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Page 1: Soil microbial biomass: its assay and role in turnover of organic matter C and N

Citation Classics

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).

Page 2: Soil microbial biomass: its assay and role in turnover of organic matter C and N

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

Page 3: Soil microbial biomass: its assay and role in turnover of organic matter C and N

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

Page 4: Soil microbial biomass: its assay and role in turnover of organic matter C and N

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.

References

Amato, M., Ladd, J.N., 1988. Assay for microbial biomass based on

ninhydrin-reactive nitrogen in extracts of fumigated soils. Soil Biology

& Biochemistry 20, 107–114.

Amato, M., Ladd, J.N., 1992. Decomposition of 14C-labelled glucose and

legume material in soils: properties influencing the accumulation of

organic residue C and microbial biomass C. Soil Biology & Biochemistry

24, 455–464.

Amato, M., Ladd, J.N., 1994. Application of the ninhydrin-reactive assay

for microbial biomass in acid soils. Soil Biology & Biochemistry 26,

1109–1115.

Anderson, J.P.E., Domsch, K.H., 1978. Mineralization of bacteria and fungi

in chloroform-fumigated soils. Soil Biology & Biochemistry 10,

215–221.

Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloro-

form fumigation and the release of soil nitrogen: a rapid direct

extraction method for measuring microbial biomass nitrogen in soil.

Soil Biology & Biochemistry 17, 837–842.

Jenkinson, D.S., 1988. Determination of microbial biomass carbon and

nitrogen in soil. In: Wilson, J.R., (Ed.), In Advances in Nitrogen

Cycling in Agricultural Ecosystems, CAB International, Wallingford,

UK, pp. 368–386.

Jenkinson, D.S., Ladd, J.N., 1981. Microbial biomass in soil: measurement

and turnover. In: Paul, E.A., Ladd, J.N. (Eds.), In Soil Biochemistry,

vol. 5. Dekker, New York, pp. 415–471.

Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on

metabolism in soil-V. A method for measuring soil biomass. Soil

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Jocteur Monrosier, L., Ladd, J.N., Fitzpatrick, R.W., Foster, R.C., Raupach,

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