilisers (Dick, 1984;
Doran et al., 1987; Powlson et al., 1987; Werner and
Dindal, 1990; Fauci and Dick, 1994). Soil microbial
biomass and activity are greatly stimulated by the
addition of manure through modi®cation of soil physical characteristics and addition of readily available C
and N sources (Reganold, 1988; Fraser et al., 1988).
0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 3 4 - 7
210
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
Since microorganisms are more responsive to changes
in the soil environment than is the soil organic matter
(Powlson et al., 1987), effects of experimental treatments are more readily detectable in the microbial
fractions (Dick, 1994). Previous studies have compared temporal responses of microbial biomass and
soil enzymes under different management or different
temperature and moisture regimes (Martyniuk and
Wagner, 1978; Lynch and Panting, 1982 Kaiser and
Heinemeyer, 1993; Joergensen et al., 1994; Ritz et al.,
1997; Jensen et al., 1997). McGill et al. (1986)
proposed that seasonal temporal changes in soil
microbial biomass are directly linked to the turnover
of organic matter and the cycling of nutrients in soil.
Temporal changes in the size of microbial pool may
enhance the ef®ciency of N use in agricultural systems
(Granatstein et al., 1987; Bonde et al., 1988; Bremer
and van Kessel, 1992).
Doran and Parkin (1994) proposed a set of soil
quality indicators that are sensitive to changes in soil
management and integrate biological, physical, and
chemical properties. Biological methods used to
assess soil quality should re¯ect cropping and management histories in long-term studies. In discussing
soil quality indicators, Karlen et al. (1997) included
soil microbial biomass C and extracellular enzyme
activity as biological indicators.
There is considerable interest in adopting alternative agricultural practices such as organic or biodynamic and low-input systems in the belief that the
present conventional agricultural system using
mineral fertilisers has detrimental effects on physical,
chemical and biological properties of soils (Nguyen
et al., 1995). Long-term bene®ts from the use of crop
rotations and animal manure include better soil tilth,
improved water in®ltration, increased soil organic
matter content, and increased biological activity
(Wardle, 1992).
Here, we report the effects of up to eight years
of high (high-OM) and low (low-OM) organic matter
input in arable farming systems on microbial biomass
carbon concentrations and selected cellulolytic
enzyme activity in the soil. The systems being compared in a long-term ®eld trial are cereal rotations
that differ in their source of fertiliser (mineral or
organic), in whether a catch crop is grown, and
whether the straw is mulched or removed. In the
high-OM system only a small part of the nutrient
supply is provided by mineral fertilisation and the
nutrient supply to crops and soil microorganisms
will be mainly from organic inputs. The objective
of this research was to determine the extent of temporal variations in microbial biomass C concentration,
cellobiohydrolase, b-glucosidase and endocellulase
activity over two successive years. A second objective
was to evaluate the effects of high and low organic
matter input on soil quality indicators after eight years
of cultivation.
2. Materials and methods
2.1. Site and soil
The study was part of a ®eld experiment on the
integrated arable farming initiated in 1987, located
at Research Centre Foulum, Denmark. It is situated
at (568300 N and 98340 E, 50 m above mean sea
level). The soil is a loamy sand with 67% sand and
8% clay. Mean annual (1961±1990) temperature
is 7.38C and ranges from ÿ0.58C in January to
15.48C in July. Average mean annual (1961±1990)
rainfall is 626 mm, of which 50% falls in autumn and
winter.
Previously these ®elds were uniformly cultivated,
and had no organic manure additions for 14 years
before the start of the experiment. In the spring of
1988, randomized blocks were established, with three
replicate blocks per treatment. The treatments differed
mainly in the form of nutrients applied (Rasmussen
and Hansen, 1994). This study included a high-OM
treatment with high inputs of organic matter in the
form of pig slurry, a catch crop and incorporation of
straw, and a low-OM treatment with mineral fertilisers, no catch crop and straw removed (short stubble
remaining) at harvest (Table 1). Both high- and lowinput treatments received a reduced input of pesticides
(Secher et al., 1995). Each spring, 30 t pig slurry haÿ1
were applied to the high-OM treatment, supplemented
by ammonium nitrate (AN) evenly spread over the
surface (Table 1). The slurry was surface-applied,
then ploughed in. Straw left after harvest was cut
before mulching. The low-OM treatment received
mineral fertilisers (NPK and AN) in the spring. In
the high-OM treatment, a four-year crop rotation of
winter wheat, spring barley with a catch crop sown
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
211
Table 1
Soil properties, treatments, crop rotation and OM inputs
Year of initiation
Soil properties in 1995
Fertilizer practice
Ploughing
Mulching
Crop rotationb
High-OM
Low-OM
1988
Total C 1.7 (%), Total N 0.13 (%),
CEC 9 (meq 100 gÿ1), pH-CaCl2 5.9
Pig slurry (30 t) and AN a,
(80 kg mineral N and 30 kg organic N haÿ1)
Mouldboard ploughed, spring
straw
winter wheat, spring barley, fodder rapec,
1988
Total C 1.6 (%), Total N 0.13 (%), CEC 6
(meq 100 gÿ1), pH-CaCl2 5.7
NPK and AN (90 kg N haÿ1)
Mouldboard ploughed, spring
Straw removed
winter wheat, spring barley, spring barley, spring rape
spring barley=ryegrassd, spring rape
Annual input of OM
e
6477 kg haÿ1
0
a
Ammonium nitrate.
Crops at sampling time underlined.
c
Catch crop.
d
Undersown catch crop.
e
Average annual input of OM in the form of straw, ryegrass and pig slurry, stubble and roots not included.
b
after harvest, spring barley undersown with a catch
crop in the spring, and pea in the ®rst and spring rape
in the second crop rotation was practised. Fodder rape
(Brassica napus L. ssp. oleifera (Metzg.) Sinsk.), or
tansy-leaf phacelia (Phacelia tanacetifolia) was sown
after harvest of the ®rst spring barley crop in the
rotation was harvested. Perennial ryegrass (Lolium
perenne L.) was undersown in the second spring
barley crop in the crop rotation. In the low-OM
treatment a four-year crop rotation of winter wheat,
spring barley, spring barley and pea in the ®rst and
spring rape in the second crop rotation was practised.
No catch crop was sown in the low-OM treatment. In
1995 and 1996 both treatments were sown to a spring
barley, as the main crop. In the high-OM treatment
fodder rape was sown in autumn 1995 as a catch crop
and perennial ryegrass was the undersown catch crop
in 1996.
Soil was sampled (0±20 cm) on 20 occasions
between May 1995 and December 1996. On each
sampling date, four soil cores (2.5-cm diameter) were
collected at random from each block and pooled to
give one sample per replicate. Freshly sampled soil
was thoroughly mixed, and roots and visible plant
residues removed. Soil was frozen (approximately
ÿ188C) for later determination of microbial biomass
C and enzyme activity. Gravimetric water content was
determined by drying duplicate 10 g portions of soil at
1058C for 24 h.
2.2. Biomass C
Microbial biomass C was determined by the fumigation±extraction method Vance et al. (1987). Soluble
C and chloroform labile C (C solubilized by CHCl3
during an 18 h fumigation period) were extracted with
0.5 M K2SO4 in a soil : solution ratio of 1 : 3 (wt : v).
The extract was centrifuged and the supernatant ®ltered through a combusted (2508C, 5 h) Whatman GF/
C ®lter and a 0.22 mm Millipore Millex-GP ®lter unit.
Organic C in all 0.5 M K2SO4 extracts was determined
by an automated UV persulphate oxidation procedure
using a Dorhrman DC-180 Carbon Analyzer (Wu
et al., 1990). Biomass C was calculated as soluble
C in fumigated minus soluble C in non-fumigated soil
using 0.45 as the kc-factor (Kaiser et al., 1992) and
determined in duplicate.
2.3. Enzyme assays
The activity of b-glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91) and endocellulase (EC
3.2.1.4) were measured in ®eld moist samples by
the use of different ¯uorogenic substrates (see below).
During hydrolysis of the substrates, the aglycone 4methylumbelliferone (4-MU) is formed, which is
highly ¯uorescent at an alkaline pH. The assays were
performed according to the methods described by
Tilbeurgh and Clayssens (1985), with the following
212
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
modi®cations. The b-glucosidase activity was determined by the addition of 2.0 ml of 0.05 M acetate
buffer, (pH 5.0) to 20±80 mg soil. The mixture was left
for ®ve minutes in polyethylene tubes at 308C. The
enzymatic reaction was initiated by the addition of
0.5 ml substrate solution, and incubated for 20 min in
a water bath at 308C. The substrate concentration of 4methylumbelliferyl b-D-glycoside was 100 mM. The
reaction was stopped by the addition of 2.5 ml 96%
ethanol (v/v). The mixture was left for 20±30 min at
58C, then transferred into centrifuge tubes and centrifuged at 58C for 10 min at 1500 g. The supernatant
(4.5 ml) was transferred to a second polyethylene
tube, and 0.5 ml 2.5 M Tris buffer (pH 10) was added
to convert 4-MU into its ¯uorescent anionic form.
Fluorescence was determined with a Perkin±Elmer
¯uorometer (LS50) at 448 nm emission and 365 nm
excitation.
The activity of cellobiohydrolase was determined
using 4-methylumbelliferyl b-D-cellobioside as substrate. Soil samples of 50 mg were pre-incubated
with 2.0 ml 0.05 M acetate buffer, (pH 5.0) for ®ve
minutes, at 308C. The assay conditions and separation
of method for 4-MU were the same as described above
for b-glucosidase. The substrate concentration in the
assay was 100 mM. Endocellulase activity was determined using 4-methylumbelliferyl b-D-cellotrioside
as the substrate. The assay was performed in the same
way as described above, but the incubation period was
increased to 50 min. Soil samples of 50 mg were
used and the substrate concentration in the assay was
25 mM. All enzyme activity measurements were determined in six replicates.
2.4. Statistical models and analysis
A split-plot design with soil treatment as the main
plot treatment and sampling date as the sub-plot
treatment was used. Within each block the 20 measurements of each of the four variables, microbial
biomass C concentration, cellobiohydrolase, b-glucosidase and endocellulase activity, were analysed for
serial correlation by means of standard semi-variogram techniques (Cressie, 1991). Based on these
analyses it was concluded that the serial correlation
was negligible for all four variables, and so it was
ignored in subsequent analyses. A linear statistical
model with systematic interaction between treatment
and sampling date and with random block effect was
accepted. The correlation analysis did, however, indicate some variance heterogeneity between treatment
groups, and even between blocks. However, only
variance heterogeneity between treatment for the variables b-glucosidase and endocellulase activity was
signi®cant (p < 0.05). The analyses were performed
by means of the SAS-procedure PROC MIXED (SAS
Institute Inc., 1996a) using the REML-methodology
(Searle et al., 1992; Diggle et al., 1994).
In the subsequent statistical analyses the systematic
part of the models, i.e. treatment and sampling date
effect were analysed. For microbial biomass C concentration and cellobiohydrolase activity for which
variance homogeneity was applicable, the classical
analysis of variance was used (PROC GLM, SAS
Institute Inc., 1989). For the effects of b-glucosidase
and endocellulase, iterative REML-methods (PROC
MIXED, SAS Institute Inc., 1996b) were applied to
allow for treatment speci®c variances. In both models,
the sampling date speci®c treatment effect and the
difference between treatments of the average level per
year of the four variables were formulated as contrasts,
and hence analysed by standard techniques. The average level per year, for the four variables, was approximated by a trapeze-sum, in order to account for the
non-equidistant measurement of time points.
In order to test for a temporal trend over the twoyear observation period, a linear time trend was added
to the systematic part of the model. This linear time
trend had a treatment speci®c intercept for all variables, whereas the slope was treatment speci®c for
microbial biomass concentration and cellobiohydrolase activity, but the same across treatments for endocellulase and b-glucosidase activity in accordance
with the statistical model derived above. Furthermore,
either the sampling date factor or the sampling
date treatment interaction factor was treated as a
random effect in order to test the signi®cance of the
added linear term.
3. Results
Mean monthly rainfall and air temperature over the
two-year observation period are shown in Fig. 1. Total
precipitation for the two-year sampling period was
1010 mm, 242 mm below the long-term average.
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
213
Fig. 1. Top: Mean monthly air temperature (line) and mean monthly rainfall (bars) from January 1995 and to January 1997 at the sampling
site. Bottom: Annual management practices in low-OM and high-OM input systems. S. barley, spring barley; CC, catch crop; T, ploughing; O,
organic fertilizers; F, mineral fertilizers; H, harvest; S, straw mulching.
Autumn 1995 and 1996 were both relatively wet,
while the spring of 1996 as well as the summers of
1995 and 1996 were extremely dry (Fig. 1). The mean
temperature for the experimental period was 7.28C,
which is comparable with the long-term average. The
soil was frozen from December 1995 to April 1996,
with a mean temperature of ÿ38C, and a mean temperature of ÿ28C in November and December 1996
(Fig. 1). The prolonged periods of frost in 1995 caused
slow and weak development of the catch crop (fodder
rape) in the high-OM treatment.
With the exception of a higher CEC for high-OM
treatment, there was little or no difference in soil
properties between treatments (Table 1). Percentage
total carbon levels were similar for the soils of both
treatments.
Signi®cant effects of both treatment and sampling
date were found for all four variables studied
(p < 0.05). For microbial biomass C concentration
and cellobiohydrolase activity the treatment and the
sampling date effects interacted signi®cantly, which
resulted in the treatment effect varying over the sampling period. The interaction between sampling date
and treatment implied that the model-predicted level
for each sampling date and treatment was equal to the
average of the true measurements for sampling date
and treatment (Fig. 2(a) and (b)). For b-glucosidase
and endocellulase activity the treatment effect was
constant over the sampling period and as a result the
predicted levels differed from the measured averages
for b-glucosidase and endocellulase activity (Fig. 2(c)
and (d)). In the treatment with high-OM input the soil
had consistently higher biomass C levels than low-OM
soil when all sampling dates were considered together
(p < 0.01). We found no signi®cant differences
between the summer and autumn sampling dates of
the respective years (Fig. 2(a)). There was no signi®cant linear temporal trend in microbial biomass C
concentration over the two-year period. Mean annual
biomass C concentration, calculated by the trapezesum on the data in Fig. 2(a), differed signi®cantly with
organic matter input (Table 2).
The high-OM treatment had consistently higher
cellobiohydrolase activity (24±92%) than the lowOM treatment at all sampling dates (p < 0.001). The
difference between treatments was signi®cant when
214
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
Fig. 2. Measured and model-predicted average levels of microbial biomass C concentration and extracellular enzyme activity in soils with
cereal rotation, with low-OM and high-OM organic matter input, April 1995±December 1996: (a) Microbial biomass C concentration; (b)
Cellobiohydrolase activity; (c) b-glucosidase activity; (d) Endocellulase activity. (*) measured and (ÐÐÐ) predicted values in low-OM
treatment, (*) measured and (- - -) predicted values in high-OM treatment.
Table 2
Mean annual microbial biomass C concentration (MB-C), and extracellular enzymes activity in 1995 and 1996 in soils with cereal rotation, as
related to low (low-OM) and high (high-OM) organic matter input
1995
Microbial biomass C (mg C gÿ1 soil)
Cellobiohydrolase activity (n mol 4-MU gÿ1 hÿ1)
b-glucosidase activity (nmol 4-MU gÿ1 hÿ1)
Endocellulase activity (nmol 4-MU gÿ1 hÿ1)
1996
Low-OM
High-OM
Low-OM
High-OM
160.9
115.1
114.5
30.6
184.6xx a
162.1xxx b
165.7xxx b
41.1xxx b
140.4
130.5
133.5
38.5
182.8xxx b
185.3xxx b
184.5xxx b
49.0xxx b
Note: Means per treatment and experimental year were estimated by trapeze-sum method. The p-values were derived from statistical tests
comparing the predicted yearly average level in low and high-OM level.
a
p < 0.01.
b
p < 0.001.
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
considering the whole entire time-course (p < 0.001),
but not on all individual sampling dates (Fig. 2(b)).
Soil with high-OM input had the greatest mean cellobiohydrolase activity in both years (Table 2). The
overall temporal trend showed fairly uniform cellobiohydrolase activity for most of the sampling dates
for both treatments, with a slight increase throughout
the experimental period (p < 0.05), indicating increasing cellobiohydrolase activity between 1995 and 1996
in both treatments.
Temporal ¯uctuations in b-glucosidase activity
were distinct in both treatments (Fig. 2(c)). There
was a signi®cant effect (p < 0.0001) of sampling date
on b-glucosidase activity, demonstrated by low activity from August 1995 to April 1996 and relatively high
activities on the other sampling dates (Fig. 2(c)). The
effect of treatment was constant and signi®cant over
all sampling dates (p < 0.001). b-glucosidase activity
followed a similar temporal pattern in the two treatments and was ca. 40% higher in the high-OM treatment than in the low-OM treatment on all sampling
dates. There was no signi®cant linear temporal trend in
b-glucosidase activity. The average difference in bglucosidase activity between high-OM and low-OM
was highly signi®cant (Table 2).
Temporal variations in endocellulase activity
showed a different pattern from those for b-glucosidase activity, with the highest activity in the autumn/
winter and early summer sampling samplings
(Fig. 2(d)). Despite temporal ¯uctuations of endocellulase activity the effect of treatment was constant and
signi®cant over all sampling dates (p < 0.001). On all
sampling dates, the endocellulase activity in the highOM treatment was ca. 30% higher in the low-OM
treatment (Fig. 2(d)) and the average level over the
two years differed signi®cantly with organic matter
input (Table 2). There was a consistent and signi®cant
temporal trend in endocellulase activity in both treatments (p < 0.05), indicating that endocellulase activity
increased during the two years.
4. Discussion
4.1. Temporal trends
Microbial biomass C concentration and extracellular soil enzymes of the cellulolytic complex displayed
215
marked temporal variability, similar in both low- and
high-OM treatments (Fig. 2). Different amounts of
annual OM input (Table 1) did not have any large
in¯uence on temporal variations of microbial biomass
C concentration and enzyme activity. This suggests
that temporal variations may have been driven by
climatic (e.g. temperature and moisture) factors and
by crop growth, i.e. by factors common to both
systems. Temporal changes in soil moisture, soil
temperature, and C input from crop roots, rhizosphere
products (i.e. root exudates, mucilage, sloughed cells,
etc.), and crop residues can have a large effect on soil
microbial biomass and activity (Ross, 1987). Crop
growth often stimulates an increase in the size of
microbial biomass during the growing season (Lynch
and Panting, 1982; McGill et al., 1986; Fraser et al.,
1988; Kaiser and Heinemeyer, 1993). Granatstein et
al. (1987) observed a signi®cant increase in microbial
biomass after harvest. Patra et al. (1990) argued that
biomass C concentration remained fairly constant
during the year due to a relatively even distribution
of litter with time. In this study, however, the peak in
microbial biomass C concentration occurred in early
May 1995 when no crop growth had yet taken place
(Fig. 2(a)). The autumn (October 1995) increase in
biomass C concentrations coincided with the onset of
rain (Fig. 1) and with the addition of new crop residues. Moisture probably became a limiting factor
during the summer 1995 (Fig. 1), with the lowest
biomass levels occurring in July (Fig. 2(a)). In contrast, a signi®cant increase in microbial biomass was
observed in July 1996 during the period of lowest soil
moisture. In many studies, the temporal variations of
microbial biomass were attributed to variations in soil
water content. Wetting and drying of soil has both
been shown to increase (Ross, 1989; Joergensen et al.,
1994), lower (Kieft et al., 1987; van Gestel et al.,
1992) or to have no effect on soil microbial biomass
levels (Ross, 1989; van Gestel et al., 1992).
Enzyme activity displayed different temporal patterns of the cellulase complex of enzymes as revealed
in different assays (Fig. 2(b)±(d)). According to Bolton et al. (1985), Martens et al. (1992) and Dick (1994)
some cellulases are closely related to inputs of fresh
organic materials, plant growth and plant residues,
while others appear to be more sensitive to soil
temperature and moisture. Temporal variations in
community structure and activity, induced by manure
216
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
decomposition and crop residue decomposition, may
modulate microbial biomass and the extracellular
enzymes that are released into the soil by the microbial
biomass (Fauci and Dick, 1994; Ritz et al., 1997;
Bossio et al., 1998).
Temporal variations in the size of microbial biomass C concentration and extracellular enzyme activities can be differentiated from that representing longterm changes in the biomass and enzyme activity in
response to inorganic fertiliser, organic manure, straw
incorporation or crop rotation with the statistical
approaches used in this study. Cellobiohydrolase
and endocellulase activity over two years showed a
consistent trend, i.e. a steady increase in the enzyme
activity in both high and low-OM treatments, but such
an increase did not appear in biomass C and bglucosidase. An increase in cellobiohydrolase and
endocellulase suggested that there were fundamental
differences in C-supplying mechanisms to the microbial community in both treatments in addition to
current-season manure or crop-derived inputs (Ritz
et al., 1997). These results suggest that a new equilibrium had not yet been attained after the change in
management. Following a sustained management
change such as the addition of organic fertiliser, crop
residues and crop rotation, it may take many years for
the soil organic matter to reach a new equilibrium (van
der Linden et al., 1987).
4.2. Soil quality and fertility
With the increasing emphasis on sustainable agriculture and soil quality it is important to determine the
effect of different organic inputs on soil organic matter
(Christensen and Johnston, 1997). Changes in the
quality and quantity of soil organic matter occur
slowly. These changes are dif®cult to quantify in
short-term studies because they are small in relation
to the large background of organic matter and the
Ê gren, 1991). Due
natural soil variability (Bosatta and A
to their dynamic nature, soil microbial biomass and
soil enzymes respond quickly to changes in organic
matter input (Powlson and Jenkinson, 1981; Powlson
et al., 1987; Dick, 1994).
In this study, no changes in total carbon levels in
soils were observed after eight years (Table 1). The
increased levels of microbial biomass C concentration
and cellulolytic enzyme activity in the high-OM treat-
ment (Table 2) re¯ect high annual inputs of organic
matter (6477 kg haÿ1) in the form of pig slurry, straw
residue and winter cover crop. Several studies have
looked at the shifts in microbial numbers and activity
in soil as related to changes in C inputs. In some
studies they were attributed to amount and diversity of
organic inputs (Fraser et al., 1988; Knudsen et al.,
1999; Martyniuk and Wagner, 1978; Powlson et al.,
1987). The importance of microbial biomass and
extracellular enzyme activity in assessment of soil
quality is established by the essential role of soil
microorganisms in nutrient cycling within agricultural
ecosystems (Rice et al., 1986). Of particular importance are free enzymes in the extracellular space, for
example for the initial depolymerization steps of
cellulose (Burns, 1982).
5. Conclusions
Large temporal variation in microbial biomass C
concentration and extracellular enzyme activity were
observed, which could be related to environmental
factors (e.g., temperature and moisture) and crop
growth but not differences in organic matter input.
Microbial biomass C concentrations and the activity
of extracellular cellulases appeared to be very good
indicators of early changes in soil biological status.
Biomass C concentration and extracellular enzyme
activity increased (30%) as a consequence of changing the cultivation system from one of low to one of
high organic matter input after eight years. In addition,
our results demonstrated that due to temporal variability repeated sampling is required to calculate annual
estimates of the size of microbial biomass C concentration and cellulolytic enzyme activity for the purpose of comparative ecosystem study.
Acknowledgements
We thank Anette Clausen, Ewa Hùrsdal, Bodil
Mùllnitz and Erling Nielsen for their ®eld, laboratory
and technical assistance and Gunnar Mikkelsen for
fruitful discussion and help in choosing the treatments.
Funding was provided by the AIR-3 Programme of
European Union, the Danish Ministry of Food, Agriculture and Fisheries Programme `Research on
K. Debosz et al. / Applied Soil Ecology 13 (1999) 209±218
Organic Farming' and the Danish Environmental
Research Programme `Centre for Sustainable Land
Use and Management of Contaminants, Carbon and
Nitrogen'.
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