III.

THE MICROBIAL BIOMASS

Required Readings:

Ley, R.E., D.A. Lipson and S.K. Schmidt. 2001. Microbial biomass levels in barren and
vegetated high altitude talus soils. Soil Sci. Soc. Am. J. 65:111–117.

Follett, R.F. and D.S. Schimel. 1989. Effect of tillage practices on microbial biomass dynamics.
Soil Sci. Soc. Am. J. 53:1091-1096.

Suggested Reading:

Jenkinson, D. S. and J. N. Ladd. 1981. Microbial biomass in soil: Measurement and turnover. p.
415-471. In E. A. Paul and J. N. Ladd (eds.), Soil Biochemistry, Volume 5. Marcel Dekker, New
York.

Tunlid, A and D. C. White. 1992. Biochemical analysis of biomass, community structure,

nutritional status, and metabolic activity of microbial communities in soil. p. 229-262. In G.
Stotzky and J.-M. Bollag (eds.), Soil Biochemistry, Volume 7. Marcel Dekker.

The soil microbial biomass acts as the transformation agent of the organic matter in soil. As

such, the biomass is both a source and sink of the nutrients C, N, P and S contained in the

organic matter. It is the center of the majority of biological activity in soil. To properly

understand biological activity in soil one must therefore, have knowledge of the microbial
biomass. Investigating the flow of C and N in the soil, from newly deposited plant or other
materials to the mineral forms of carbon dioxide and ammonium or nitrate ions, clearly shows

the central role of the microbial biomass.

The definition of the soil microbial biomass is the living portion of the soil organic matter,

excluding plant roots and soil animals larger than 5 x 10-3 um3. The microbial biomass generally

comprises approximately 2% of the total organic matter in soil and it may be easily dismissed as

of minor importance in the soil. However, this chapter will introduce the biomass as an important

agent in controlling the overall biological activity of the soil.

Methods of Measuring Microbial Biomass Pool Sizes. Microbial biomass has been measured in

soil by a variety of methods. The ideal method would be rapid, sensitive, and distinguish living

and active microbial cells from the microbial biomass that is essentially dormant or unresponsive

to the addition of a readily available C source to the soil system. The classical procedure for the

estimation of microbial biomass is by direct microscopy. Biovolume of microbial cells in a

microscopic field is determined and the biovolume is then converted to biomass. For conversion

of biovolume data to biomass C values, it is necessary to know cell density, cell matter content,

and the C content of the dry matter. A conversion factor of 0.13 has been determined assuming

the wet density of soil microorganisms is 1.1 g cm-3, a dry matter content of 0.28 g g-1, and a C

content of microbial cells of 0.47 g g-1. Using these assumptions we obtain

(1.1 g cm-3)(0.25 g g-1) 0.47 g g-1) = 0. 13 g cm-3

and

biovolume (in cm3 g-1) x 0.13 g cm-3 = biomass C (g g-1).

A second procedure is to extract a specific component of the microbial biomass and use the

concentration of this component to estimate the concentration of the total biomass. For a specific

biomass component to accurately reflect the quantity of the microbial biomass in soil it must
meet the following requirements.
 1. It must be present in all of the biomass at a known concentration.

2. It must be present in living organisms only and must not accumulate in the soil in

nonliving material.

3. It must be extracted quantitatively from soil.

4. It must have a reliable and accurate method of analysis once it is extracted.

Several components have been proposed to estimate and evaluate the microbial biomass in soil.

The chapter by Tunlid and White (1992), listed above, provides an overview of these various

components and their relative strengths and weaknesses for such purpose. One advantage of a

chemical component is that, in contrast to other methods, it can also give information on biomass

content, community structure and the metabolic activity of the microflora.

Probably the most commonly used classical technique for determining the microbial biomass

size is that of chloroform fumigation. Fumigation ruptures microbial cells and releases cell walls

and cellular contents into the soil. If incubation follows the fumigation, a "flush of

decomposition" of the soil organic matter occurs that is due to the decomposition of

microorganisms killed during fumigation. When the fumigation and subsequent incubation

procedures are conducted under a strict set of conditions, the size of the biomass pool of C can

be determined by the size of the carbon dioxide flush using the equation

B = F/kc

where B is the concentration of biomass C in the soil, F is the amount of carbon dioxide evolved

from the fumigated soil minus that from the unfumigated soil, and kc is the fraction of killed

biomass C which subsequently is evolved as carbon dioxide. Five assumptions are implicit in the

calculation of biomass C from the carbon dioxide flush associated with chloroform fumigation.

1. The C in killed microorganisms is mineralized to carbon dioxide more rapidly than that in

living microorganisms.
2. The kill caused by the fumigation is essentially complete.
 3. The fraction of microorganisms that die in the unfumigated control is negligible

compared to those killed in the fumigated sample.

4. The fraction of the killed biomass that is mineralized (kc) is the same in different soils,

i.e. a single kc value can be used to estimate biomass C in a wide variety of soils.

5. Fumigation has no effect on the soil other than the killing of the microbial biomass.

Both chloroform carbon dioxide and methyl bromide have been used as fumigants. Generally

with chloroform, the kill is 99% or greater but enough microorganisms survive for an inoculum

not to be required. However, an inoculum is required when methyl bromide is used.

A variation of the fumigation method is to do an extraction of the soil after chloroform

fumigation. The C content in the extractant is then compared to the amount of C in a similar soil

that has not been fumigated. The difference is due to the C released from the microbial biomass.

An extraction efficiency (Kec factor) of 0.45 (or a close similar value) is often used to calculate

the microbial biomass C value using the fumigation-extraction method.

The chloroform fumigation technique and the application of equations similar to that used for

biomass C determinations have also been used to determine the pool sizes of microbial biomass

N, P, and S. Microbial S and P measurements have the advantage in that the extraction of the

mineral forms of S and P can be carried out immediately after chloroform treatment, eliminating

the lengthy delay and unknown mineralization and immobilization rates that occur during

incubation. Extraction of C and N constituents within the microbial biomass immediately after

chloroform fumigation has not proven as successful.

The k factors determined for C, N, P, and S are all of the same order of magnitude. However,

exact agreement is not observed and would not be expected because of the different experimental
protocols used for each element. Differences in incubation times after fumigation, differences in
extractability of the mineral forms of the nutrients, and the relative ratio of the extracted form of

the nutrient compared to the total concentration of the nutrient in the biomass fraction all work to

cause variation in the k factors.

Another method of determining the size of the microbial biomass includes the initial respiratory

response method. This method measures the initial maximum respiration rate in a soil upon

addition of an easily degraded C source such as glucose. Microcalorimetry determines the

microbial biomass by measuring the rate of heat output from soil and relating this heat output to

the size of the microbial biomass.

Dynamics of the Microbial Biomass in Soil. When discussing the microbial biomass in soil, two

different situations may be explored. In the first, the biomass size is considered to remain

relatively constant over time and C plus other nutrients simply flow through the biomass. Carbon

and other nutrients enter the biomass as complex organic forms and leave as carbon dioxide or

mineralized forms of the elements.

Organic C, N, P, S <------> microbial <-------> CO2, mineral N,

biomass mineral P, mineral S

The second case involves the addition of a substrate to soil which stimulates the growth of the

microbial biomass and the fate of the nutrients in the organic material may result in their release

to the mineral form or they may become incorporated in the expanding microbial biomass pool.

Each case must be considered separately.

If the first case is investigated, the mathematical equation describing the turnover of the

microbial biomass can be described using the first-order Michaelis-Menten equation.

k = (2.303/t)log(x/x-a)
where k is the turnover factor, t is time, x is the dry weight of the microbial biomass pool size,

and a is the production of new material per hour. The turnover time of the microbial biomass

during a steady state condition, i.e. where there is no change in the biomass size with time, can

be given by

t = 2.303 log (x/x-a)]/k

The validity of this approach can be determined by adding a pulse of 14C-labelled substrate to

soil. The amount of C must not be too great so that the system is perturbed as little as possible.

The movement of the labelled substrate through the microbial biomass can be followed by

measuring 14CO2 output and the amount of 14C in the biomass by the biomass measurement

procedures mentioned previously.

The second case involves stimulation in the size of the microbial biomass pool itself by the

addition of substrate. The relationship between the growth in the biomass and the breakdown of

the substrate is

Bt = Bto ekt

where Bt is the biomass at time (t) and Bto is the initial amount of biomass present in the soil, and

k is the rate constant.

Similar equations, but with a negative sign before the rate constant (k) could be used to follow

the decrease in the microbial biomass size. The time required for the microbial biomass to fall to

one-half its initial value is the half-life of the biomass in the soil. Using the above equation but

with a negative rate constant and equating Bt equal to 0.5Bto, the half life (t1/2) can be expressed

as ln 2/k or 0.693/k.
As with the steady state condition mentioned previously, the relationship between microbial

biomass and substrate utilization is most easily studied under laboratory conditions. With 14C

and 15N-labelled substrate, changes in the organic and inorganic components of the soil system

can be followed. Using 14C-labelled acetate and 15N-labelled (NH4)2SO4, the following concepts

were developed. Applying these concepts to a computer model provided results that agreed

closely with observed results.

1. Two biochemically separate populations develop sequentially.

2. The primary microbial population serves as a sink for the added acetate C and other more

complex compounds.

3. The secondary population utilizes microbial metabolites and soil organic C but not added

C.

4. All populations undergo some cryptic growth (i.e. growth due to unidentified means or

use of C for maintenance).

5. The quantity of C and N entering a given population is a function of the size of that

population.

6. The quantity of C or N released from a given population is dependent on the amount

present in the population.

7. Organic N turnover is strictly dependent on C turnover.

8. Microbial biomass increases in size to reach a maximum after 10 day of incubation. A

longer time is required to reach optimal size for more complex substrates.

Studies to understand the role of the microbial biomass other than as a source-sink for nutrients
involve knowledge of the activity of the biomass. The term "activity" includes the many

processes carried out by microbial enzymes. However, not all of the microbial biomass is

operating at the same level of activity and sometimes a direct correlation between the size of the

microbial biomass and its activity, as measured by some index, cannot be observed. This is

because three different fractions of the microbial biomass, which differ in their activity, exist

within the soil. One, the active biomass fraction is that which is capable of growth and all

metabolic functions. Two, there exists a sustainable portion of the biomass which is nongrowing

but which can degrade readily available compounds and resume growth under favorable

conditions. The third fraction exists of dormant spores or other long-term resting structures.

Estimates of the active biomass fraction range from 10 to 40% of the total identifiable biomass.

Application of Microbial Biomass Measurements to Soil Problems. Information concerning the

size of the soil microbial biomass has been used to study soil problems relating to (i) the

degradation, stabilization, and incorporation of plant C and N into the biomass, (ii) the effects of

freezing and thawing, (iii) the effect of tillage, (iv) the role of soil sampling, mixing, and

grinding, (v) the effect of climatic variations, (vi) biomass in forest floors, and (vii) the effect of

faunal feeding.

Management techniques such as no-tillage, crop rotations, and intercropping influence the size of

the microbial biomass. If better management of this biomass could be obtained, improved

utilization of the soil and fertilizer nutrients would result. The biomass is large enough, under

temperate conditions, to act as both a significant temporary storage pool and as a source of

nutrients. In a study of 100 soils from Saskatchewan, Canada, a close relationship was observed
between the size of the microbial biomass and the amount of soil N mineralized during a

laboratory incubation. Using 15N, it was clearly demonstrated that the N mineralized moved

through the biomass on its way to the ammonium ion, which is the initial mineral N compound

that forms as a result of mineralization.

Although improved methods for measuring microbial biomass is soil are needed, existing

procedures coupled with pulse-tracer techniques and mathematical modeling have provided

knowledge of this very important component of the soil. This knowledge will, in turn, be applied

to improving agricultural soil management and ecosystem functioning.