Geoderma 100Ž. 2001 389–402 www.elsevier.nlrlocatergeoderma

Developments in since the mid 1960s

Heribert Insam) Institute of Microbiology, UniÕersity of Innsbruck, Technikerstrabe 25, A-6020 Innsbruck, Austria Received 10 January 2001; received in revised form 19 February 2001; accepted 22 February 2001

Abstract

Since the 1960s, soil microbiology underwent major changes in methods and approaches and this review focuses on the developments in some selected aspects of soil microbiology. Research in cell numbers of specific bacterial and fungal groups was replaced by a focus on biochemical processes including soil activities, and flux measurements of carbon and . Ecologists focused on soil microbial pools whereas soil microbial biomass as an important source and sink of nutrients were recognized in agriculture. Soil microbiologists started to use structural components like phospholipid fatty acids for quantification of specific microbial groups without the need to cultivate them. In the last decade, molecular approaches allowed new insights through the analysis of soil extract DNA showing an unexpected diversity of genomes in soil. At the end of the review a brief outlook is given on the future of soil microbiology which ranges from in situ identification of , to routine assays of microbial communities by microarray technology. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Soil microbiology; Review; ; Microbial biomass; N turnover; Molecular ecology

1. Introduction

Microbial ecology, and more specific, soil microbiology is a dynamic and growing subdiscipline of . There are a number of new journals in this field but also traditional soil science journals increasingly attract soil microbiological papers. In 1967 the first issue of Geoderma was published. Thirty-four years is a long time span in most scientific fields and during these years soil microbiology has developed from a playground of soil scientists, microbiologists and soil chemists to an own subdiscipline. This review on soil microbiology on the occasion of 100 volumes of

) Tel.: q43-512-507-6009; fax: q43-512-507-2928. E-mail address: [email protected]Ž. H. Insam .

0016-7061r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved. PII: S0016-7061Ž. 01 00029-5 390 H. InsamrGeoderma 100() 2001 389–402

Geoderma aims to introduce to soil scientists the importance of microbiological aspects of the soil and the new insight offered by new techniques. It gives a brief historical overview of the developments within soil microbiology, and some thoughts on future prospects.

2. Driven by methods Soil is very complex with diverse niches offered to soil . Having to deal with solid, liquid and gaseous phases, soil microbiologists were in need of suitable methods for studying their subject since the beginnings of this field. ‘Ecology of soil microorganisms’Ž. Parkinson et al., 1971 was the standard reference for soil microbiolo- gists 30 years ago and it was a relatively thin booklet with 116 pages. Nowadays, methods books in soil microbiology are abound and they usually exceed 500 pagesŽ e.g., Weaver et al., 1994; Alef and Nannipieri, 1995; Schinner et al., 1996; Van Elsas et al., 1997; Hurst et al., 1997.Ž. . New windows into the black box are being opened Fig. 1 . In soil microbiology it was realised that simply extracting and counting microorganisms is not enough to characterise the soil and its significance for the functioning of soilsŽ. e.g., Macura, 1974 . Prevalent methods of cell enumeration account for a very small fraction of the total number of microbes, and reveal no information about the activity of the counted organism. Both methods of extraction and enumeration are subject to bias and differences may be as large as three orders of

Fig. 1. The soil as a Black Box. Soil microbiologists open up new windows to investigate soil microbial communities, their composition and functionsŽ CLPP, Community level physiological profiles; PLFA, Phospholipid fatty acids; details see text. . H. InsamrGeoderma 100() 2001 389–402 391

Table 1 Ž.a Effect of extraction method on total microbial counts Ž Smith and Stribley, 1994 . Method of extraction Bacterial numbers per gram inorganic matter Dispersed soilŽ. control 8.5=108 Repeated centrifugation and resuspension 1.5=109 Ž.Hopkins et al., 1991 Aqueous two-phase partitioning 1.3=1010 Ž.Smith and Stribley, 1994

Ž.b Total, viable, and culturable soil bacteria of a barley field soil Ž Winding et al., 1994 .Ž simple extraction. Method of counting Bacterial numbers gy1 soil Acridine orange direct countŽ.Ž. AODC total bacteria 1=109 CTC reducing bacteriaŽ. metabolically active bacteria 3.5=107 Microcolony-forming unitsŽ. micro-CFU 2.5=107 Žbacteria that are able to perform a few, but not more cell divisions on agar. Ž.viable but not culturable bacteria a Colony-forming unitsŽ. CFU on agar plates 7=106 Ž.culturable bacteria a

a Longer incubations, up to 64 days, increased the numbers of CFUs. magnitudeŽ. Table 1 . Despite this shortcoming, cell enumeration methods have long been the prime choice for soil microbiologists to obtain quantitative data. Counting methods do have virtue in cases when a specific organism is studied but it is advisable to crosscheck the figures on microbial numbers in relation to the applicability of the analytical method. The data in Table 1 show that with traditional cultivation methods on agar plates only a small percentage of the total microflora is accounted for, and for the remaining 99% physiologic and taxonomic information is lacking. For most soil microbiologists such data were not satisfactory for understanding soil functioning and the consequence was to attempt to measure processes like enzyme activities or N fixation and nitrification, or more unspecific processes like CO2 evolution or heat generation.

3. Soil enzymes Enzymes in soil may be extra- or intracellular. Extracellular enzymes are necessary for the breakdown of organic macromolecules, like cellulose, hemicelluloses or lignin whereas intracellular enzymes are responsible for the breakdown of smaller molecules like sugars or amino acids. Soil enzymes are predominantly of microbial origin and are closely related to microbial abundance andror activity. Biochemical tools that allowed rapid measurement of soil enzyme activities made soil enzymology fashionable in the late 1960s, and such tools remained widely used for 20 years. Numerous enzymes have been tested on their suitability for soil investigations; the choice of method will largely depend on the geochemical cycleŽ. e.g., C, N, P or S under investigation. Using 392 H. InsamrGeoderma 100() 2001 389–402 databases from the Institute of Scientific InformationŽ. ISI it has been attempted to quantify which enzyme tests are most frequently used today and these are urease Ž.Ž.Tabatabai and Bremner, 1972 , phosphatase Hoffmann, 1967 and dehydrogenase Ž.Trevors, 1984 , indicating the acceptance of the methods in the scientific community. A major weakness of enzyme tests is that the actual microbial activity of a soil is not well reflected. Moreover, the tests show ‘historic’ features of enzymes bound to or minerals. Therefore, Visser and ParkinsonŽ. 1992 disputed the suitability of enzyme assays for microbial activity and assessments, with the exception of dehydrogenase because its biological properties make it unlikely to be present in soil in an extracellular stateŽ. Skujins, 1978 . To overcome the interpretation problems of single enzyme tests, BeckŽ. 1984 proposed a soil microbiological index calculated from microbial biomass, reductase and hydrolase activities. This index, however, never became popular. In another attempt to propose such an index, Trasar-Cepeda et al.Ž. 1998 found a very close relation of total N with a linear combination of soil microbial biomass C, mineralised N, phosphomo- noesterase, b-glucosidase and urease activity. This test set was proposed as a soil quality index closely related to soil sustainability. The problem with both Beck’sŽ. 1984 and Trasar-Cepeda et al.’sŽ. 1998 approaches is that the usefulness of the enzyme tests used as components of their indices is disputed, and universally accepted enzyme tests are still lacking. The use of fluorogenic substrates has recently been proposed for studying microbial activities. Miller et al.Ž. 1998 used 4-methylumbelliferyl N-acetyl-b-D-glucosaminide, that, when hydrolysed, releases 4-methylumbelliferone which fluoresces and can be detected in nanomolar concentrations. This test specifically determines fungal chiti- nolytic activities. Assays using labelled substrates directly test for substrate degradation and may be performed in microplates, they need smaller sample sizes than traditional enzyme tests and allow measurement of many parallels in one assay. Community-level physiological profilingŽ. CLPP , which is another type of ‘enzyme assay’, has been proposed by Garland and MillsŽ. 1991 . With this method, the ability of microbial communities to degrade a set of up to 95 different substrates is tested in one single assay. A redox indicator in the Biolog w microtiter plates indicates if the specific substrate is used as an energy source. CLPPs have been shown to be very sensitive indicators of disturbancesŽ. e.g., Mayr et al., 1999; Yan et al., 2000 and microplates Ž.EcoPlates especially designed for environmental applications Ž Insam, 1997 . are now available. Summarizing, soil enzyme assays, including CLPPs, are never stand-alone parame- ters. They are quick monitoring tools, but cannot give answers on functionality. To characterize the soil, or to understand differences among soils, additional methods Ž.addressing pools and fluxes are necessary.

4. Fluxes

Enzyme tests cannot be used to estimate in situ matter fluxes as they are an estimate of the potential to use a certain substrate under optimised conditions. In ecology, the H. InsamrGeoderma 100() 2001 389–402 393 work of OdumŽ. 1969 has been very influential. Pools and fluxes were measured and calculated for catchments and whole ecosystems and this caused direct flux measure- ments to become important for soil microbiologists. In the 1970s and 1980s, respiratory

CO2 evolution was indispensable for soil microbiological work in ecosystem research. Carbon dioxide evolution, a traditional method since the early days of soil microbiology is still the best index of gross metabolic activity of mixed microbial populations Ž.Stotzky, 1997 . While in situ determinations are preferred for ecosystem studies, in vitro tests have proven to be sufficient for other purposes like decomposition studies or more general soil characterisation.

Concerns about caused by atmospheric inputs of N, NO3 , move- ments from soil to , and the contribution of N gasesŽ. e.g., N2 O and NO to the climate change and ozone destruction have triggered detailed investigation on N transformation in soilsŽ. Myrold, 1997 . Biological has been considered as a factor to increase agricultural production and to mitigate hunger in the world. Both symbiotic and nonsymbiotic nitrogen fixation have been studied intensely since the 1970s, and the significance of nitrogen fixation for the yield of several crops and for the sustainability of farming systems has been emphasised repeatedlyŽ e.g., Paul and Clark, 1996. . Today, soil microbiologists work on genetically engineering plants and microor- ganisms to further improve the benefits of biological nitrogen fixation. Nitrogen turnover measurements are essential for understanding ecosystem dynamics and agricultural productivity, because N occurs in soils, oceans and the atmosphere and its cycling has global implications. Nitrogen is also often the limiting factor for microbial activity. Many traditional methods, like N mineralisation tests, potential nitrification, or the acetylene inhibition method for denitrification are still widely used. As with C turnover studies, the use of an isotope tracerŽ15 N. may give detailed insight in the allocation of N within the soil, or even within the soil microbial community. It is, however, beyond the scope of this review to address all the different methods for measuring rates of N cycle transformations which can be found in Weaver et al.Ž. 1994 or Alef and NannipieriŽ. 1995 . Most flux measurements are not performed in the field but in the laboratory. The problem of in vitro tests and plot investigations is the extrapolation of measured values. Spatial heterogeneity is a considerable problem in soil microbiology but excel- lent geostatistical tools are available to deal with this problemŽ. Goovaerts, 1998 . Bruckner et al.Ž. 1999 studied physico-chemical and soil biological parameters Ž respira- tion, microbial biomass and N-mineralisation. and detected three different scales of spatial variability in a temperate coniferous forest:Ž. 1 a fine-scale pattern -1m due to retarded decomposition of Picea abies Ž.spruce litter, and lacking bioturbation by earthworms,Ž. 2 a mesoscale pattern Ž 1.0–1.5 m . , reflecting the influence of individual trees, andŽ. 3 unexplained long-range trends of Nmin and water content exceeding the transect length of the study. Specific turnover processes do occur in specific niches, e.g. in certain aggregate size classes onlyŽ.Ž. Stemmer et al., 1999 . Rasiah and Kay 1999 found that management-induced changes in compaction, spatially variable textural characteristics and soil organic matter had strong influence on microbial biomass N. This effect was stronger on soils amended with red clover shoot biomass than in unamended soils. Stenberg et al.Ž. 1998 showed that the variability of most microbiolog- 394 H. InsamrGeoderma 100() 2001 389–402 ical parameters within one single agricultural field was as large as for a set of 26 different Swedish soils.

5. Pools

BiomassŽ. e.g., plant and shoot biomass, faunal biomass and other organic pools Ž.litter, soil organic matter are important components for the functioning of an ecosys- tem. Due to a lack of suitable and sufficiently standardised methods in soil micro- biology, the microbial biomass pool was long neglected or estimated based on microbial counts. This largely changed through the work of JenkinsonŽ. 1976 , who proposed a method for indirect microbial biomass determination encompassing ecological and microbiological thinking. The idea was to kill and lyse microbial cells in a soil sample by chloroform fumigation. Following re-inoculation with soil, the respiration is mea- sured for some 10 days. Compared to an unfumigated control the fumigated sample shows an enhanced CO2 production which is attributed to the killed and subsequently decomposed microbial biomass C. The method was named fumigation–incubationŽ. FI method. Another physiological approach was proposed by Anderson and DomschŽ. 1978 , the so-called substrate-induced respirationŽ. SIR method. The idea was derived from pure culture studies. Microorganisms respond to the supply with a readily available substrate, i.e. glucose, with an immediate response in respiration that was supposed to be linearly correlated with biomass C. Both the FI and SIR methods have drawbacks. The FI method takes 10 days and is generally unsuitable for acid soils. SIR has originally been calibrated against FI and several different calibration factors have been proposed. The calculation of microbial biomass C from the SIR data is therefore often disputedŽ. e.g., Sparling, 1995 . A more direct way to estimate microbial biomass P and N has been proposed by Brookes et al.Ž. 1982, 1985 . After chloroform fumigation soil samples are extracted ŽŽ..fumigation–extraction FE method and biomass P or N are measured directly. The method has also been adapted for biomass C measurementsŽ. Vance et al., 1987 , and has become the most frequently used method for microbial biomass determination. The FE method requires correction factors for different soils and for the different elements analysed. The determination of these correction factors is laborious for routine analyses. The different methods to estimate microbial biomass are frequently used as revealed by a survey of recent publicationsŽ. Fig. 2 . Apart from their use in scientific studies, fumigation–extraction and SIR have also been adopted by national authoritiesŽ e.g., in Germany. for routine soil surveys. Advantages and disadvantages of the methods are summarised in Table 2. One of the main concepts of Odum’s theory on ecosystem development was the energetic optimisation during successionŽ. Odum, 1969 . According to Odum, the ecosystem respiration-to-biomass ratio decreases during maturation of an ecosystem. Since all C from primary production eventually reaches the soil C pool, this concept was adopted for the soil compartment of an ecosystemŽ Anderson and Domsch, 1986; Insam and Haselwandter, 1989. . For many environmental studies, the so-called metabolic H. InsamrGeoderma 100() 2001 389–402 395

Fig. 2. Number of SCI citations of original papersŽ Jenkinson and Poulsen, 1976; Anderson and Domsch, 1978; Vance et al., 1987; Brookes et al., 1982, 1985. on microbial biomass determination during the last 3 years. This gives a rough estimate on the relative frequency of use of the four selected methods of biomass determination.

y1 y1 quotient Žmg CO2mic –C respired g C h.Ž Anderson and Domsch, 1986 . has proven to be more sensitive than the measurement of microbial biomass or respiration alone. Microbial pool sizes have proven to be reliable indicators of soil quality, and contribute to the understanding of nutrient dynamics, both on the long term and season by season. Microbial biomass is a robust parameter that may rapidly, and reproducibly, be determined. It allows gross comparisons of soils, and reflects changes, or pollution impact. If, however, only certain functions or specific microorgan- isms are affected, microbial biomass is not a sufficiently sensitive parameter. In that case, fluxes, or the community composition need to be investigated in more detail.

Table 2 Advantages and disadvantages of the most frequently used methods of microbial biomass determination in soils Method Advantages Disadvantages Fumigation–incubation Direct, biological measurement, Long durationŽ. 10 days no toxic chemicals needed Not suitable for acidŽ. pH-6.0 soils Substrate induced Good reproducibility Calibration with another method respiration necessaryŽ. indirect estimate No toxic chemicals needed Not suitable for soils that received fresh organic amendments Rapid Ž.-8h Fumigation–extraction Good reproducibility C measurement requires expensive equipment Rapid Ž.-24 h 396 H. InsamrGeoderma 100() 2001 389–402

6. Recent developments Despite all attempts to measure fluxes and gross microbial pools, the soil and its microbiota still remained a black box, with only a few tiny windows opened up for the organisms that were isolated, cultured and identified. Actors determining the mass and nutrient flows were unknown. The change in approaches for studying the soil microbiota is reflected by the change of keywords found in the titles of publicationsŽ. Fig. 3 . While ‘enzyme’ and ‘microbial biomass’ are found in ISI databases from 1991 to 2000 in constant frequency, ‘respiration’ and, first of all, ‘microbial community’ occur increas- ingly since a few years. Initial attempts in the 1970s for opening up the black box were the separation of bacterial and fungal biomass, for example by the selective inhibition techniqueŽ Ander- son and Domsch, 1975. . In this method, fungal or bacterial growth was inhibited by the addition of specific and the fungalrbacterial ratio was calculated from the extent of inhibition. More successful was the determination of ergosterolŽ West et al., 1987. in soils, which is still used if fungal biomass is to be quantified. Ergosterol is the main sterol of most Ascomycetes, Basidiomycetes and Fungi imperfecti, and thus an indicator of fungal biomass. This was recently disputed by Ruzicka et al.Ž. 2000 . They found that a universal conversion factor of ergosterol content to fungal biomass remains elusive and problematic and is not a measure of fungal biomass, but rather an indicator of the extent of fungal membranes in soils. Still, ergosterol is able to give a an estimate on the extent of fungal colonization of a soil. Muramic acid, in contrast to ergosterol, is only found in and has been suggested as an indicator for bacterial and cyanophyte biomassŽ Millar and Cassida, 1970. but the method has not received wide acceptance because the extraction parame- tersŽ. HCl concentration, time need to be optimised for each soil, and because of the

Fig. 3. Frequency of publications that have the word soil and, in addition, certain other keywordsŽ search terms. in their title. These few examples show a constancy in papers on microbial biomass and enzymes, and a clear increase in studies on microbial communities during the last 10 years. Unfortunately, the Science Citation Index does not date back further than 1991. H. InsamrGeoderma 100() 2001 389–402 397

Table 3 Phospholipid fatty acids may be used to determine the community composition of the soil microbiotaŽ Zelles and Alef, 1995. . This table shows a summary of the most important signature phospholipid fatty acids. Microbial group Phospholipid fatty acid signatures Archaebacteria Fatty residues are ether-linked to glycerol Anaerobic bacteria Contain sphingolipids which are largely absent from aerobes Bacteria, in general Saturated or monounsaturated fatty acids ester-linked to glycerol Gram-negative bacteria Contain more hydroxylated fatty acids Gram-positive bacteria Contain more branched fatty acids CyanobacteriaŽ. and eucaryotes Lipids containing polyunsaturated fatty acids Fungi A specific PLFA, 18:2v6Ž. Frostegard and Baath,˚˚ 1996 suspicion that muramic acid might mainly be present in dead cell materialŽ Zelles and Alef, 1995. . For the microbiologist a differentiation between bacteria and fungi is not enough because within these two groups many taxonomic and functional subgroups do exist that deserve special attention. Certain functions, like nitrification, may be impaired by some management measure like the use of nitrogen , herbicides or pesticides, while the total bacterial or fungal biomass remains unaffected. A breakthrough in the use of biomarkers for community characterisation were the phospholipid fatty acidsŽ. PLFA , which are found in the membranes of all living cells and can be used for the determination of the community compositionŽ Zelles et al., 1992; Guckert and White, 1986; Petersen et al., 1997. . Bacterial groups and fungi can be characterised according to their lipid compositionŽ. Table 3 . All biomarkers have in common the problem of extractability and unknown stability in the soil. Therefore, it is not easy to judge if data derived from biomarkers are related to living cells only, or that they also include dead cell material. The stability of biomarkers in the soil largely depends on temperature, moisture and other conditions directly related to degradation processes. Furthermore, the physiological status of microorganisms often determines the content of different components. Summarizing, biomarkers were an acceptable tool as long as no better alternatives were available, but in the near future, biomarkers for community characterization are likely to be replaced by molecular approaches.

7. Soil microbiology and DNA

In her seminal paper, TorsvikŽ. 1980 was able to obtain DNA from soil sufficiently pure to allow hybridisation techniques, and in 1990 they showed by DNArDNA hybridisation that 1 g of soil contained more than 4000 different genomes of bacteria Ž.Torsvik et al., 1990 . Most of the diversity was found in the fraction that could not be isolated and cultured by standard and sophisticated plating techniques. For obtaining pure DNA from the soil, two procedures may be followed:Ž. 1 initially, cells are extracted by repeated suspension–centrifugation steps and then the cells are 398 H. InsamrGeoderma 100() 2001 389–402 lysed for DNA recovery;Ž. 2 the DNA is directly extracted from the soil after lysis of the cells. The advantage of the first procedure is that the DNA is purer whereas in the second procedure a more complete extraction is obtained which is more likely to represent the total community. Currently, most researchers extract the DNA directly from the soil because improved purification procedures are available that avoid inhibi- tion of polymerase chain reactionŽ. PCR of humic acids. Several methods are used today to study soil DNA or RNA. Soil molecular ecologists use the unique feature of 16S rDNA that contains conserved and variable regions. The 16S rDNA is a strand of about 1500-bp length. The conserved regions deviate only among taxonomically distant groups, while the variable regions may show differences even among different strains of a single species. The extracted DNA is purified, and amplified by polymerase chain reactionŽ. PCR . The primers determine the region that is amplified, so that an analysis may either give a rough overview or detailed insight into a specific group. The amplification products are analysed by gel elec- trophoresis. One of the approaches is denaturing gradient gel electrophoresisŽ Muyzer et al., 1993. that allows the separation of amplificates according to their GqC content. On a formamide denaturing gradient gelŽ.Ž DGGE or, alternatively, a temperature gradient gel, TGGE. genes with a lower GqC content are denatured earlier and do not travel far. Genes with different GqC contents yield distinct bands. The pattern of the bands is different in different communities. If more detailed information is required, bands may be excised, cloned, sequenced and compared to known organisms in databases. Results of such investigations showed that many of the detected sequences were not matched by any known isolate and they represent new, uncultured species. A similar approach has been proposed by Schwieger and TebbeŽ. 1998 , using a method called single strand conformational polymorphism, which is superior when sequences are different but GqC contents are similar among species. These methods are, however, qualitative. Quantitative PCR methods are tedious and still need to be improved for environmental applicationsŽ. Ogram, 2000 . Recent publications have shown that soil science and molecular ecology are inti- mately linked. An example is the growing concern about environmental and health effects of genetically engineered crops, particularly in Western Europe. Saxena and StotzkyŽ. 2000 and Saxena et al. Ž. 1999 demonstrated that insecticidal toxin in root exudates from genetically modified maize plants is strongly bound to soil clay minerals and may persist for a long time. The unpredictable release of the toxin may constitute a hazard to non-target organisms. It seems that the DNA encoding for this toxin may bind to expandable clay minerals like montmorilloniteŽ. Stotzky, personal communication , and upon their expansion the DNA is rendered susceptible to uptake by competent bacteria. It is unresolved if these genes, after renewed uptake, may then render weeds resistant.

8. Future outlook

Perspectives for soil microbiologists are bright because new, mainly molecular techniques offer new insight into the soil black box so that microbial community H. InsamrGeoderma 100() 2001 389–402 399 composition and microbial activities can be investigated, and even localized on a microscale. In situ hybridisation is able to show where bacteria and fungi are existing Ž.e.g., Lubeck¨ et al., 2000 , and even where they are active. Of particular interest for the future are the study of microbial hot spots, as they do occur in the guts of soil microfauna, or around root surfaces. It is assumed that in hotspots the majority of turnover processes takes place, and microbial loops are formedŽ. Clarholm, 1994 . Microbial loops are long known from marine waters. For soils, the has been described as the flora, where bacteria that utilize root-derived carbon, bind inorganic nutrients transported to the rootsŽ. by mass flow and diffusion . The bacteria are grazed by , who then release one third of the bacterial N as ammonium available for plant uptake, while two thirds form protozoan biomass or are egested in organic formsŽ. Clarholm, 1994 , and thus become inaccessible for plant uptake. Microarray technology will soon enable us to assess community diversity in soils by directly exposing and hybridising oligonucleotides fixed on membranesŽ Guschin et al., 1997; Ogram, 2000. . Another aim will be to relate community structure with community function by using messenger RNA. Due to the short half-life of mRNA this is currently not possible, but it has several advantages. If combined with PCR amplification it will be more sensitive than any enzyme test, it provides information about the metabolic state at the moment of testing, and, if combined with analysis of rDNA, very detailed information may be obtained about the involvement of certain populations in a particular metabolic activityŽ. Gottschal et al., 1997 . The very small sample sizes Ž in the range of a few milligrams. allow a very small-scale spatial resolution. Soil will no longer be a black box, but we will be able to see where the microbes live, what their role in soil processes is, and how their abundance and activity is influenced by soil physical and soil chemical properties. Thus, today, in soil microbiology questions like who is active and where are the activities located are answered that have been asked many years ago. Soil management may aim to successfully establish desired microbial populations. Such microorganisms may be degraders of xenobiotics, nitrogen fixers, or pathogen antago- nists. In the not-so-far future a single keyŽ. biological player in soil may be altered in a desired way, thus altering in a beneficial way to man. This will hopefully increase the sustainability of agricultural systems on the long run, and also enable us to successfully remediate polluted soils and protect natural ecosystems.

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