Geoderma 100 Ž2001.

389–402
www.elsevier.nlrlocatergeoderma

Developments in soil microbiology since the

mid 1960s
Heribert Insam )
Institute of Microbiology, UniÕersity of Innsbruck, Technikerstra b e 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 enzyme activities, and flux measurements of carbon and nutrients.
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 bacteria, to routine assays of microbial communities by microarray technology.
q 2001 Elsevier Science B.V. All rights reserved.

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

1. Introduction

Microbial ecology, and more specific, soil microbiology is a dynamic and growing
subdiscipline of soil science. 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: Heribert.insam@uibk.ac.at ŽH. Insam..

0016-7061r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 1 6 - 7 0 6 1 Ž 0 1 . 0 0 0 2 9 - 5
390 H. Insamr Geoderma 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 microorganisms. 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’ ŽParkinson 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 pages Že.g.,
Weaver et al., 1994; Alef and Nannipieri, 1995; Schinner et al., 1996; Van Elsas et al.,
1997; Hurst et al., 1997.. New windows into the black box are being opened ŽFig. 1..
In soil microbiology it was realised that simply extracting soils and counting
microorganisms is not enough to characterise the soil microbiota and its significance for
the functioning of soils Že.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 functions ŽCLPP, Community level physiological profiles; PLFA,
Phospholipid fatty acids; details see text..
 H. Insamr Geoderma 100 (2001) 389–402 391

Table 1
Ža. Effect of extraction method on total microbial counts ŽSmith and Stribley, 1994.
Method of extraction Bacterial numbers per gram
inorganic matter
Dispersed soil Žcontrol. 8.5=10 8
Repeated centrifugation and resuspension 1.5=10 9
ŽHopkins et al., 1991.
Aqueous two-phase partitioning 1.3=10 10
ŽSmith and Stribley, 1994.

Žb. Total, viable, and culturable soil bacteria of a barley field soil ŽWinding et al., 1994. Žsimple
extraction.
Method of counting Bacterial numbers gy1 soil
Acridine orange direct count ŽAODC. Žtotal bacteria. 1=10 9
CTC reducing bacteria Žmetabolically active bacteria. 3.5=10 7
Microcolony-forming units Žmicro-CFU. 2.5=10 7
Žbacteria that are able to perform a few,
but not more cell divisions on agar.
Žviable but not culturable bacteria. a
Colony-forming units ŽCFU. on agar plates 7=10 6
Žculturable bacteria. a
a
Longer incubations, up to 64 days, increased the numbers of CFUs.

magnitude ŽTable 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 CO 2 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 cycle Že.g., C, N, P or S. under investigation. Using
392 H. Insamr Geoderma 100 (2001) 389–402

databases from the Institute of Scientific Information ŽISI. it has been attempted to
quantify which enzyme tests are most frequently used today and these are urease
ŽTabatabai and Bremner, 1972., phosphatase ŽHoffmann, 1967. and dehydrogenase
ŽTrevors, 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 soil
organic matter or clay minerals. Therefore, Visser and Parkinson Ž1992. disputed the
suitability of enzyme assays for microbial activity and soil quality assessments, with the
exception of dehydrogenase because its biological properties make it unlikely to be
present in soil in an extracellular state ŽSkujins, 1978..
To overcome the interpretation problems of single enzyme tests, Beck Ž1984.
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. Ž1998. 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’s Ž1984. and
Trasar-Cepeda et al.’s Ž1998. 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. Ž1998. 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 profiling ŽCLPP., which is another type of ‘enzyme
assay’, has been proposed by Garland and Mills Ž1991.. 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 disturbances Že.g., Mayr et al., 1999; Yan et al., 2000. and microplates
ŽEcoPlates. especially designed for environmental applications ŽInsam, 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
Žaddressing 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. Insamr Geoderma 100 (2001) 389–402 393

work of Odum Ž1969. 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
CO 2 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
ŽStotzky, 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 soil acidification caused by atmospheric inputs of N, NO 3 , move-
ments from soil to groundwater, and the contribution of N gases Že.g., N2 O and NO. to
the climate change and ozone destruction have triggered detailed investigation on N
transformation in soils ŽMyrold, 1997.. Biological nitrogen fixation 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 repeatedly Že.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 tracer Ž15 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. Ž1994.
or Alef and Nannipieri Ž1995..
Most nutrient 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 problem ŽGoovaerts, 1998..
Bruckner et al. Ž1999. studied physico-chemical and soil biological parameters Žrespira-
tion, microbial biomass and N-mineralisation. and detected three different scales of
spatial variability in a temperate coniferous forest: Ž1. a fine-scale pattern - 1m due to
retarded decomposition of Picea abies Žspruce. litter, and lacking bioturbation by
earthworms, Ž2. a mesoscale pattern Ž1.0–1.5 m., reflecting the influence of individual
trees, and Ž3. 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 only ŽStemmer et al., 1999.. Rasiah and Kay Ž1999.
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. Ž1998. showed that the variability of most microbiolog-
394 H. Insamr Geoderma 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

Biomass Že.g., plant root and shoot biomass, faunal biomass. and other organic pools
Žlitter, 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 Jenkinson Ž1976., 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 CO 2 production which is attributed to the killed and subsequently
decomposed microbial biomass C. The method was named fumigation–incubation ŽFI.
method.
Another physiological approach was proposed by Anderson and Domsch Ž1978., the
so-called substrate-induced respiration ŽSIR. 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 disputed Že.g., Sparling, 1995..
A more direct way to estimate microbial biomass P and N has been proposed by
Brookes et al. Ž1982, 1985.. After chloroform fumigation soil samples are extracted
Žfumigation–extraction ŽFE. method. and biomass P or N are measured directly. The
method has also been adapted for biomass C measurements ŽVance 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 publications ŽFig. 2.. Apart from their use in scientific studies,
fumigation–extraction and SIR have also been adopted by national authorities Že.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 succession ŽOdum, 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 ecosystem ŽAnderson and Domsch, 1986; Insam
and Haselwandter, 1989.. For many environmental studies, the so-called metabolic
 H. Insamr Geoderma 100 (2001) 389–402 395

Fig. 2. Number of SCI citations of original papers ŽJenkinson 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.

quotient Žmg CO 2 –C respired gy1 C mic hy1 . ŽAnderson 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 soil management
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 duration Ž10 days.
no toxic chemicals needed
Not suitable for acid ŽpH -6.0. soils
Substrate induced Good reproducibility Calibration with another method
respiration necessary Žindirect estimate.
No toxic chemicals needed Not suitable for soils that received
fresh organic amendments
Rapid Ž -8 h.
Fumigation–extraction Good reproducibility C measurement requires expensive
equipment
Rapid Ž - 24 h.
396 H. Insamr Geoderma 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 publications ŽFig. 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 technique ŽAnder-
son and Domsch, 1975.. In this method, fungal or bacterial growth was inhibited by the
addition of specific antibiotics and the fungalrbacterial ratio was calculated from the
extent of inhibition. More successful was the determination of ergosterol ŽWest 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. Ž2000.. 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 Prokaryotes and has been
suggested as an indicator for bacterial and cyanophyte biomass ŽMillar and Cassida,
1970. but the method has not received wide acceptance because the extraction parame-
ters ŽHCl 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 keywords Žsearch
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. Insamr Geoderma 100 (2001) 389–402 397

Table 3
Phospholipid fatty acids may be used to determine the community composition of the soil microbiota ŽZelles
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
Cyanobacteria Žand eucaryotes. Lipids containing polyunsaturated fatty acids
Fungi ˚˚ 1996.
A specific PLFA, 18:2v6 ŽFrostegard and Baath,

suspicion that muramic acid might mainly be present in dead cell material ŽZelles 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 fertilizers, 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 acids ŽPLFA., which are found in the membranes of all living cells
and can be used for the determination of the community composition ŽZelles et al.,
1992; Guckert and White, 1986; Petersen et al., 1997.. Bacterial groups and fungi can be
characterised according to their lipid composition ŽTable 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, Torsvik Ž1980. 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
ŽTorsvik 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: Ž1. initially,
cells are extracted by repeated suspension–centrifugation steps and then the cells are
398 H. Insamr Geoderma 100 (2001) 389–402

lysed for DNA recovery; Ž2. 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 reaction ŽPCR. of humic acids.
Several methods are used today to study soil DNA or RNA. Soil molecular ecologists
use the unique feature of prokaryote 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 reaction ŽPCR.. 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 electrophoresis ŽMuyzer et
al., 1993. that allows the separation of amplificates according to their G q C content. On
a formamide denaturing gradient gel ŽDGGE. Žor, alternatively, a temperature gradient
gel, TGGE. genes with a lower G q C content are denatured earlier and do not travel
far. Genes with different G q C 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 Tebbe Ž1998., using a method called single strand
conformational polymorphism, which is superior when sequences are different but
G q C contents are similar among species. These methods are, however, qualitative.
Quantitative PCR methods are tedious and still need to be improved for environmental
applications ŽOgram, 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
Stotzky Ž2000. and Saxena et al. Ž1999. 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 montmorillonite ŽStotzky, 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. Insamr Geoderma 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
¨
Že.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 formed ŽClarholm, 1994..
Microbial loops are long known from marine waters. For soils, the microbial loop has
been described as the rhizosphere flora, where bacteria that utilize root-derived carbon,
bind inorganic nutrients transported to the roots Žby mass flow and diffusion.. The
bacteria are grazed by protozoa, 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 forms ŽClarholm, 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 membranes ŽGuschin 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 activity ŽGottschal et al., 1997.. The very small sample sizes Žin 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 key Žbiological. player in soil may be altered in a
desired way, thus altering soil functions 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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