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  • Nutrient Cycling in Agroecosystems 61: 4151, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands. 41

    The role of soil microorganisms in soil organic matter conservation in thetropics

    David S. Powlson, Penny R. Hirsch & Philip C. BrookesSoil Science Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK; Author for correspondence (e-mail: [email protected])

    Received 3 November 1999; accepted in revised form 7 September 2000

    Key words: bacteria, carbon, diversity, fauna, fungi, microbial biomass, mineralization, nitrogen, soil organicmatter

    Abstract

    Soil is a large sink for organic carbon within the terrestrial biosphere. Practices which cause a decline in soilorganic matter cause CO2 release, in addition to damaging soil resilience and, often, agricultural productivity.The soil micro-organisms (collectively the soil microbial biomass) are the agents of transformation of soil organicmatter, nutrients and of most key soil processes. Their activities are much influenced by soil physico-chemical andecological interactions. This paper addresses two key issues. Firstly, ways of managing, and the extent to whichit is possible to manage, soil biological functions. Secondly, the methodologies currently available for studyingsoil micro-organisms, and the functions they mediate, are discussed. It is concluded that, as the world populationdevelops in this new millennium, there will be an increased dependence upon biological processes in soil to provideadequate crop nutrition for the majority of the worlds farmers. Although a major increase in the use of artificialfertilisers will be necessary on a global scale, this will not be an option for large numbers of farmers due to theirpoverty. Instead they will rely on recycling of nutrients from animal and vegetable composts and urban wastes,and biological cycling from nitrogen fixation and mycorrhizae. The challenge is to select the most appropriatetopics for further research. Not all aspects are likely to lead to significantly improved agricultural productivity, orsustainability within the foreseeable future.

    Introduction

    Soil serves a range of different functions that cansometimes conflict. It is the basis for agriculture andforestry and the importance of this role will increasethroughout the next millennium if the food, fibre andfuel needs are to be met for a population of 8-10 billionexpected by 2050 (Fischer and Heilig, 1998). Theseneeds cannot be met by a major expansion of the areaunder cultivation so intensification of currently man-aged land is inevitable. Agricultural intensificationcarries dangers including the possibility of damagingsoil functions and thus threatens the sustainability ofthe agroecosystem (Lal, 1998) and risks altering otherparts of the environment through emissions to wateror air (Powlson, 1997). Soil is also used for wastedisposal, so detoxifying and filtering functions are im-

    portant. A vast range of organic wastes are appliedto soil including sewage sludge, composted municipalwaste and effluents from biologically-based industriessuch as the processing of oil palm. Such materials canbe beneficial to soil properties because of their organiccarbon and nutrient content but they may also containpollutants such as heavy metals that damage soil mi-crobial processes (e.g. Brookes, 1994). Soil organicmatter represents a major proportion of the organiccarbon within the terrestrial biosphere. An accumu-lation of organic matter is not only beneficial to soilfunctions related to agriculture but also represents asequestration of carbon from atmospheric CO2. Con-versely, management practices leading to a decline insoil organic matter content release CO2, the majorgreenhouse gas, in addition to generally having a det-rimental impact on agricultural productivity and soil

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    resilience (Lal, 1998). Soil also has a range of otherenvironmental functions such as water catchment andthe regulation of fluxes of trace greenhouse gases (e.g.N2O, CH4) between land surface and the atmosphere.These functions can be greatly modified by agricul-tural practices, sometimes in completely unexpectedways. One example is the bacterial oxidation of meth-ane in aerobic soil (Goulding et al., 1995) which isgreatly decreased by conversion of forest or grasslandto arable cropping and further decreased by the use ofN fertiliser.

    Virtually all of the key processes are mediated byorganisms whose activities are greatly influenced bysoil physico-chemical conditions and by ecologicalinteractions. This paper addresses two issues: first,the extent to which soil biological functions can bemanaged in agricultural soils and, second, the meth-odologies available for studying soil microorganismsand the functions they mediate.

    Direct management of soil biological processes

    As a habitat for microoganisms, soil is probably themost complex and diverse on the planet. Conventionalapproaches based on culturable microbes and newertechniques based on analysis of DNA in soil show anenormous diversity at the genetic level (Torsvik et al.,1990; Borneman et al., 1996). This diversity arises,in part, through the wide variety of incoming sub-strates but the major factor is probably the spatiallyheterogeneous nature of soil. It comprises mineralfragments covering a range of sizes that span severalorders of magnitude. The mineral particles have differ-ing chemical composition and surface properties thatinfluence microbial survival and activity and soil solu-tion composition. Soil also contains organic debris ofplant, animal or microbial origin, again having a widerange of chemical properties and potential for biolo-gical decomposition (reviewed by Foster, 1988). Solidparticles are associated in various ways and are sep-arated by either water or air. The size and distributionof spaces between particles controls the transport anddiffusion of solutes and gases, especially oxygen. Allof these factors lead to great variability in the environ-ment at the microscopic scale so, for example, oxygenconcentration can vary by several orders of magnitudeover a distance of micrometres (Macdonald, 1986).Although much mixing of soil occurs on a larger scalethrough physical processes, water movement, rootgrowth, and turbation by soil fauna, it is also likely

    that microbial populations suited to very different en-vironments co-exist in close proximity and mediatedifferent processes simultaneously. For example, ni-trification (an aerobic process) and denitrification (ananaerobic process) can occur simultaneously in thesame soil (Macdonald, 1986). In evolutionary termsit seems likely that this enormous variation in environ-ment over short distances has been a major factor inleading to the observed genetic diversity of soil mico-organisms (Foster, 1988; Stotzky, 1997). Populationsrelatively close to each other may have developed inisolation; this contrasts with the situation in water orsediments where a greater degree of mixing occurs. Afurther source of complexity in soil biological activityis the existence of exocellular enzymes, presumablyderived from past populations of organisms but stabil-ised by sorption on mineral surfaces and retaining atleast part of their activity (Burns, 1978).

    In view of this complexity and diversity it is per-haps not surprising that attempts to alter biologicalactivity by inoculating soil with specific organisms arerarely successful. Any added organism has to com-pete for substrates and an ecological niche with anative population that is likely to be better suited tothe environment through past selective pressure. Forexample, most attempts to accelerate decompositionof organic pollutants in soil by inoculating with spe-cific organisms are not overwhelmingly successful.In some cases, the white rot fungus Phanerochaetehas been used to decontaminate polyaromatic hydro-carbons (PAHs), but the most promising approachesinvolve soil farming via the provision of nutrients,water and aeration to stimulate degradation by the in-digenous microbial populations (Balba, 1993). Thiscontrasts with the relative success of inoculants inother situations such as silage where the material ismore thoroughly mixed and less heterogeneous (Caiet al., 1997) or in biomining, where ore-containingrocks can be treated with Thiobacillus ferrooxidans orsimilar acidophillic bacteria which oxidize sulphatesto release metal ions in leachate (Brock, 1988).

    In the early part of the century there were many ex-periments in which free-living N2-fixing organisms, inparticular Azotobacter, were added to soil (Rubenchik,1960) but despite reports of increased crop yields, thepractice ceased and it is now generally agreed that thisapproach does not result in significant N2-fixation. Be-nefits may have been derived from plant nutrients suchas N or P released from the bacterial biomass itselfor from the effects of phytohormones on root growthand plant development (Bashan and Holguin, 1997).

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    Similarly, phosphate-solubilizing bacterial inoculantshave been used in the past (Cooper, 1959) and are stilldiscussed although the most probable mechanism forany yield increase is bacterial production of phytohor-mones resulting in increased root growth (de Freitas etal., 1997).

    Direct intervention can be successful if a pro-cess is mediated by only a few species of organisms;an example is nitrification. In oxic agricultural soils,autotrophic nitrifiers of the family Nitrobacteraceaeare the major contributors (Underhill, 1990). Only afew genera are known to oxidize ammonia to nitriteand only one to oxidize nitrite to nitrate in soil. Thefirst step appears to be rate limiting for the overallprocess and can be inhibited by several compounds,the best known of which is nitrapyrin (2-chloro-6-trichloromethylpyridine) which is available as a com-mercial product. In contrast, the mineralisation oforganic compounds to ammonium and CO2 is medi-ated by a very wide range of soil organisms. Even instrongly polluted soils in which the total populationhas been halved in size due to heavy metals, there israrely a decrease in the rate of C or N mineralisationdue to loss of microbial groups (Chander et al., 1995).

    A similar situation concerns the biological controlof soil-borne plant pathogens and pests. Successfulmicrobial antagonists are usually very specific in theiraction and in some circumstances can replace chem-ical pesticides (Jones, 1993). Likewise, inoculationof soil or seeds with symbiotic bacteria or fungi isan established practice especially with rhizobia andlegumes such as soya beans. Where no native rhizobiaare present, inoculation has a dramatic effect, allevi-ating the need for N fertiliser (Jones, 1993). Rhizobiaproliferate in the rhizosphere in addition to the rootnodules. In many situations in temperate regions theysurvive in sufficient numbers to nodulate compatiblecrops in subsequent years, but in tropical environ-ments it is more common for inoculation to be requiredfor each crop if maximum nitrogen fixation is to beachieved. However, this varies greatly between differ-ent soil types and legume species; even sub-optimalfixation can be highly beneficial in conditions of lowsoil fertility. The interaction between rhizobia andhost is highly evolved and requires specific signal mo-lecules to be produced by both rhizobia and host plant(Hirsch, 1996). The root nodules on the host plantprovide a unique niche for rhizobia where bacteria canmultiply without competition from other members ofthe soil community.

    Mycorrhizal fungi also form symbiotic relation-ships with host plants, increasing the effective surfacearea of the roots, improving P nutrition in particular.Arbuscular mycorrhizal fungi do not show significanthost specificity and are ubiquitous in most temper-ate soils (Harley and Smith, 1983), thus inoculationis rarely beneficial (Jones, 1993). In tree and shrubnurseries seedlings are often inoculated with ectomy-corrhizal fungi because survival after transplantationis improved.

    It is well known that the soil microflora and faunacan be manipulated indirectly through crop rotationsand management practices: pathogen and pest popula-tions increase when susceptible plants are grown andpopulations of antagonistic microbes usually followthese, to create disease-suppressive soils (Davison,1988). Similarly, cultivation of legumes leads to in-creased numbers of their compatible rhizobia in thesoil (Hirsch, 1996). Less well-defined effects are seenduring crop rotations, because of differences in thetypical rhizoflora of each plant type (Joos et al., 1988).

    Indirect manipulation of soil biological processesfor improved nutrient utilisation

    As discussed above, opportunities for directly manip-ulating soil organisms are limited to a small groupof organisms (albeit extremely important ones) thatform symbioses with plants and, perhaps, some non-symbiotic groups finding a niche within the rhizo-sphere. In the case of the general heterotrophic soilpopulation, manipulating processes such as the miner-alisation or immobilisation of N or C can only be doneby extremely indirect means. They generally rely onthe impacts of agronomic practices on the soil envir-onment and are based on empirical observations ratherthan an understanding of microbial ecology.

    The impacts of management on processes suchas N mineralisation or accumulation are totally gen-eric and it is not necessarily helpful to distinguishbetween tropical and temperate situations. Differencesresult from altered environmental conditions and inthe practicalities of particular agronomic practices,not because the processes themselves are different. Inwarm humid climates (except flooded conditions), de-composition of incoming organic materials proceedsrapidly as conditions for microbial activity are oftenideal throughout much of the year. One result of this isthat it is difficult to accumulate high concentrations oforganic matter in soils under these conditions, even if

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    inputs are large. Wu et al. (1998) give examples of theextent to which soil organic carbon could be increasedby introducing grass or legumes into an arable systemgrowing cassava.

    There are also implications for the time course ofN mineralisation and whether or not it is well syn-chronised with crop uptake. Conditions for microbialactivity in warm humid environments contrast stronglywith those in dry tropical regions or those with dis-tinct wet and dry seasons. In addition to the obviousconstraint to microbial activity in very dry soil, therecan also be a stimulation of activity, and N miner-alisation, due to drying/wetting cycles. These are ofmore significance to short-term nutrient dynamics thanto the long-term level of soil organic matter that isattained. In rice-based cropping systems there is theadditional influence of prolonged anaerobic conditionsand alternating anaerobic/aerobic cycles on microbi-ally mediated processes. In the most intensive ricesystems in which soils is flooded almost continually toachieve two or three crops per year, there are indica-tions that chemical reactions between ammonium andphenolic groups within soil organic matter may con-vert N into organic forms which are highly resistant tomicrobial decomposition (Cassman et al., 1995; Olkand Senesi, 2000).

    Mineralisation of organic N to inorganic formsis a key process which is reviewed fully by Jarviset al., 1996. The process is not totally microbial asinvertebrate fauna make important contributions in-cluding: (i) redistributing organic materials over arange of spatial scales, (ii) enhancing the rate ofcycling through chemical change during metabolism,and (iii) altering microbial populations themselves bycreating or removing appropriate conditions for theirvarious activities (Woods et al., 1982). Soil inverteb-rates thus contribute to N fluxes by changing micrositeenvironments and controlling populations of other or-ganisms, and through trophic transfers in food websand turnover of tissues (Anderson, 1988). Althoughthere is no clear consensus about the net effects offeeding and other activities of micro-, meso-, or mac-rofauna, it is clear that they have substantial impacts.Thus earthworms can increase CO2 evolution, de-crease microbial biomass, and increase mineralisation(Ruz Jerez et al., 1988). Higher mineral N levels anddenitrification activity have been measured in earth-worm casts than in surrounding soil (Scheu, 1987;Elliott et al., 1990).

    The role of microbivorous fauna (e.g. protozoa,nematodes) has been extensively studied (Bouwman

    et al., 1994) and many studies indicate that predationstimulates N turnover since more N was mineralisedwhen protozoa were present (e.g. Woods et al., 1982;Kuikman and van Veen, 1989). However, this is notuniversally observed (Hassink et al., 1993).

    Returning crop residues to soil is an importantmeans of maintaining soil organic matter and recyc-ling nutrients. The N contained in organic moleculesin plants may either be rapidly mineralised or initiallyimmobilised into microbial cells and later releasedby mineralisation; often both processes occur con-currently. The carbon-to-nitrogen (C/N) ratio of thematerial is often a useful guide to which process willdominate. It is often observed that materials with aC/N ratio20) often cause immobilization of soil inorganic Nfor a period before mineralization begins (e.g. Jen-kinson, 1984; Marstorp and Kirchmann, 1991). Thisis a vast oversimplification as any plant residue willcontain a wide variety of different molecules includingcellulose and cell wall constituents that are low in N(wide C/N ratio) and proteins or amino compound richin N (narrow C/N ratio). Some highly decomposablecompounds such as proteins can be stabilised by as-sociation with lignin. Especially with plant materialsfound in tropical agriculture, the content of lignin orpolyphenols has been found to be a useful indicator ofwhether mineralisation or immobilisation will domin-ate in the initial stages of decomposition (e.g. Palmand Sanchez, 1991).

    The quantities of nitrogen returned in crop residues(including roots) can often be large, typically between50 and 150 kg N/ha for a range of temperate arablecrops in the UK (Jarvis et al., 1996) and >200 kgN/ha for some horticultural crops (Rahn et al., 1992).The time course of N release is crucial in determiningthe amounts of N that will (1) be available to the nextcrop and permit a decrease in fertiliser N application,(2) be mineralised more slowly and become availablein decreasing quantities to subsequent crops, and (3)be mineralised at times when crop uptake is small butthe risk of loss (e.g. due to nitrate leaching or deni-trification) is high. For tropical situations a decisiontree approach has been developed (Palm, 1997) basedon empirical rules relating residue composition (C/Nratio, lignin or polyphenol content) to mineralisability.The decision tree is designed for use by smallholderfarmers so residue composition is related to character-istics that are detectable without the need for chemicalanalysis. The categories of plant material range from

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    those that decompose, and mineralise N, so rapidlythat they are almost equivalent to inorganic N fertiliserto those with such slow N release rates that they are ofvirtually no value as a short term N source but add tothe stock of soil organic matter.

    Computer based decision support systems basedon N cycle models are being developed to providefarmers and their advisers with advice on N fertil-iser requirements for crops and ways of decreasing Nlosses. A central factor in these systems is the predic-tion of net N mineralisation from native soil organicmatter, crop residues or manures. One system is basedon the SUNDIAL model (Smith et al., 1996). Thedevelopment and testing of such systems for use intropical agriculture lags behind progress in temperatesituations. It is interesting that descriptions of micro-bial activities in such models is usually implicit ratherthan explicit and is empirical rather than mechanistic:Smith et al. (1998) discussed this issue in the contextof soil C turnover models. They compared compart-mental process-based models with food web modelsin which microbial and faunal interactions are repres-ented explicitly. They concluded that the latter werevaluable as research tools where the aim was to under-stand processes at the organismal level; they were alsonecessary as a means of simulating feedbacks based onmicrobial interactions. However, in practice they areextremely difficult to use because so many parametersare required to describe the response of each organismor group to environmental factors.

    A possible example of microbial adaption to in-put composition was reported by Wedin and Tilman(1990). After five species of grasses were grown in thesame soil for 3 years there were 10-fold differences innet mineralisation rate which corresponded to speciesdifferences in quantity of roots, C/N ratio and lignincontent of material entering the soil.

    Many agronomic practices influence the rate ortime course of N mineralisation. These include thetime of tillage or residue incorporation, application ofmanures, the use of catch crops or green manures tocapture N that would otherwise be lost by leaching anddiverse ways of introducing legume plant material intocropping systems. Although the impacts on N availab-ility to crops depend on changes in microbial activity,these are managed in a very coarse and indirect waybased on empirical observations of N fluxes.

    Methodologies for investigating soilmicroorganisms

    The soil microbial population and its activities canbe studied in different degrees of detail. Each ap-proach has advantages and limitations so the choiceof methodology must be guided by the objectives ofthe particular research being undertaken. At the leastdetailed level, overall processes such as CO2 evolu-tion, or the activities of widely distributed enzymessuch as dehydrogenase or phosphatase, can be usedas indicators of microbial activity. Also at the leastdetailed level, a range of techniques are available tomeasure the size of the whole microbial population,termed the soil microbial biomass. At an intermediatelevel the population structure can be investigated usingvarious chemical markers such as fatty acid methyl es-ters (FAMEs) and phospholipid fatty acids (PFLAs).An intermediate level tool for classifying componentsof the population on the basis of function is the useof substrate utilisation typically using the BiologTMmethod.

    In situ assays can provide realistic information onthe activity of soil microbes belonging to some spe-cific functional groups, including methane oxidizers,diazotrophs and nitrifiers. The easiest approaches in-volve supplying gaseous substrates and measuring theend-products, as in methane oxidation and nitrogenfixation. The degradation of unusual compounds suchas pesticides, which have readily-identifiable break-down products, can also be measured relatively easily.Measuring the dynamics of isotopically labelled sub-strates, including the dilution of labelled compoundsin product pools, are valuable approaches but limitedto well-defined processes.

    Much more detailed information on microbial di-versity and/or the presence of specific groups can nowbe obtained using molecular biological techniquesbased on DNA or RNA and the polymerase chain re-action (PCR). Some of these approaches are discussedbelow.

    Process measurements

    Measurements of the final product of microbial activ-ity, such as CO2 evolution or the formation of in-organic N, are well established as a means of mon-itoring overall activity. Until recently measurementsof N mineralisation were limited to the net resultof mineralisation and immobilisation, even though it

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    was clear that the two processes occurred simultan-eously. Gross mineralisation is the total release ofNH+4 through microbial activity, i.e. before any im-mobilisation back into microbial cells. The differencebetween gross rates of mineralisation and immobilisa-tion is net mineralisation or, in some circumstances,net immobilisation. If only the net rates are measuredit is difficult to draw conclusions regarding the con-trolling factors or to extrapolate to other conditions. Inrecent years 15N methodologies based on isotopic pooldilution have been developed to measure gross ratesof mineralisation (e.g. Myrold and Tiedje, 1986; Bar-raclough and Smith, 1987; Powlson and Barraclough,1993; Murphy et al., 1999) and nitrification (Willisonet al., 1998). Although the practical development ofthe technique has occurred over the last decade or so,the principles were established almost 50 years ago(Kirkham and Bartholemew, 1954, 1955). For meas-uring gross mineralisation, 15NH+4 is added to soil tolabel the NH+4 pool. As unlabelled N from organicmatter is mineralised the 15N enrichment of the pooldecreases and the rate of decrease can be used to cal-culate the rate of gross mineralisation. Removal ofNH+4 (e.g. due to nitrification or plant uptake) altersthe size of the pool but not its 15N enrichment. Themethodology involves a number of assumptions andlimitations but the ability to obtain values for grossrates of mineralisation and immobilisation separatelyhas transformed understanding of N turnover. For ex-ample, gross rates of mineralisation at least four timesgreater than net rates have been measured (Murphyet al., 1999). Such results indicate that a large pro-portion of the N released by gross mineralisation isreassimilated into microbial cells. This in turn raisesquestions regarding the source of C to provide thenecessary energy. There are also spatial issues; doesa large proportion of the NH+4 released remain veryclose to the site of release (e.g. from a dead cell) tobe rapidly assimilated by a neighbouring cell and notenter the pool of inorganic N in the bulk soil (Davidsonet al., 1990; Drury et al., 1991)?

    Microbial biomass measurements

    The concept of the soil microbial biomass is that, forsome purposes, the entire population can be treated asa single entity. The concept was put forward by Jen-kinson (1966) and a practical method for estimatingthe quantity of carbon held in the biomass publishedby Jenkinson and Powlson (1976). The original meth-

    ods were based on the use of chloroform fumigation,to kill living cells in soil, followed by an incuba-tion during which a new population decomposes thekilled cells and a proportion of their C is evolved asCO2. This is now termed the fumigation-incubation(FI) method. More recent developments have led tomethods in which soil is extracted with appropriatesolutions following fumigation, termed fumigation-extraction (FE); e.g. Vance et al. (1987). Methods areavailable for measuring the quantities of C, N, P andS in the microbial biomass see Powlson (1994) for areview.

    Although these techniques give no information onthe diversity within the biomass or the fractions ofspecific groups, they have proved to be extremelyvaluable in studies concerned with SOM managementor nutrient dynamics. They have proved equally use-ful in tropical and temperate soils (Grisi et al., 1998;Haron et al., 1998) and the methodology has beenmodified for use in flooded rice soils (Inubushi etal., 1991; Gaunt et al., 1995). Biomass C values ob-tained by FE, FI and related techniques are reasonablyclosely correlated with biovolume values obtained us-ing direct microscopic counts (Lin and Brookes, 1996)and with ATP measurements on soil (e.g. Ocio andBrookes, 1990; Powlson, 1994).

    One application of microbial biomass measure-ments is their use to detect slow changes in total SOMcontent resulting from changes in management. Forexample, in an experiment in sub-tropical Australiaabove-ground residues of sorghum had been either re-moved or retained for 5 years. The impact of residueretention, under conventional tillage, on total soil or-ganic C was barely measurable (an increase of only8% above the residue removed treatment) but the in-crease in biomass C was proportionately much larger(15%; Saffigna et al., 1989).

    In addition to the FI and FE methods, the substrateinduced respiration method (SIR) is also available, ori-ginally introduced by Anderson and Domsch (1978).This has proved to be very useful for estimating bio-mass C content. It is based on the short-term effecton soil respiration of an addition of readily decom-posable substrate. If an appropriate concentration ofglucose is added, under specified conditions, a shortperiod of constant respiration is observed before theonset of rapidly increasing respiration due to micro-bial proliferation. This is interpreted as representingthe response of the initial population. The rate of CO2evolution during this short period is reasonably wellcorrelated with biomass C content as measured by

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    FE (Lin and Brookes, 1996) and FI (Anderson andDomsch, 1978).

    Combining microbial biomass measurements withactivity measurements, such as CO2 evolution,provides a more sensitive index of microbial activ-ity than either used alone. Rates of biomass specificrespiration, expressed as CO2C respired per unitof biomass C per unit time, have been used to de-tect stress such as that caused by metal pollution(e.g. Brookes and McGrath, 1984). From such ob-servations, Killham (1985) developed a simple bioas-say procedure based on proportioning 14C-labelledglucose between biomass-14C and 14C evolved. Heshowed, for a given increase in stress, that the ratio:[(respired 14C):(biomass 14C)] was, on average, twiceas great as the magnitude of the decrease in eitherrespiration or dehydrogenase activity.

    Chemical markers to investigate soil microbialcommunity structure

    The ester-linked fatty acids in the phospholipids(PLFAs) are considered the most sensitive and usefulchemical measures of microbial community structure.The fungal and bacterial components of the micro-bial biomass can be determined by specific signaturePLFAs. For example, bacteria characteristically con-tain odd-chain, methyl-branched and cyclopropanefatty acids. The PLFAs in fungi are typically saturated,even-chained, polyenoic fatty acids. Many actinomy-cetes contain methyl-branched tuberculostearic acid(Tunlid and White, 1992). Fatty acid methyl esters(FAMEs) and PLFAs can also be derived from soilsand used as indicators of community structure. Thereis some evidence to suggest that changes in FAMEsand PFLA profiles indicative of stress can be identified(Parker et al., 1982; Frostegrd et al., 1993). FAMEscan arise from any organic material in soil includ-ing plant and animal debris and microfauna whereasPLFAs are believed to be derived only from living mi-crobes. Thus the interpretation of FAME data is moredifficult.

    Ergosterol (ergosta-5,7,22-trien-3B-ol) is the pre-dominant sterol in most fungi (Tunlid and White,1992) but is not present in bacteria. In a metal pollutedsoil three independent biomass measurements (bio-mass C by fumigation-extraction, substrate-inducedrespiration and ATP) closely followed decreases in soilergosterol content along a heavy metal gradient froma CuNi smelter (Fritze et al., 1989). Soil ergosterolmay therefore have potential as an indicator of fungal

    biomass in metal-contaminated soils but this requiresfurther evaluation.

    Direct microscopy

    The number of microbes in soil can be estimated frommicroscopic counts on soil suspensions treated withfluorescent dyes. Depending upon the dye used, thetotal number of cells present, the number that are vi-able (i.e. retain membrane potential), and even thenumbers that are Gram-positive or Gram-negative, canbe counted (Bloem et al., 1995; Braux et al., 1997).

    Culturing microorganisms

    There are many growth media that are selective, orsemi-selective, for different groups of bacteria andfungi (Alef and Nannipieri, 1995). Most soils contain105107 culturable, heterotrophic bacteria per g soil,whereas the total number of cells seen by direct micro-scopy may exceed 108 per g soil, although generallyonly 0.11% of cells are culturable. Many importantbacterial groups, in particular autotrophs such as ni-trifiers and methane oxidisers, are known to be verydifficult to culture in laboratory conditions. Thus res-ults from culturing approaches need to be interpretedwith caution as they almost certainly select a smallsub-set of the total population.

    Substrate utilisation

    The response to particular substrates can be measuredby respiration (CO2 evolution) or by reduction of a tet-razolium dye (BiologTM). The Biolog system allowsthe rate of growth on 95 different carbon substrates(compared to a water control) to be measured concur-rently on an automated plate reader. It was designedoriginally to provide diagnostic metabolic profiles oflaboratory-cultured bacteria, but can be used with di-lute soil suspensions to provide a profile of metaboliccapability in the population (Garland and Mills, 1991).The system has two significant limitations. First, it isbased on the ability of organisms to grow on the sub-strate provided so non-culturable organisms (or thoseunable to use the particular substrates) are excluded.Thus, only a small proportion of the population canbe studied. Second, the activity of the most numerousheterotrophs dominate the system so information on

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    the metabolic capability of slower growing membersof the population is lost. Nevertheless, it remains aconvenient method for comparing the functional di-versity of the microbial populations in different soils(Nicholson and Hirsch, 1998). A more conventionalapproach is to provide soil with a range of C and Ncompounds and measure CO2 evolution (Degens andHarris, 1997). This method, substrate induced respir-ation (SIR), may give a more realistic measure of soilmicrobial activity but the number of substrates that canbe compared is limited.

    Methods based on DNA and RNA

    Over the past decade, methods for identifying partic-ular microbes, that avoid the need for culture, havebeen developed. These can discriminate between re-lated groups more accurately than is possible withPLFAs, because they rely on the basis of diversity, thegenetic material. DNA can be extracted from both soilbacteria and fungi in situ, and subjected to analysis bypolymerase chain reaction (PCR). PCR relies on theamplification of specific regions of the genome flankedby primers that have been designed to recognise andhybridise to unique sequences within the DNA ofthe target organism. Thus, some prior knowledge ofthe target organism is needed in order to design theprimers. However, the rapid expansion of DNA se-quence databases means that the relevant informationat the genus or family level is available for manygroups, to design universal primers for many genes.A recent example is amplification of the gene forammonia monooxygenase from a community of soilnitrifiers. Subsequently the amplified products weresequenced and the information used to design primersmore suitable for the strains that were present. It wasthen possible to obtain quantitative data on popula-tion fluctuations using competitive PCR (Mendum etal., 1999). The ribosomal genes, in particular the 16srRNA gene in prokaryotes, have been very useful forsuch studies: parts of the gene for this essential cel-lular component are highly conserved at the kingdomlevel, whereas others vary at the family, genus or spe-cies level (Woese, 1987). Thus, primers with differentdegrees of specificity can be designed for PCR ampli-fication of particular groups. The diversity of the mostprevalent individuals within these groups can then beexamined by separating out and sequencing the amp-lified fragments. These will have identical ends (i.e.primer binding sites) but variable internal sequences,

    and can be separated by molecular cloning or physicalmethods such as denaturing or temperature gradientgel electrophoresis (DGGE/TGGE). The properties ofthe rRNA genes have also been exploited in design-ing fluorescent oligoprobes that can be used to bind tothe ribosomal RNA and identify specific strains for insitu detection (Amann et al., 1995). There are practicalproblems in using such methods with soil but they arelikely to become more important in the future. Anotherfeature of the ribosomal RNA is that it is much moreabundant in metabolically active cells than in restingcells (by a factor of 103), although the number of genesin the DNA (usually 110 copies, depending on thespecies) remain constant. If RNA rather than DNA canbe extracted from soil, and then subjected to reversetranscriptase PCR (RT-PCR), the fraction of the pop-ulation that is active can be examined. These methodsare also in their infancy but show great promise.

    Future directions

    To feed the worlds population in the next millennium,a large-scale intensification of agriculture will be re-quired in Latin America, Africa and much of Asia.This will require considerable increases in the use ofinorganic fertilisers but this does not imply decreaseddependence on soil biological processes in fact theopposite is true. In the foreseeable future the majorityof the worlds farmers will remain too poor to purchasethe quantities of fertiliser required to achieve anythingapproaching the crop yields that are achievable andnecessary. Recycling of nutrients from crop residues,animal manures and urban wastes will be of increas-ing importance as a means of meeting the shortfall innutrients. In addition, well informed management oforganic materials is necessary to maintain soil organicmatter levels which, in turn, contribute to the sustain-ability of soils and the ecosystems of which they arepart. There is evidence from Africa, Asia and Europethat the combined use of organic inputs and inorganicfertilisers is more successful in achieving and sustain-ing satisfactory crop yields than inorganic fertilisersalone (Pieri, 1992; Greenland, 1997). Achieving anefficient, integrated and environmentally acceptablemanagement of nutrients will require increased fun-damental understanding of soil biological processes,using the full range of techniques becoming avail-able, and the novel application of this knowledge todesign practical management techniques. This will in-clude a more specific and mechanistic understanding

  • 49

    of mineralisation processes, and their representationin models, to provide more precise prediction of N, Sand P release. This is necessary if inorganic fertiliserapplications are to be adjusted accordingly and lossesto the environment minimised.

    It will also be necessary to capitalise on the oppor-tunities for improved nutrient cycling offered by bio-logical nitrogen fixation and mycorrhizal associations.In the case of nitrogen fixation, the emphasis shouldno longer be on the process of fixation but rather theimproved utilisation of N after fixation through min-eralisation of above- and below-ground plant parts;losses at this stage can be very large. There are almostcertainly indirect effects of rhizosphere processes, yetto be discovered, that could be utilised to increase theefficiency of nutrient cycling or enhance crop growthin other ways. Agroforestry, often using nitrogen-fixing trees, offers considerable potential as a meansof introducing or recycling nutrients and achieving arange of beneficial impacts on soil properties (Sanchezet al., 1998).

    Managing soil biological processes is a key as-pect of sustainable development. Soil scientists nowhave many opportunities to better understand soil or-ganisms, their functions and their interactions withthe chemical and physical environment. They alsocarry a great responsibility to select research topicsand methodologies in a rational way and to apply theresults in the development of sustainable agriculturalsystems that genuinely meet the needs of societiesworldwide. Many aspects of soil biology and ecologyare worthy of research in view of their fundamentalscientific interest and their role in ecosystem function-ing. However, not all aspects are equally likely to leadto improved agricultural productivity or sustainabilitywithin the foreseeable future. Selecting the most ap-propriate topics for research represents a considerablechallenge.

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