soil microbial biomass: its assay and role in turnover of organic matter c and n
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Soil microbial biomass: its assay and role in turnover
of organic matter C and N
Jeffrey N. Ladda,*, Maurice Amatoa, Hans A. van Veenb
aCSIRO Land and Water, Private Bag 2, Glen Osmond, South Australia 5064, AustraliabCentre for Terrestrial Ecology, Netherlands Institute of Ecology—KNAW, P.O. Box 40, 6666 ZG Hateren, The Netherlands
1. An assay for soil microbial biomass
1.1. Assay of fumigated-incubated (FI) soils
Throughout the 1970s and 1980s, we were involved in
studies on the decomposition of 14C- and 15N-labelled plant
residues, as part of a massive program on symbiotic nitrogen
fixation in cereal/legume rotations, and the supply of
legume N to crop and soil. Studies inevitably included
measurements of soil microbial biomass C and N (both
labelled and unlabelled) using the exciting new technique of
Jenkinson and Powlson (1976). As all soil microbiologists
now know, the basis of their assay was to measure CO2
evolved from soils that had been chloroform-fumigated for
1 day then, after chloroform removal, incubated aerobically
for 10 days. This is commonly referred to as the FI
treatment. The CO2 values were corrected for the amounts
evolved from nonfumigated control soils and biomass C
calculated.
This assay seemed to work well enough for biomass C
and 14C with the small number of soils used in our early
studies. Later, using an extended range of soils, we found
that CO2 release from the control soils commonly exceeded
50% of that from the FI-treated soils, which may have given
rise to underestimations of biomass C values. On occasions,
we calculated, as did others, negative values for biomass.
Jenkinson (1988) has appraised the conditions under which
underestimations of biomass C values were most likely and
has discussed the use of alternative controls. The problem of
choosing a ‘best option’ control treatment might be lessened
by basing the assay on net N mineralization rather than on C
mineralization. This approach had appeal but it too had
problems.
1.2. Assay of fumigated-only (F) soils
Other approaches have sidestepped the decision on the
most appropriate treatment for control soils by using soils
that were fumigated for 1 day (or longer), but without the
subsequent aerobic incubation. The amounts of total C or N
of soil extracts could then be measured and corrected for the
amounts in soil extracts prior to fumigation, thus forming
the basis for biomass calculations (Brookes et al., 1985;
Vance et al., 1987; Tate et al., 1988). Our contribution was
to base measurements on ninhydrin-reactive N, formed and
extracted from fumigated soils under conditions which were
shown to curb oxidative reactions and N immobilization
processes. Evidence for such conditions was two-fold. (i)
Protease/peptidase activities towards substrates (sodium
caseinate, a substituted amide or substituted dipeptides)
were either not or were only marginally affected by soil
fumigation (even up to 10 days), whereas dehydrogenase
activities were irreversibly inactivated even within 1 day of
fumigation. (ii) Glucose and ammonium N and nitrate N
amendments were completely recovered from soils held
under chloroform for up to 10 days but were readily
metabolised with complete net immobilization of amend-
ment N by nonfumigated soils incubated for a similar
period.
Extracted ninhydrin-reactive N of fumigated-only soils
was attributed mainly to amino acid N and ammonium
N. Ninhydrin-reactive N from 10-day fumigated soils
usually accounted for about 40–60% of ammonium N
extracted from soils fumigated for 1 day then incubated
without fumigant for a further 10 days. In some circum-
stances, viz., when soils were pretreated with glucose or
wheat straw amendments, ninhydrin-reactive N from
fumigated-only soils exceeded that from the respective
fumigated-incubated soils.
0038-0717/$ - see front matter q 2004 Published by Elsevier Ltd.
doi:10.1016/j.soilbio.2004.05.001
Soil Biology & Biochemistry 36 (2004) 1369–1372
www.elsevier.com/locate/soilbio
* Corresponding author.
E-mail address: [email protected] (J. N. Ladd).
1.3. FI and F treatments of ‘reference’ microbial cells
Research at Rothamsted (Jenkinson and Powlson, 1976)
and Braunschweig (Anderson and Domsch, 1978) had
allowed calculation of microbial biomass C of soils by
utilizing the mean percentages of cell C released as CO2
from suites of organisms, which had been fumigated then
incubated under stated procedures. The percentages of cell
C so converted varied somewhat but were such that a mean
could be struck and used in a general measure of biomass
C. Cell C content and the percentage mineralization of cell
C appeared to range less widely than did cell N content and
the percentage mineralization of cell N. In the case of the FI
treatment, the C:N ratios of the decomposing dead cells
were considered to influence the relative extent to which
dead cell N was mineralised and immobilized during the
decomposition processes (Jenkinson and Ladd, 1981).
A defining moment for us was reached in 1979 when we
compared specific microorganisms of different C:N ratios
for their relative release of (i) ammonium, after fumigation
with chloroform for 1 day followed by chloroform removal
then aerobic incubation for 10 days, and of (ii) ninhydrin-
reactive N after 10 days fumigation with chloroform. Our
aims were to demonstrate, first, under the FI treatment a
range of percentage mineralization of cell N which was
inversely related to cell C:N ratio, and second, under the F
treatment with N immobilization curbed, a much narrower
range of percentage release of ninhydrin-reactive N. We
used as ‘reference’ organisms a Streptomyces sp., C:N ratio
3.8:1, a Pseudomonas sp., C:N ratio 4.1:1, a Bacillus sp.,
C:N ratio 4.7:1, a Penicillium sp., C:N ratio 8.3:1 and a
Rhizoctonia sp., C:N ratio 11.3:1. Pure cultures of these
organisms were freshly harvested and added as washed cell
suspensions to a sand of low C and N contents.
After the FI treatment all organisms were extensively
decomposed, as judged by the large conversion of cell C to
CO2 C (51–60%). As predicted, the percentage release of
cell N as ammonium N was inversely related to the C:N
ratio of the added organisms, ranging from 66% (Pseudo-
monas sp.), to 33% (Penicillium sp.), to only 16%
(Rhizoctonia sp.). By contrast, under the F regime, maximal
release of ninhydrin-reactive N (approximately 30% cell N),
was achieved with Rhizoctonia sp. and Bacillus sp.,
followed by 20% with Penicillium sp. In the case of
Rhizoctonia sp., the percentage release of ninhydrin-
reactive N from the F-treated cells far exceeded the very
low release of ammonium N achieved with the FI-treated
cells and was consistent with an improved net release of the
fungal cell N when N immobilization was curbed. However,
the unexpected and very disappointing results were
provided by the behaviour of the Streptomyces sp. and
Pseudomonas sp., both of low C:N ratio, both giving high
release of cell N under the FI regime (50% and 66%,
respectively), but both giving very low release of cell N
under the F regime (9% and 8%, respectively). The results
with these admittedly few microbial species served to
indicate that whereas differences between organisms in the
percentage release of cell N under the FI regime were
probably due to differences in the extent of N immobiliz-
ation linked to cell C:N ratios, differences under the F
regime were due to other factors.
1.4. Correlation between microbial biomass C
and ninhydrin-reactive N
Further investigations with a greater range of microbial
species were desirable but were not possible at that time due
to other pressures, in particular the ever-increasing demands
of our field-based program, The biomass methodology
project was shelved and not revisited for five or so years. In
1984, one of us (JNL) visited the UK and had very useful
discussions with David Jenkinson and Phil Brookes at
Rothamsted, who described the development of their assays
for soil microbial biomass C and N, based on analysis of
extracted C and N from soils that had been fumigated-only.
This was the stimulus we needed and we (JNL and MA)
decided to expand our studies of ninhydrin-reactive N of
extracts of fumigated-only soils to develop an analogous
assay. When time eventually permitted, we demonstrated
strong and direct correlations ðP , 0:001Þ between
microbial biomass C (based on net CO2 evolution from 23
FI-treated soils), and ninhydrin-reactive N of both FI-treated
and F-treated soils. It is the latter that is described in the
highly cited paper by Amato and Ladd (1988). Our earlier
plans to develop an assay of microbial biomass based on the
mean of a narrow range of values of ninhydrin-reactive N
released from fumigated ‘reference’ organisms were
abandoned.
Our assay routinely involved a 10-day fumigation period
of soils to ensure a slowing, near-maximal release of
ninhydrin-reactive N and, importantly, to permit a direct
comparison with C and N mineralization from FI-treated
soils. Reducing the soil fumigation period from 10 days to 1
week (or 1 day) may be advantageous if there is no loss of
sensitivity or accuracy and where routine analyses of large
numbers of samples are required. We recognised that values
for soil microbial biomass C calculated from the ninhydrin-
based assay of F-treated soils were a general measure only
and, as with other assays, depended upon the relationship
established with the FI assay, which was itself subject to
some uncertainties, as mentioned above. General values for
soil microbial biomass N could be calculated from estimates
of microbial biomass C and a recommended value for the
C:N ratio of the soil biomass e.g. 6.7:1 (Shen et al., 1984).
Two of the attractions of the ninhydrin-based assay of
fumigated-only soils are its simplicity and its speed of
analytical procedures. Furthermore, the assay is precise and
sensitive and has wide applicability. For example, it can be
used to measure microbial biomass in soil fractions, e.g.
microaggregates of particle sizes within the 2–250 um
diameter range (Jocteur Monrosier et al., 1991). Also, the
ninhydrin-based assay for biomass C can be successfully
J. N. Ladd et al. / Soil Biology & Biochemistry 36 (2004) 1369–13721370
applied to whole, unbuffered acidic soils of pH levels at
which the CO2-based assay of FI-treated soils fails
completely (e.g. Amato and Ladd, 1994). Nevertheless,
caution is also required using fumigated-only soils. For
example, Ladd et al. (1994) showed that an apparent decline
in microbial biomass C and N in soils of increasing acidity
in response to high fertilizer N treatments, was due in part,
to a reduction in the assay performance at lower pH values.
2. Simulation modelling of C and N behaviour in soils
2.1. Microbial biomass: a focus of C and N flow in soils
During the period of intensive appraisal of the various
assays of soil microbial biomass, models of soil organic
matter dynamics were also being devised to simulate the
flows of C and N through soil organic matter pools of
different size and stability. Most emphasis was placed on
pools that reflected the availability of organic constituents
for biochemical transformations, e.g. soluble organics,
chemically resistant or physically protected organic
materials. Microbial biomass C and N, a relatively small,
active pool accessible to experimental measurement,
provided the focus for C and N flow in soils. Several
models contained an explicit description of C and N
turnover through microbial biomass. One such model was
developed by Hans van Veen (then at Research Institute
ITAL, Wageningen, The Netherlands), to describe the
turnover of C and N in Dutch and Canadian soils.
We were very pleased when, in 1982, the opportunity
arose for Hans to visit Adelaide and work in our laboratories
for about 6 months. Our aim then was to simulate, with
minimal changes to model parameters, the behaviour of 14C
and 15N in two soils of contrasting clay contents, following
soil amendment with three isotope-labelled substrates—
glucose/ammonium, live microbial cells and legume
residues. Whereas the development of the ninhydrin-based
biomass assay rested on several small experiments carried
out spasmodically over a number of years, the modelling of
the short-term metabolism of C and N of added substrates
resulted from an intensive program of sampling and analysis
of incubated, amended soils and model adjustment.
The latter study resulted in three papers published in Soil
Biology and Biochemistry, the first of which was the highly-
cited paper by van Veen et al. (1985) on the turnover of
glucose 14C and ammonium 15N.
At the time, it was well recognised that clay or clay-
related factors had a stabilizing and protective influence on
soil organic matter and microbial cells and biomass (Paul,
1984). Our later studies with 23 soils demonstrated a strong,
direct correlation between soil clay content and microbial
biomass 14C, residual in soil after 44 weeks of 14C-glucose
decomposition (Amato and Ladd, 1992). The key exper-
iment with Hans included a treatment where isotope-
labelled glucose-ammonium substrate was added to two
soils, a sandy loam and a highly aggregated clay, and were
incubated under constant temperature and moisture con-
ditions for up to 101 days. Microbial biomass 14C was
assayed by the FI treatment, the assays with fumigated-only
soils not being finalised at the time. Experimental 14C data
showed a large consistent soil effect on microbial biomass
formation and turnover. The model was reformulated to
include unique features to allow explicit modelling of the
soil effects.
First, it was proposed that for a given set of conditions a
soil had a characteristic capacity to protect and preserve its
microbial biomass. If the amounts of microbial biomass in a
given soil were less than or equal to the protection capacity
of the soil, then the biomass was treated by the model as
totally protected and decay rates were relatively low. In our
experiment, the protective capacities of each soil were set as
the mean value for soil sampled under young (,3 months)
pastures, i.e. for soil which had been neither recently
disturbed nor recently amended with plant residues.
Microbial biomass generated in excess of the soil’s
protective capacity was considered to be unprotected, for
which decay rates were set much higher. Second, it was
proposed that micro-organisms and their immediate organic
products of decay formed a semi-closed system from which
materials leaked out to become temporarily unavailable,
according to soil clay content and soil moisture conditions.
Input data for this were obtained by trial and error to fit the
model output to the data set of the clay soil, after which the
same input data were maintained throughout the entire
modelling exercise. Third, soils may influence the efficiency
of substrate metabolism.
2.2. Model outputs: 14C and 15N
Thus, three features of the model were proposed to
explain the differences in 14C turnover in the two soils, viz.,
the clay soil (i) had a greater capacity than the sandy loam to
protect microbial biomass, (ii) provided a sorptive, high
surface area environment for closer interaction between
micro-organisms and their immediate products of decay,
and (iii) promoted a higher efficiency of utilization of
glucose and metabolic products for biomass production. The
fit of model output and experimental data for 14CO2,
residual organic 14C and microbial biomass 14C was very
close.
Modelling 15N behaviour was less successful than 14C.
A further modification to the model, viz., adjusting, for
each soil, the proportions of organic N and 15N allocated in
the model to the old fraction and active protected fraction,
resulted in a fair description of inorganic N and 15N
immobilization and remineralization in the high-clay soil.
The match in the sandy loam was less satisfactory. For
both soils, amounts of biomass 15N, as model outputs, were
substantially greater than amounts estimated by utilizing
published information on kN values. These results
presented us with a delicious (and still unsolved) dilemma.
J. N. Ladd et al. / Soil Biology & Biochemistry 36 (2004) 1369–1372 1371
In the model the fate of N and 15N was determined by C
turnover. Using C:N ratios of the different organic pools,
including biomass, flows of N were calculated according to
the flow of C from one pool to the other. Either the model
overestimated the amounts of microbial biomass 15N and
needed further changes, perhaps to the present built-in
relationship whereby the C:N ratio of microbial biomass also
varied with inorganic N concentration; or alternatively,
perhaps biomass 15N was underestimated, when calculated
using kN values published in procedures more suited to
general assessments. Soil biota are very diverse. Their
locations in soil and their generalised properties, including
their reaction to soil fumigation, likely may differ from those
of the primary and secondary populations of organisms
growing in soils in response to addition of specific substrates.
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