Abstract: Microbial biomass is a small but labile component of soil organic matter and plays an important role in cycling and other nutrient elements. Because of its importance in the functioning of different ecosystems, synthesis/dynamics of microbial biomass and its role in plant nutrition under different ecosystem conditions has assumed greater significance. In the same perspective, developing quick and convenient methods of determination have been of immense research interest for the least few decades. As a result significant improvements have thus far been made although no single method is devoid of snags. This paper presents I) a critical evaluation of methods to determine microbial biomass and ii) a review on the formation/dynamics of microbial biomass and its role in plant nutrition. Since carbon © and nitrogen (N) are the major elements, special attention has been given to these while discussing microbial biomass.
Introduction
Microbial biomass is a small but labile and living component of soil organic matter that is involved in most biogeochemical processes in terrestrial ecosystems (Paul and Voroney, 1980). It interacts with ecosystem productivity by regulating nutrient availability, determining soil C storage, and contributing to the atmospheric CO2 from respiration. As such, microbial biomass is the main agent that controls the flow of C and cycling of nutrient elements in terrestrial ecosystems. The large size of the soil microbial biomass implicates it as a major nutrient sink during C immobilization (growth) and as a source during mineralization (decay). It consists of bacteria, fungi, actinomycetes, and protozoa etc. However, fungi and bacteria are the dominant organisms both with regards to biomass and metabolic activities (Anderson and Domsch, 1973). Jenkinson and Ladd (1981) have defined biomass as the living part of soil organic matter excluding plant roots and soil animals larger than 5 x 103 um3. The importance of microorganisms in ecosystem functioning has led to an increased interest in determination of soil microbial biomass. As a result, a wide variety of methods have been proposed and practiced over the last 3-4 decades. This paper presents I) a critical evaluation of methods of determining microbial biomass and ii) a review on the formation/dynamics of microbial biomass and its role in plant nutrition. Since carbon 8 and nitrogen (N) are the major elements, special attention has been given to these while discussing microbial biomass.
Methods of determining microbial biomass: The increased interest in the role of soil microorganisms in nutrient and energy flow relationships in natural as well as man-manipulated environments has emphasized the need for simple and objective methods for measuring the size of microbial populations in soils. Among the methods currently available are those involving I) direct counting, in which microorganisms are variously stained (Babiuk and Paul, 1970) and observed by different optical methods (Frankland, 1974), ii) soil extractions in which enzymes (Skujins, 1967), cell components peculiar to cell walls of certain groups of microorganisms (Miller and Casida, 1970), or components characteristic to living cells (Lee et al., 1971) are extracted and quantitatively assayed, iii) disintegration of the soil followed by separation and collection of the microbial cells (Balkwill et al., 1975), iv) measurement of respiratory activities of substrate-supplemented habitats and thus the relative proportions of actively metabolizing biomasses are used to (Anderson and Domsch, 1978), v) ultra-violet absorption (Turner et al., 2001), vi) microwave radiation that leads to the release and determination of microbial C and N, vii) measuring heat output or microcalorimetry as an index of initial and final energy states of the system irrespective of the types of organisms or reaction pathway (Sparling, 1981), viii) ninhydrin reaction (Amato and Ladd, 1988), ix) chloroform fumigation incubation (CFIM) in which C bound in microbial biomass is released by microbial mineralization and thereby provides a means for calculating its biomass (Jenkinson and Powlson, 1976), x) chloroform fumigation extraction (Brookes et al., 1985; Vance et al., 1987) in which C and N that becomes extractable following fumigation is measured, and finally xi) chloroform extraction in which soil samples are extracted with K2SO4 + CHCl3 and the extractable N determined as a measure of biomass N (Azam et al., 1989a). Different methods available for quantitative determination of microbial biomass in soil and their advantages/disadvantages have been reviewed by Jenkinson and Ladd (1981) and Martens (1995). In this paper, a brief account of the commonly used and convenient methods is being provided.
Substrate-induced respiration method (SIRM): This method involves incubation of soil samples with an excess of easily oxidizable C source (25-4000 ug g–1 soil) and measuring the respiratory activity (CO2 evolved) after one hr of incubation in all soils at 22 °C (Anderson and Domsch, 1978). A conversion factor of 1 ml CO2 hr–1 to 40 mg biomass C was found to be realistic. Short period of incubation ensures the determination only of the prevalent and not the newly developed biomass. The authors claimed to obtain reliable estimates of biomass from soils of widely different origin e.g., from forests and agricultural sites. It was emphasized, however, that temperature of incubation plays a crucial role in final estimates and ay deviation from 22 °C will require fresh standardization of the method.
Substrate-induced respiration method is based on the premise that at increasing levels of easily degradable substrate like glucose and casamino acid the initial respiration rate of a soil increases in parallel to the biomass content. The fundamental assumption is that I) initial respiration rate in glucose-amended soil is the same for different sections of the microbial population and ii) biomass measurements by CFIM are correct. This method gives quick and direct information on the comparative estimates of biomass in different soils. However, quantitative measurement of the biomass is not possible. However, one of the advantages of SIRM is the apportioning of biomass into fungal and bacterial components by using inhibitors specific to either group; fungal and bacterial contributions to soil respiration being estimated at 22 and 78% (Anderson and Domsch, 1973). The method can be applied to hundreds of samples simultaneously within a day and is well suited to determine biomass C in acid forest soils as well as for soils rich in organic matter content. The disadvantages include I) an intricate respirometery system that requires continuous flushing of the incubation vessels for the determination of CO2 at short intervals (e.g., an infrared gas analyzer) ii) the fact that the method does not give direct estimates of nutrients contained in the biomass, iii) indirect estimates of nutrients contained in biomass are generally erroneous, and iv) the need for determining the concentration of substrate to achieve maximum initial respiration response for each soil. In spite of such shortcomings, SIRM can conveniently be used for comparing microbial activity of soils especially for assessing the effect of soil additives. A semi- or fully automated respirometric system may, however, be required for the ease of determinations and data handling. This factor alone makes the method unsuited for most labs especially in developing countries.
Chloroform fumigation-incubation method: The method involves CHCl3 fumigation of moist soil samples for 0-20 days at 25°C and determining CO2-C evolved, O2 consumed or N mineralized in fumigated versus unfumigated soil (Jenkinson and Powlson, 1976b). Briefly, the soil samples are placed in a desiccator that contains acid washed CHCl3 (ethanol free) and the desiccator evacuated to an extent that vigorous bumping of CHCl3 is achieved. After 24 hrs under vacuum in dark, CHCl3 is removed by repeated evacuations and the soil samples incubated for 0-10 or 10-20 days without or with the addition of ca 1% unfumigated moist soil to revive microbial activity. The incubation is carried out in a way to trap CO2 evolved from the soil. A similar set of unfumigated samples is also incubated. The difference of CO2-C evolved, O2 consumed, or NH4-N accumulated in fumigated and unfumigated samples is used to calculate biomass C and biomass N by using the expression: B = F/K, where B is the biomass, F is the flush or difference in fumigated and unfumigated soil samples in terms of CO2-C evolved or N mineralized, and K is the proportion of biomass C or N mineralized (KC and KN, respectively).
This has been the most widely used method and hence subject to immense scrutiny as well. It is based on the observations that moist soil samples subjected to wetting-drying, freezing-thawing, physical disruption, irradiation or fumigation show a flush of CO2 evolution and N mineralization upon incubation under laboratory conditions (Jenkinson, 1966; Powlson and Jenkinson, 1976; Marumoto et al., 1977). Such an effect is reportedly substantial following fumigation of soil with chemicals to control soil-borne plant pathogens (Jenkinson and Powlson, 1976a,b). When a soil is exposed to a volatile fumigant, the fumigant removed, and the soil incubated, respiration rate of the fumigated soil is greater than that of the unfumigated control. This effect has been attributed to I) exposure of otherwise inaccessible organic matter to microbial attack (Rovira and Graecan, 1957), ii) release of cellular components (Marumoto et al., 1977), and iii) partial or complete sterilization followed by mineralization of dead microbial cells by surviving or newly inoculated microorganisms (Jenkinson, 1966). Thus over a short period, fumigated soil consumes more O2 and evolves more CO2 than untreated soil. According to Jenkinson (1966) the flush of activity following fumigation is mainly due to the decomposition of dead microorganisms and is related to the size of soil biomass.
One of the limitations of the CFIM may be the qualitative change in the microbial population following fumigation (Martin, 1963). Shift in species composition is also exhibited through a change in C/N ratio of the biomass; narrowing of C/N ratio means more bacterial biomass and vice versa (Grace et al., 1993). The major assumption in CFIM that mineralization of non-biomass material is unaffected by fumigation has generally been found invalid especially for soils having a higher basal respiration. Shields et al. (1974) suggested that fumigation (with chloroform e.g.) not only killed the microorganisms but altered certain extracellular microbial metabolites rendering them susceptible to decomposition. Using 15N methodology, Azam et al. (1989b) observed significant increase in the extractability of non-biomass N (unlabelled N) following fumigation suggesting that CFIM may give over-estimates of biomass.
The fraction of biomass C and N mineralized following fumigation/incubation i.e., KC and KN is the single most important in biomass determinations by CFIM. However, it varies widely depending upon the incubation conditions and the type of soil. To begin with, Jenkinson (1966) proposed a KC factor of 0.3 that was later modified to 0.5 (Jenkinson, 1976). Adams and Laughlin (1981) reported value of 0.55 based on several organisms, while a weighted average of 0.41 was reported by Anderson and Domsch (1978). Like KC, KN is also reported to vary over a wide range depending upon the type of microorganisms studied. Generally, 24-59% of the bacterial N may be mineralized under aerobic conditions yielding KC factor of 0.24-0.59, while for fungal biomass, net N mineralization is not observed and hence the contribution to flush of mineral N may be negligible or even negative (Jenkinson, 1976; Adams and Laughlin, 1981; Marumoto, 1984; Voroney and Paul, 1984). Under anaerobic conditions, Voroney and Paul (1984) determined KN factor of 0.2-0.3 depending on the C/N ratio of decomposing biomass. Inubushi and Watanabe (1987) found this factor to be 0.43 and attributed this to higher C/N ratio of the decomposing biomass in anaerobic than aerobic soil understandably because of the preponderance of bacterial biomass in the former and fungal biomass in the later. Grace et al. (1993) used KC and KN factors of 0.21 and 0.22, respectively, while Voroney and Paul (1984) used KN of 0.3.
In most of the studies, KC or KN factor has been determined by subjecting the laboratory-raised microbial material to decomposition/mineralization and hence may not represent the native microflora of a particular test soil. Voroney and Paul (1984) were the first to determine KC or KN of biomass developed in soil in situ by incubating soil samples with 14C-labelled glucose and 15N-labelled KNO3. They assumed (and rightly so) that at the time of subjecting the treated soils to CHCl3 fumigation, the entire 14C and 15N in soil was in microbial cells and associated metabolites. The authors determined a KC and KN of 0.41 and 0.42, respectively. Azam et al. (1988) used NH4-N rather than NO3-N for in situ determination of KN in soil containing variable amounts of biomass. The values of KN thus determined varied from 0.19 to 0.42 and increased with the amount of glucose and N applied.
In spite of the limitations discussed above, CFIM has been the most widely employed method of determining biomass in samples originating from diverse ecological conditions. It has prompted extensive research on microbial biomass vis-à-vis its role in ecosystem functioning. In addition, the underlying principles helped develop new and less time-consuming methods of biomass determination that are described below.
Chloroform fumigation-extraction method (CFEM): Some of the shortcomings of CFIM for the determination of biomass N, particularly those arising from changes that occur during incubation as a result of immobilization-remineralization turnover, were apparently overcome by the method of Brookes et al. (1985). This method involves direct extraction of CHCl3-fumigated as practiced in CFIM. Total N and NH4-N extracted after CHCl3 fumigation is closely related with mineral N flush obtained with CFIM, while a KN factor of 0.54 can be used for calculating biomass N. Cheng and Virginia (1993) suggested that CFEM works better for biomass N than biomass C. Recently, Bailey et al. (2002) compared CFEM with extractable phospholipids fatty acids and found a good correlation between the two.
This method has also been used for the determination of biomass C using K2SO4 or water as extractant. Jenkinson (1966) reported CHCl3 fumigation led to an increase in the amount of C extractable with 0.5 M K2SO4. A close correlation between microbial biomass in soil and the extra C extractable due to fumigation was confirmed in subsequent studies of Vance et al. (1987) who proposed the relationship: biomass C = KEC x EC where KEC is the extractability factor for C and EC is organic C extracted from fumigated soil. They used a KC factor of 0.38. Nunan et al. (2000) determined biomass indirectly from the difference of organic C and organic N in K2SO4 extracts of fumigated and unfumigated soil samples (used CFEM). Biomass C was calculated as 2.64 x EC (extractable C in fumigated minus unfumigated soil) and N as 2.22 x EN. Gregorich et al. (2000) found K2SO4 and water to be equally efficient in extracting biomass C. The problem could be the artifacts resulting from incomplete removal of CHCl3 after fumigating the soil samples and thus leading to overestimates of biomass C. However, the method can conveniently be used in studies aimed at determining incorporation of 14C from diverse plant residues into the microbial biomass and its subsequent dynamics. In such studies, 14C-labelled plant material produced under controlled conditions is incorporated into the soil and biomass 14C determined at desired time intervals (Smith et al., 1995; Malik and Azam, 1980).
Like CFIM, CFEM is beset with the problem of increased susceptibility of non-biomass materials to extraction following fumigation leading to inflated values of nutrients contained in the microbial biomass. Studies by Azam et al. (1989b, referred to above) clearly demonstrated the release of non-biomass entities due to fumigation. In addition, the data obtained does not necessarily indicate the exact proportion of biomass N that is recovered by fumigation-extraction because the biomass content of the soils used in the study of Brookes et al. (1985) was not known. In addition, chloroform fumigation may work best for soils at lower moisture content in which case CHCl3 is distributed fairly uniformly in the soil sample. In soil samples at higher moisture (e.g., flooded rice soils) biomass may not be exposed uniformly to the fumigant leading to underestimates. In spite of these short comings, CFEM followed CFIM closely as a method of choice for determining biomass and prompted further improvements as discussed below.
Chloroform extraction method (CEM): As a logical extension to CFEM, Azam et al. (1989a) suggested a chloroform extraction method for aerobically incubated soils on the pretext that if CHCl3 vapours can extract microbial contents and render them degradable, CHCl3 may be more efficient in doing so. They incubated a silt loam soil from Pakistan with increasing amounts of 15N-labelled ammonium sulphate and glucose (C/N ratio of the additives being 30) in order to generate different levels of microbial biomass in soil in situ. At a stage when all of the applied 15N was in organic forms (microbial biomass and products), the soil samples were analyzed for biomass N by CFEM. In comparison, soil samples were extracted for 1 hr with K2SO4 containing different amounts of CHCl3. Biomass N determined with CFEM ranged between 14.6 and 30.6, while that extracted with 20% CHCl3 was 21-65%. The difference in the extractability of biomass N as well as extractability ratios revealed that CEM is more selective than the CFEM. In addition, CEM is simpler and rapid as compared to CFEM while retaining the advantages of the later. Azam et al. (1989c) used CEM for the determination of biomass N in 18 agricultural soils from Punjab, Pakistan. The values obtained with CEM were significantly correlated (r = 0.89; P < 0.01) with those by CFEM. The biomass N determined by CEM accounted for 2.8 to 13.8% of the total soil N; an almost similar variation (2.6 to 14.8%) was observed for CFEM. Biomass N estimated by CFIM under anaerobic conditions accounted for 6.1 to 13.6% of the soil N and showed no significant correlation with values obtained by CFEM and CEM. With CEM it was possible to specifically differentiate between microbial and non-microbial origin of the extracted N. The results clearly demonstrated the extractability of unlabelled N that increased with the amount of C added to the soil. Hence, overestimates of biomass are obtained especially in the presence of increasing levels of an easily oxidizable C source. In spite of this deficiency, however, methods involving CHCl3 in one or the other way still remain the most popular and widely used methods of determining microbial biomass.
Inubushi et al. (1991) introduced a modification of the CEM with specific objective of determining biomass in flooded (anaerobic) rice soils. A rigorous shaking of anaerobic soil samples with 10 Fl CHCl3 g–1 soil was proposed to facilitate uniform exposure of soil to the fumigant. This treatment was superimposed with 24 h fumigation in a desiccator as proposed in CFIM and the samples extracted with 0.5 M K2SO4 for the determination of extractable C and N for subsequent calculation of biomass values as suggested in CFIM and CFEM. According to Inubushi et al. (1991), the method is useful for determining microbial biomass in flooded or anaerobic soils. The method was further simplified by Gaunt et al. (1993) for use with waterlogged soils. One-step fumigation with CHCl3 liquid in closed bottles was found as effective as fumigation in a desiccator under vacuum. Further improvements in the method for the sake of convenience and rapidity were proposed by Witt et al. (2000) for use with flooded rice soils and termed CFAP (chloroform-fumigation extraction at atmospheric pressure). In this method, the soil samples (35 g) are fumigated (so it is termed) by adding 2 ml of CHCl3. and agitating for 24 h in the dark at 25°C followed by removal of the fumigant through evaporate in the fume hood at 25°C and extraction with 140 ml of 0.5 M K2SO4 and determination of C and N in the extract. Unfumigated samples were extracted straight away with 140 ml of 0.5 M K2SO4 Biomass C and N was calculated as the difference in extractable fractions between fumigated and unfumigated soil. Based on a series of laboratory experiments, Witt et al. (2000) found CFAP to be a reliable alternative to the CFEM for measuring biomass C and N; the two methods showed highly significant correlation.
Ninhydrin reaction method: In the CFIM, biomass is measured on the basis of CO2-C evolved and NH4-N accumulated in fumigated versus unfumigated soil samples. As a change in approach, Amato and Ladd (1988) proposed to use ninhydrin reactive C and N compounds released during fumigation-incubation as a measure of biomass. They specifically determined that fumigated soils retained protease but lost dehydrogenase activity as well as the ability to decompose glucose and immobilize NH4-N during 10 days of incubation. They proposed to quantify ninhydrin reactive N compounds released in CFIM (10 days incubation at 25°C, extraction with 2N KCl) and determine biomass N by using a multiplication factor of 21. Thus the method differs from original CFIM in that ninhydrin reactive C and N compounds rather than NH4-N (or total mineral N) and CO2 are taken into consideration while calculating biomass. Ocio and Brookes (1990) considered the method suitable for freshly amended soils (CFIM gives unreliable results for such soils) and found good correlation with CFEM and SIRM (next section). Sparling et al. (1994) concluded that the ninhydrin method can give a reliable estimate of biomass in organic materials as well as mineral soils. They determined a biomass C factor of 21 x NPC (ninhydrin positive carbon) and biomass N of 3.5 x NPC. They preferred to calculate biomass C by this method and biomass N from the soluble N flush (KEN = 0.38). Van Gestel et al. (1993) determined biomass C indirectly by multiplying ninhydrin reactive extractable N of fumigated soils with 21 (Amato and Ladd, 1988); they used 2N KCl for extraction.
As with original CFIM, the disadvantage of ninhydrin reaction method is the length of time (at least 10 days) required for obtaining biomass values, while it has the advantage of giving reliable results for freshly amended soils or soils rich in easily oxidizable C. However, the premise that fumigated soils retain protease activity but loose the ability to decompose organic matter and immobilize N would seem contradictory to the basis of CFIM that relies on microbial activities including C mineralization. These difficulties are fairly overcome if ninhydrin reactive compounds are measured using CFEM rather than CFIM. However, the values obtained are significantly lower compared to total extracted N (Inubushi et al., 1991).
Microcalorimetry: Sparling (1981) proposed microcalorimetry as a method to assess microbial metabolism in soil on the pretext that the heat produced depends only on the initial and final energy states of the system and is independent of the types of organisms or reaction pathway. In addition, the total catabolic activity in the soil is closely related to the heat production, anabolic processes normally contribute a little to the heat. Sparling (1981) studied heat output from 12 soils and compared the results with CFIM and SIR, ATP, dehydrogenase and amylase, and basal respiration. Correlation of heat with some but not all methods was obtained. The rate of heat output from soil is closely related with the rate of respiration. However, microcalorimetric method has not achieved popularity to significant extent.
Microwave irradiation: Microwave irradiation is an effective biocidal treatment of soil which kills weeds, nematodes and microorganisms; the effect on microorganisms being probably entirely thermal (Vela and Wu, 1979), fungi being more susceptible (Wainwright et al., 1980). Spier et al. (1986) were probably the first to use as microwave radiation as a soil treatment to measure microbial biomass, an approach akin to CHCl3 fumigation. They found that both eukaryotes and prokaryotes are equally susceptible to killing effect of radiation. Generally 90 sec irradiation was enough to get an effect similar to that of CHCl3 fumigation in terms of mineralization/extractability of C and N. In spite of its simplicity, this method has not found a wide spread acceptability.
Cellular contents: In theory, the soil biomass can be determined by measuring its own constituents. but there is some difficulty in the choice of constituent. Jenkinson and Ladd (1981) suggested that a suitable constituent should meet four conditions at the same time: I) it should be uniformly present at the same concentration in all biomass, ii) it should be present in the living cells alone and should not be adsorbed on soil organic matter/clay or if adsorbed should be readily degradable, iii) it should be extractable reproducibly and quantitatively, and iv) the method of its determination should be accurate and reproducible. No cell constituent fully meets these conditions. The constituents tried include, ATP, muramic acid, nucleic acid bases and N-acetylglucosamine (Jenkinson and Ladd, 1981). Of these constituents, ATP has been the most frequently used. It has been used as an indicator of microbial activity (Lee et al., 1971) and as a means of calculating microbial biomass (Oades and Jenkinson, 1979; Jenkinson et al., 1979). Jenkinson et al. (1979) and Sparling and Eiland (1983) found a close relationship between ATP and biomass as measured by CFIM. Nannipieri et al. (1978) found soil P to be an important factor in determining ATP. Extractability and reliable determination have been the main limitations in using ATP as a measure of microbial biomass. However, Verstraete et al. (1983) further evaluated the procedure and found it 80% efficient for ATP added to the soil. They found the method rapid, specific for microbial biomass, and applicable to all soils. ATP is assayed from the amount of light emitted by the luciferin luciferase system. The procedure is extremely sensitive and 10-14 mol of ATP can be measured.
ATP contents vary widely according to the growth conditions of microorganisms. For example, under P deficiency (Nannipieri et al., 1978), ATP content of biomass will be low, while it may rise in glucose-amended soils during and shortly after the period of maximum biological activity (Oades and Jenkinson, 1979). Values ranging 3 to 6 Fmol g–1 dry biomass have been reported (Lee et al., 1971, Oades and Jenkinson, 1979; Jenkinson et al., 1979). According to Jenkinson and Ladd (1981), close agreement between the mean values for the ATP contents of prokaryotic and eukaryotic organisms grown in vitro and the measured values for the soil biomass strongly suggests that the concentration of ATP in the largely resting soil population is little different from that in actively growing organisms.
Concluding remarks: Each of the biomass methods mentioned has its limitations. The microscopic methods require skilled personnel and often present difficulties in distinguishing between living and dead cells. In addition a number of assumptions have to be made before the counts are converted into weights. The extraction methods often fail to fully extract the component of interest and the recovery often depends on the characteristics or type of extraction procedure used. Also, the quantity of particular cell component can vary considerably with growth conditions and within different members of the microbial community. The ratio of specific component to the actual biomass is therefore not necessarily constant. Jenkinson and Ladd (1981) suggested that more than one method may be used for biomass determination in acid soils or those undergoing rapid metabolic changes.
Synthesis and dynamics of microbial biomass from different substrates: Availability and use of isotopic techniques have greatly helped in studying the formation and breakdown of microbial biomass in soil. For example, it has become quite convenient to study the fate (including incorporation into the biomass) of 14C-labelled plant residues and components added to the soil. Similarly, microbial biomass can be isotopically labelled under laboratory conditions or in soil in situ to study its further transformations. Indirect evidence of the synthesis and breakdown of biomass can also be obtained by following immobilization-remineralization turnover of N, a process that is governed to a significant extent by the nature of carbonaceous materials available to soil microorganisms. With the background knowledge of C/N ratio of the microbial biomass, the later can be quantified at any stage of the study from the amount of inorganic N transformed into organic forms (courtesy microorganisms). A major assumption while making such calculations will be that microbial immobilization is the main, if not the sole, process of consuming the applied N. The methods described above have been of great help to study the synthesis of microbial biomass and its subsequent fate in soil.
Generally 2-3% of the soil C is found in microbial biomass (Jenkinson and Ladd, 1981). However, values higher than this have often been reported (Azam et al., 1989c). Relationship of other nutrient elements to C being known, quantitative information on other nutrients (e.g., N and P) contained in microbial biomass can easily be gathered as reported by Anderson and Domsch (1980). In most studies, not more than 60% of the substrate C is converted into biomass. For plant materials, the rate of synthesis of microbial biomass depends on the chemical constituents (Amato and Ladd, 1980). Substrates like glucose are rapidly metabolized within 1-2 days followed by a quick decline in the biomass which itself becomes a substrate for the succeeding microflora (Azam et al., 1985; Haider and Azam, 1982). Stott et al. (1983) followed the fate in soil of 14C-labelled lignins, polysaccharides and protein fractions for one year and determined 4.9-7.8 and 4.6-13.4% of the residual C from polysaccharides and proteins, respectively, in the biomass, while a clear decrease with time was evident. Nicolardot et al. (1994) studied carbon and nitrogen cycling through soil microbial biomass at various temperatures (4-28°C) for 140 days. Before incubation the soil was amended with K15NO3 and either 14C-glucose or 14C-holocellulose. More 14C was found in biomass at lower temperature probably because of higher mineralization of biomass at elevated temperature. Some studies have indeed been reported on the synthesis in situ and breakdown of microbial biomass taking immobilization-remineralization of N as an index of biomass dynamics (Azam et al., 1988, 1989a,b). In some other studies, the build-up and decrease of microbial biomass has been studied using 14C and 15N isotopes (Azam et al., 1985; Haider and Azam, 1982; Ladd et al., 1985; van Veen et al., 1985). From these studies, it appeared that the immobilization and release of nutrients e.g., N could serve as an indirect index of the persistence of the newly synthesized microbial biomass. In studies using 14C-labelled lignins, polysaccharide and protein fractions 4.9-7.8% and 4.6-13.4% of the residual C from polysaccharides and proteins was found to be present in biomass after one year, a decrease with time was observed (Stott et al., 1983).
While studying transformation of added C into biomass, attention needs to be given to the native organic C content of the soil. For example, Cerri and Jenkinson (1980) studied the transformation of 14C-labelled plant residues into biomass and found less incorporation in acid than neutral soils. They attributed this difference to pH and clay but overlooked the significant effect of native organic matter that was 3 times higher in acid clay soil. These authors attributed difference in the transformation of added 14C-labelled ryegrass to soil pH. However, a critical evaluation of their data will suggest that it was the amount of native soil C in the two soils that mattered. Acid soil had higher organic matter than neutral soil and thus less decomposition and incorporation of added material into microbial biomass. Net decomposition was similar in the two soils. There are indications that the CFIM cannot be applied to strongly acid soils with pH 4.5 and less (Jenkinson and Ladd, 1981). A strongly acid soil used by Powlson and Jenkinson (1976) did not give a flush and yet contained appreciable amount of biomass, while a similar soil used by Cerri and Jenkinson (1980) gave a flush.
Residence time of microbial biomass in soil may vary from days to years depending upon the quantity and quality of the carbonaceous substrate available and the soil conditions (Jenkinson and Ladd, 1981). Biomass developed at the expense of easily oxidizable C like glucose and sucrose being more labile compared to that synthesized from more complex materials (Azam et al., 1986,1988; Stott et al., 1983). Decomposition of microbial biomass and cells will mainly depend on the community structure that changes with the chemical composition of the substrate. For example, biomass of hyaline fungi developed at the expense of easily oxidizable C compounds will decompose rapidly as compared to melanoid mycelium. Indeed, the pure culture studies served as the basis for the development of CFIM of determining microbial biomass (Jenkinson, 1976; Jenkinson and Powlson, 1976b), the proportion of biomass C mineralized (K value) being an important part. As such, the earlier discussion related to K factor would suffice to understand at least the rate of decomposition of microbial material under different conditions. However, it varies from 0.21 to 0.55 depending upon the incubation conditions and the type of soil (Jenkinson, 1966, 1976; Adams and Laughlin, 1981; Anderson and Domsch, 1978; Grace et al., 1993). As mentioned earlier, in most of the studies, KC factor has been determined by subjecting the laboratory-raised microbial material to decomposition/mineralization and hence may not represent the native microflora of a particular test soil. Voroney and Paul (1984) were the first to determine KC in soil in situ by incubating soil samples with 14C-labelled glucose and determined a factor of 0.41.
Marumoto et al. (1982) studied the decomposition and mineralization of 14C- and 15N-labelled dried biomass from several bacterial and fungal species. After 10 days, up to 43% of the labelled C was released as CO2 and up to 50 % after 28 days; up to 33% of microbial N was mineralized. In a 14C-labelled soil, the amount of labelled 14C mineralized during incubation after fumigation was greater than could be accounted for by the decrease in soil biomass (Shields et al., 1974), suggesting that CHCl3 fumigation caused the release of some non-biomass C.
The persistence of microbial biomass depends, like any other organic matter entity, on the phyico-chemical characteristics of the soil and management practices. For example, soil porosity also affects the decomposition of organic matter and the build-up of microbial biomass due to spatial distribution of substrate and microbes (Killham et al., 1993). Ploughing and other soil operations involving physical disruption of soil are usually considered to accelerate the decomposition of soil organic matter including microbial biomass. Cultivation may cause a rapid increase in the size of microbial population that is quickly reduced as the sources of available C decrease and the soil dries out. Likewise, an increase in mineral N content following soil disturbance is due to the mineralization of biomass (Powlson, 1980; Sorenson, 1983). Cultivation also promotes the decomposition of recently synthesized microbial metabolites and enhances substrate depletion (Grace et al., 1993).
Increased respiration or mineralization of N has been observed after compression and shearing, grinding, ultrasonic treatment, and chemical treatments like fumigation etc. (Jenkinson, 1976). Grinding more than doubled the respiration rate in two soils (Powlson, 1980); the increase being smaller in previously fumigated soil suggesting that the flush of decomposition is caused in part from killed organisms and in part from enhanced decomposition of non-biomass organic matter. Increased in mineralization following grinding is attributed to an enhanced exposure of substrates (Rovira and Greacen, 1957). The non-biomass material released by grinding contains a large proportion of polysaccharides, which are thought to stabilize soil aggregates and have a high C/N ratio (Powlson, 1980).
Soil drying causes a rapid decrease in biomass, while drying and rewetting promote the turnover of C derived from added plant material mainly as a result of enhanced turnover of microbial products (Van Gestel et al., 1993). Short-term increase in the C and N mineralization in soil following physical treatments is believed to have a biological origin. This may primarily be due to a physical effect of desiccation upon soil organic matter getting exposed to microbial action because of aggregate fragmentation or increased porosity. Marumoto et al. (1977) demonstrated that cell cytoplasmic compounds become easily mineralizable when active organisms are dried at low temperatures, whereas the cell walls are more resistant to degradation. Factors other than soil characteristics that may determine decreases in biomass by drying are the inherent properties of soil microbial populations. Soil microorganisms are not homogeneous as they differ in age, type, physiological, and metabolic state. Sorenson (1983) studied the influence of stress treatments on the dynamics of microbial biomass and humus in soils with variable clay content. He used 14C-labelled glucose, cellulose, hemicellulose, maize straw and barley straw. After 2 years 10% of the 14C was in biomass determined by CFIM. Heating, grinding, drying etc. enhanced the mineralization of C and biomass.
Van Veen et al. (1985) observed decreases in biomass C and flushes of mineralization after desiccation and remoistening of soils. They found clays to exert a positive effect on the formation and persistence of biomass. Ladd et al. (1985) also suggested that heavier soils (clayey) retained higher biomass. This would suggest that factors other than clays may be involved. Sorenson (1983) studied the influence of stress treatments on the dynamics of microbial biomass and humus in soils with variable clay content. He used 14C-labelled glucose, cellulose, hemicellulose, maize straw and barley straw. After 2 years 10% of the 14C was in biomass determined by CFIM. Heating, grinding, drying etc. enhanced the mineralization of C and biomass.
Microbial biomass and plant nutrition: Soil microorganisms play a major role not only in the decomposition of organic matter but also serve as a sink for plant nutrients in soil that are made available slowly during the cropping period. Despite the small size, biomass makes a contribution to plant nutrition far greater than could be anticipated because of its labile nature. Portions of microbial biomass are always killed by the changing environmental conditions as discussed in the previous section. Since cells are readily mineralized by the surviving microflora (Jenkinson, 1976; Anderson and Domsch, 1978), it has been suggested that they contribute substantially to the pool of mobile plant nutrients in soil (Anderson and Domsch 1980). Studies have indicated that N from microbial cell walls and cell contents makes an essential contribution to the readily mineralizable organic N in soil (Marumoto et al., 1977).
Anderson and Domsch (1980) indirectly estimated the amount of nutrient elements in the microbial biomass of 26 soils. The average amount of N, P, K and Ca stored in the vegetative cells of microflora of arable soils (upper 12.5 cm) was estimated to be about 108, 83, 70 and 11 kg ha–1, respectively. Jenkinson and Ladd (1981) determined biomass N and biomass P to be 95 and 11 kg ha–1 with a flux of 38 and 5 kg ha–1 yr–1, respectively. They found a close relationship between biomass N and plant uptake of N; a similar relationship was found for P taking C/P ratio as 50. Azam et al. (1989c) found biomass N to be 64-186 kg ha–1 and it showed a significant correlation with N uptake by wheat plants. Witt et al. (2000) demonstrated a clear indication that biomass underwent a transition from sink to source of plant nutrients, flooded rice being the indicator crop.
Except for the studies just referred there are hardly any estimates of a direct impact of microbial biomass on plant nutrition. Nevertheless, sufficient indirect evidence is available to suggest a significant role of microbial nutrients in plant nutrition. For example, biomass C represents 2-3% of the total soil C that is equivalent to 44-66 tons ha–1. A nitrogen equivalent of this amount will be 440-660 kg ha–1. In view of the labile nature of microbial biomass, especially of the newly synthesized fraction, a reasonable proportion of the crop N may actually come from the biomass.
Microbial biomass is a small but labile component of soil organic matter and makes a significant contribution to the plant available nutrients. The most important aspect of microbial biomass is its dynamic nature that facilitates recycling of nutrients in the soil-plant system. Being the living component of soil matrix, microbial biomass may serve as a good index of biological status of a soil. In view of the importance of microbial biomass in organic matter dynamics and nutrient cycling, a variety of methods have been developed and used over the past 3 decades. The methods differ in reproducibility and convenience, but quite a few of them show reasonably comparable results. Of the available methods, those using CHCl3 have been most popular and used extensively. Several modifications/ refinements have been made in the original CFIM and it appears that direct incorporation of CHCl3 in the extractant may ultimately be the method of choice for extracting nutrients from the biomass and their subsequent quantification by appropriate methods.