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aSee Jenkinson et al. (1976) for method of calculation. 'Calculated using K of 0.45, not 0.5 as in the original paper. cTemperate soils from the United Kingdom. dSubhumid tropics, Nigeria.

"Arable cropping for 2 years after clearing secondary forest, Nigeria. Source: From Powlson, 1994. Adapted from Jenkinson et al., 1976.

aSee Jenkinson et al. (1976) for method of calculation. 'Calculated using K of 0.45, not 0.5 as in the original paper. cTemperate soils from the United Kingdom. dSubhumid tropics, Nigeria.

"Arable cropping for 2 years after clearing secondary forest, Nigeria. Source: From Powlson, 1994. Adapted from Jenkinson et al., 1976.

The Chloroform Fumigation and Extraction (CFE) Technique

Vance et al. (1987) noted that low pH soils, particularly those in the range below pH 5.0, including many forest soils, were not well characterized for microbial biomass using the CFI procedure. They modified the CFI procedure (Jenkinson and Powlson, 1976) to the chloroform fumigation and extraction (CFE) procedure as follows (Vance et al., 1987): soil samples are fumigated with chloroform for 48 hours, the fumigated and nonfumigated control samples are extracted with 0.5 M K2SO4, and the resulting organic extracts are measured for carbon, nitrogen, and other elements. The difference between the total organic carbon from the chloroform-fumigated soils minus the nonfumigated controls, multiplied by the kec factor (see Chapter 9 for details) is the microbial biomass carbon. For soils with pH values less than 4, the kec values are usually lower, from 0.2 to 0.35kec (Jenkinson, 1988). The CFE method has proven quite successful, and enables one to obtain microbial biomass values for carbon, nitrogen, phosphorus (Hedley and Stewart, 1982), and sulfur (Gupta and Germida, 1988).

Afew authors have expressed concern about the extent of faunal contributions to the fumigation "flush." Protozoan biomass may be a significant contributor in some soils (Ingham and Horton, 1987), but usually constitutes less than 2% of total microbial carbon.

Some general comments on the methodology of the microbial biomass method are necessary. The CFI and CFE methods should be emp loyed within the context or intent of the methods originally described. Because they are bioassays, and not general chemical assays, they are not as robust as the latter. They can be misused, particularly if a great deal of organic matter substrate, waterlogging, or very low pH conditions are encountered (Powlson, 1994). However, the microbial biomass values are useful in the development and exercising of simulation models of labile carbon and nutrient turnover in a wide range of ecosystems (e.g., Parton et al., 1987; Parton et al., 1989a, 1989b; Jenkinson and Parry, 1989). Jenkinson et al. (2004) provide a helpful review of the chloroform fumigation techniques. Interestingly, they consider the fumigation and incubation (FI) technique to be obsolete, and urge caution in the usage of the k values, as had been noted by several investigators.

For reviews of biochemical methods to estimate microbial biomass, see Sparling and Ross (1993) and Alef and Nannipieri (1995a). For more specific details, see Chapter 9 (laboratory exercises) for comments on details of the microbial biomass estimation procedure. The ultimate "take home message" in studies of microbial biomass is the necessity to use more than one method to have some confidence in the numbers and hence biomass of the microorganisms measured. Although more timeintensive, it is advisable to compare biomass of microorganisms to direct counts made microscopically (Parkinson and Coleman, 1991).

Physiological Methods: SIR Technique

Additional methods for measuring microbial biomass include the substrate-induced-respiration (SIR) technique, first developed by Anderson and Domsch (1978). The SIR technique involves adding a substrate such as glucose to soil, and measuring the respiration resulting from the stimulated metabolic activity in the experimental soil sample, versus control treatments that received no carbon substrate. It is possible to measure the relative contributions of bacteria and fungi by using inhibitors (e.g., cycloheximide to inhibit fungal activity or streptomycin to inhibit bacterial activity). The assumption is that one measures only bacterial activity when fungi are inhibited, and vice versa. The technique requires some care, because soil texture may affect the apparent "resistance" to biocides. Further details of the technique are given by Beare et al. (1990, 1991), Insam (1990), Kj0ller and Struwe (1994), and Alphei et al. (1995).

Additional Physiological Methods of Measuring Microbial Activity

There is a large body of literature dealing with the indirectly measured signs of metabolic activity, namely CO2 output or oxygen uptake. The ratio of the two gases, in terms of either uptake or output, is very informative about the principal sources of carbonaceous compounds being metabolized. The ratio of CO2 evolved to O2 taken up, known as RQ, is lowest for carbohydrates, intermediate for proteins, and highest when lipids are the principal substrate being metabolized (Battley, 1987). In several studies, the microbial respiration per unit microbial biomass (qCO2 = pg CO2-C/mgCmic/h) (Anderson and Domsch, 1978; Insam and Domsch, 1988; Anderson and Domsch, 1993; Anderson, 1994) was measured and found useful as an indicator of the overall metabolic status of a given microbial community. Additional metabolic quotients have been used to study influences of climate and temperature, soil management, heavy metals, and soil animals in ecosystems, notably the ratio of microbial carbon to organic carbon, expressed as a percentage of microbial carbon to total organic carbon, or Cmic/Corg (Table 3.2) (Anderson, 1994; Joergensen et al., 1995). This follows from the assumption that terrestrial ecosystems in a near-steady state are characterized by a constant flow of nutrients and energy into and out of the ecosystem on a yearly basis, and entering and leaving the microbial biomass pool as well (Fig. 3.7) (Anderson, 1994). All of the foregoing is based on aerobic conditions. The extent of anaerobicity can be important at certain times, and needs to be carefully measured.

Enough data sets on microbial biomass carbon and nitrogen have accumulated by now that an extensive synthesis of temporal and latitudinal variation was carried out on data from more than 58 studies worldwide. For the entire data set, temporal variability was best predicted by

TABLE 3.2. Examples of Studies in Soil Microbiology in Which Metabolic Quotients Have Been Applied

Field of study

Metabolic quotienta

Maintenance carbon requirement

m, qCO2

Carbon turnover

qCO2, m, KmGLUCOSE, Y, m, qD, Cmic/Corg

Soil management

qD Vmax Cmic/Corg

Impact of climate and temperature

qCO2, Cmic/Corg, qD

Impact of soil texture and soil compaction

qCO2, qD

Impact of heavy metals

qCO2

Ecosystems, ecosystem theory

qCO2, qD, Cmic/Corg

Impact of soil animals

qCO2

am = maintenance coefficient; qCO2 = metabolic quotient or specific respiration rate; M = specific growth rate; Km = Michaelis-Menten constant; Vmax = maximum specific uptake rate; Y = growth yield; qD = specific death rate; Cmlc/Corg = microbial carbon to organic carbon ratio expressed as a percentage of microbial carbon to total organic carbon.

See Anderson (1994) for specific references pertaining to usage of particular metabolic quotients.

Modified from Anderson, 1994.

am = maintenance coefficient; qCO2 = metabolic quotient or specific respiration rate; M = specific growth rate; Km = Michaelis-Menten constant; Vmax = maximum specific uptake rate; Y = growth yield; qD = specific death rate; Cmlc/Corg = microbial carbon to organic carbon ratio expressed as a percentage of microbial carbon to total organic carbon.

See Anderson (1994) for specific references pertaining to usage of particular metabolic quotients.

Modified from Anderson, 1994.

Compartment microbial biomass /¿jjPff^q.^q.ff.^gfe

> c& S »P-ftP^Ofl Sa So tP^&è&tâï

Defining general physiological performances

Maintenance

Nutrient

Growth/

Degradation

requirements

requirements

death

ability

Based on microbial biomass and time

Based on microbial biomass and time

The prevailing assumption is that terrestrial ecosystems in a quasi-steady state are characterised by a constant flow of nutrients and energy, entering and leaving the system on a yearly basis, a well as entering and leaving the microbial biomass compartment. Microbial biomass communities adapt to the flow rate specific to the system.

Cmic/Corg ratio = percentage of microbial carbon to total soil carbon Metabolic quotient for CO2 (qCO2) = |ig CO2 - C/mg Cmic/h

FIGURE 3.7. Working hypothesis for the application of metabolic quotients in ecosystem development at the synecological level (from Anderson, 1994).

a three-component model incorporating pH, soil carbon, and latitude (Wardle, 1998). The increasing latitude reflected higher interseasonal variations in temperature, causing greater interseasonal flux of the biomass. A majority of these studies provided data showing less than one turnover of the entire microbial biomass per year, reflecting the extreme scarcity of food for most of the microbial populations much of the time (reasons for this are discussed earlier in this chapter).

Enzyme Assays and Measures of Biological Activities in Soils

Numerous soil biologists/ecologists have used enzyme assays to measure soil biological activity (Coleman and Sasson, 1980; Nannipieri,

1994; Alef and Nannipieri, 1995b). Oxidoreductases, transferases, and hydrolases have been most studied. These assays have been considered of questionable value, mostly because of misapplication of the techniques and misinterpretation of the resulting data. The principal objection to soil enzyme assays is that the activities are substrate specific, and hence related to specific reactions and do not necessarily reflect organismal activities (Nannipieri et al., 1990; Nannipieri, 1994). This concern is very well expressed by Nannipieri et al. (2002), who note that enzymes in soils can be in six different locations: (1) active and present intracellularly in living cells, (2) in resting or dead cells, (3) in cell debris, (4) extracellularly free in the soil solution, (5) adsorbed by inorganic colloids, or (6) associated in various ways with humic molecules. The preferred situation is being able to assay enzymes that are active and present intracellularly in living cells. The array of extracellular and intracellular distributions in the soil environment is expressed in Figure 3.8 (Nannipieri et al., 2002), which depicts various aspects of overall enzyme diversity related to microbial functional diversity in soil.

It should be noted that enzymes related to particular target substrates, such as ligno-cellulases in leaf litter, may be relatively good predictors of mass loss. After early stages of mass loss caused by leaching and mineralization, the "middle stage" is often strongly correlated with enzyme activity. In the final stages, with less than about 25% of initial

Abiotic transformations or enzymelike reactions

Abiotic transformations or enzymelike reactions

Free extracellular enzymes

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