III. THE MICROBIAL BIOMASS

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1 III. THE MICROBIAL BIOMASS Required Readings: Ley, R.E., D.A. Lipson and S.K. Schmidt Microbial biomass levels in barren and vegetated high altitude talus soils. Soil Sci. Soc. Am. J. 65: Follett, R.F. and D.S. Schimel Effect of tillage practices on microbial biomass dynamics. Soil Sci. Soc. Am. J. 53: Suggested Reading: Jenkinson, D. S. and J. N. Ladd Microbial biomass in soil: Measurement and turnover. p In E. A. Paul and J. N. Ladd (eds.), Soil Biochemistry, Volume 5. Marcel Dekker, New York. Tunlid, A and D. C. White Biochemical analysis of biomass, community structure, nutritional status, and metabolic activity of microbial communities in soil. p 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

2 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 um 3. 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 ) = g cm -3 and biovolume (in cm 3 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.

3 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/k c 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 k c 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.

4 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 (k c ) 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 (K ec 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

5 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)

6 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 = 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 14 CO 2 output and the amount of 14 C 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 B t = B to e kt where B t is the biomass at time (t) and B to 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 0.5B to, the half life (t 1/2 ) can be expressed as ln 2/k or 0.693/k.

7 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 15 N-labelled substrate, changes in the organic and inorganic components of the soil system can be followed. Using 14 C-labelled acetate and 15 N-labelled (NH 4 ) 2 SO 4, 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

8 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

9 between the size of the microbial biomass and the amount of soil N mineralized during a laboratory incubation. Using 15 N, 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.