240

Soils sustain an immense diversity of microbes, which, to a

large extent, remains unexplored. A range of novel methods,
most of which are based on rRNA and rDNA analyses, have
uncovered part of the soil microbial diversity. The next step in
the era of microbial ecology is to extract genomic, evolutionary
and functional information from bacterial artificial chromosome
libraries of the soil community genomes (the metagenome).
Sophisticated analyses that apply molecular phylogenetics,
DNA microarrays, functional genomics and in situ activity
measurements will provide huge amounts of new data,
potentially increasing our understanding of the structure and
function of soil microbial ecosystems, and the interactions that
occur within them. This review summarizes the recent progress
in studies of soil microbial communities with focus on novel
methods and approaches that provide new insight into the
relationship between phylogenetic and functional diversity.
Addresses
Department of Microbiology, University of Bergen, Post Box 7800,
Jahnebakken 5, N-5020 Bergen, Norway
*e-mail: [email protected]

e-mail: [email protected]
Current Opinion in Microbiology 2002, 5:240245
1369-5274/02/$ see front matter
2002 Elsevier Science Ltd. All rights reserved.
Published online 13 May 2002
Abbreviations
BAC bacterial artificial chromosome
BrdU 5-bromo-2-deoxyuridine
DGGE denaturant gradient gel electrophoresis
FI SH fluorescent in situ hybridization
GC guanine + cytosine
PCR polymerase chain reaction
PLFA phospholipid fatty acid
Introduction
Microbial diversity in soil ecosystems exceeds, by far, that
of eukaryotic organisms. One gram of soil may harbor
up to 10 billion microorganisms of possibly thousands
of different species [1

]. As less than 1% of the micro-

organisms observed under the microscope (Figure 1) is
cultivated and characterized, soil ecosystems are, to a large
extent, uncharted. Microbial diversity describes complexity
and variability at different levels of biological organization.
It encompasses genetic variability within taxons (species),
and the number (richness) and relative abundance
(evenness) of taxons and functional groups (guilds) in com-
munities. Important aspects of diversity at the ecosystem
level are the range of processes, complexity of interactions,
and number of trophic levels. Thus, measures of microbial
diversity should include multiple methods integrating
holistic measures at the total community level and partial
approaches targeting structural or functional subsets [2,3].
Diversity may also be considered to be the amount and
distribution of information, which is directly applicable to
the total genetic diversity or complexity in a community.
The genetic complexity or genome size of microbial
community genomes can be assessed by re-association of
community DNA. Such broad-scale analysis has revealed
[4,5] that the community genome size equals the size of
600010 000 Escherichia coli genomes in unperturbed
organic soils, and 3501500 genomes in arable or heavy-
metal-polluted soils. These values may be conservative, as
genomes representing rare and unrecovered microorgan-
isms are probably not included in the analysis. In contrast,
the genomic complexity recovered by culturing methods
was less than 40 genomes. The total genomic complexity
denotes the confines of diversity in terms of genetic infor-
mation present and provides information about the overall
(potential) taxonomic and functional variability at the com-
munity level. Because this represents an average diversity
value, no detailed information about taxon and functional
diversity at lower levels of biological organization is
obtained. A number of methods with higher resolving
power have been developed for characterization of micro-
bial communities that includes both cultured and
uncultured microorganisms. Most of them are based on
analyses of ribosomal RNA genes (rDNA). They have
uncovered part of the microbial diversity in soil, yielding
sequences from many novel phylogenetic lineages.
Measures of microbial patterns (fingerprinting) and
taxonomic variability have been coupled with analysis of
functional genes and activity measurements. Such investi-
gations aim to reveal and understand the relationship
Microbial diversity and function in soil: from genes to ecosystems
Vigdis Torsvik* and Lise vres

Figure 1
Epifluorescence micrography of soil microorganisms stained with
4,6-diamidino-2-phenylindole (DAPI). The total bacterial count was
4.2 10
10
cells gram
1
soil (dry weight) by fluorescent microscopy,
and 4.2 10
6
colony-forming units gram
1
soil (dry weight) by plating.
Current Opinion in Microbiology
Microbial diversity and function in soil: from genes to ecosystems Torsvik and vres 241
between structural and functional diversity in soil
microbial ecosystems.
In this review, we summarize the recent progress in studies
of soil microbial communities. We present an overview of
novel molecular methods for studying all the microorganisms
in soil, including those uncultured, and approaches for
obtaining information from community genome analysis.
The review highlights some recent studies that link phylo-
genetic groups to function and may contribute to exciting
new insight into the relationship between community
composition and function. Finally, we discuss the effect of
soil physical and chemical conditions on microbial diversity
and the importance of functional diversity for soil ecosystem
stability and resilience.
Methods to describe diversity
The indications of the vast diversity of uncultured life in soil
have stimulated development of methods for culture-
independent study of microbial communities. These methods
have employed a combination of nucleic acid characterization
and microscopy. Over the past two decades, molecular methods,
especially 16S rRNA gene sequencing, have become very
popular to help identify unknown bacteria [6,7]. In turn, this
has led to community analysis using total community DNA
extracted from the environment. Broad-scale analysis of com-
munity DNA, using techniques such as DNA re-association,
provides information about the total genetic diversity of a
given bacterial community [4]. A shift in guanine + cytosine
(GC) content can be used to detect changes in microbial
community structure, but does not tell us anything about the
other diversity parameters, which are richness, evenness and
composition. Polymerase chain reaction (PCR)-based finger-
printing techniques give a higher resolution and provide
information about changes in the whole community structure.
These fingerprinting techniques, such as phospholipid fatty
acid (PLFA) analysis, denaturant gradient gel electrophoresis
(DGGE), amplified rDNA restriction analysis (ARDRA), ter-
minal restriction fragment length polymorphism (T-RFLP)
and ribosomal intergenic spacer analysis (RISA), provide
information on the species composition, and can be used to
compare common species present in samples. However, there
are some problems and biases in the PCR amplification step
and, therefore, these methods cannot be used as definite
indicators of species richness. Despite the PCR problems, a
combination of the techniques mentioned above can reveal a
great deal about the microbial community diversity. Recently,
a method based on integron-targeting PCR assays has been
developed to recover genes from environmental DNA
without the necessity to know their sequences [8

]. A
comprehensive description and discussion of the potential
and limitations of the methods are given in the overviews of
Kozdrj and van Elsas [2] and Johnsen et al. [3].
Single genome versus community genome
analysis
Although the complete sequences of more than 60 microbial
genomes have provided critical insights into the individual
microbial cell, the study of the collective genomes in a
community the metagenome promises to unlock
the secrets of community life. Approximately 60 microbial
genomes have been completely sequenced, and hundreds
more are in the process of being sequenced. Complete
microbial genome sequencing has made it possible to
identify and characterize all genes present in a species.
This means that we get information about novel metabolic
pathways, gene regulatory elements, genes of unknown
function, and genes for pathogenesis, virulence and drug
resistance [9]. This information also provides insights into
the evolution of genes and species. Furthermore, the
understanding of species diversity based on comparative
genomics has led to a new epoch for biological investiga-
tions, using techniques such as microarray analysis [10].
The next step in the era of microbial genomics is to extract
functional and evolutionary information from these large
datasets and to apply genomics technology to relevant
questions in microbial ecology [11

]. Thus, new technology

will have an important impact on the understanding of soil
microbial ecology. Recently, soil community genomes have
been cloned using a bacterial artificial chromosome (BAC)
vector. Together, all the genomes of soil microbes can be
considered to be one large soil microbial community
genome the metagenome [12

]. Combined compara-
tive analyses of core housekeeping genes like rDNA and
functional genes may provide information on both phylo-
genetic diversity and the potential functional diversity of
microbial communities. There are several ways in which
environmental microbiology will benefit from microbial
genomics. Comparative genomic analysis and microarray
technology may be used to determine patterns of gene
expression, and to detect novel metabolic pathways. This
offers a quick method to access functional information for
genes of unknown function. Such information is very useful
in functional diversity studies to track highly expressed
genes and genes critical in biogeochemical pathways.
It is also important to understand how microbial cells are
regulated under varying conditions such as carbon supply,
energy source and electron acceptor availability, thereby
obtaining information on microbial community response to
environmental changes. The new tools may also increase
our understanding of the process of genome evolution and
the factors that regulate diversity.
Novel methods linking phylogenetic groups to
their activities and function
Perhaps the greatest challenge facing microbiology today is
the problem of linking phylogeny and function. The methods
based on 16S rRNA analysis provide extensive information
about the taxa present in an environment, although they
provide little insight into the functional role of each
phylogenetic group. Metagenomic analysis provides some
functional information through genomic sequence and
expression of traits, but other methods are required to link
specific functions with the group responsible for them.
The concomitant quantitative and comparative analyses
of expressed rRNA genes and genes for key enzymes in
242 Ecology and industrial microbiology
relation to environmental factors can be used to obtain
information about the phylogeny and ecology of functional
bacterial groups responsible for processes like denitrification,
nitrification and methane oxidation.
Environmental functional gene arrays could be constructed
using oligonucleotid probes to target gene expression or
genes coding for key enzymes in all biogeochemical cycles.
These can be used for specific detection of gene expres-
sion in the environment. Investigations in which attempts
have been made to associate specific microorganism or
microbial groups with their ecological function have been
performed on functional groups of bacteria, using genes for
key enzymes such as nitrate reductase [13], ammonia
monooxygenase [14] and methane monooxygenase [15].
Another strategy to look for specific functional groups is to
use microsensor measurements of chemical profiles in
relation to the distribution of different bacterial taxa to
identify environmental conditions favored by particular
bacteria [16,17

]. It remains, however, a major challenge in

soil microbial ecology to ascribe microbial processes to
specific microorganisms. Before the microorganisms are
cultivated, their ecological functions must be elucidated
through culture-independent characterization that links
phylogenetic and functional genetic analyses to metabolic
activities in soil.
Microradioautography, using the uptake of specific radio-
labeled substrates by individual cells, can be used to
detect and quantify the active populations utilizing this
substrate. In order to link the uptake of a specific substrate
with the phylogenetic identity of a specific bacterial cell,
microautoradiography has been used in combination with
fluorescent in situ hybridization (FISH) of microbial cells
using fluorescent, group-specific phylogenetic probes
(targeting rRNA) and fluorescence microscopy. Studies in
which microautoradiography and FISH are combined in
natural and engineered environments are rapidly increasing
[14,16,18,19]. The application of stable isotope probing
(SIP) and phospholipid fatty acid (PLFA) labeling to
determine functionally active components of microbial
communities is also becoming increasingly used in micro-
bial ecology studies [20,21

]. In SIP,
13
C-DNA produced
during the growth of metabolically distinct microbial
groups on a
13
C-enriched carbon source can be resolved
from
12
C-DNA by density gradient centrifugation. The
DNA can then be fractionated, and each fraction can be
further analyzed taxonomically and functionally by gene
probing and functional analysis. This method, therefore,
offers a powerful technique for identifying microorganisms
that are actively involved in specific metabolic processes.
Another method in which activity can be linked to phylo-
genetic information is the incorporation of 5-bromo-
2-deoxyuridine (BrdU) into DNA to detect metabolically
active community members in response to substrate or
other stimuli. BrdU can be added to a culture of microbial
cells, and metabolically active members of a community
will then incorporate BrdU into their DNA [22,23].
Yin et al. [24

] recently used this technique to determine

the extent of functional redundancy along a soil reclamation
gradient in a highly contaminated mine spoil. Different
carbon amendments were used and significant differences
were detected in the microbial populations that had
incorporated BrdU in their DNA, indicating a bacterial
functional redundancy within this microbial community. A
problem using this technique is that selective stimulation
of bacteria that are not actually active before substrate
amendment may occur.
The effect of soil structure and environmental
conditions on microbial diversity
Soil is a very complex system that comprises a variety of
microhabitats with different physicochemical gradients and
discontinuous environmental conditions. Microorganisms
adapt to microhabitats and live together in consortia with
more or less sharp boundaries, interacting with each other
and with other parts of the soil biota. A number of investi-
gations emphasize the impact of soil structure and spatial
isolation on microbial diversity and community structure
[11

,25,26]. Analysis of the spatial distribution of bacteria at

microhabitat levels showed that, in soils subjected to differ-
ent fertilization treatments, more than 80% of the bacteria
were located in micropores of stable soil micro-aggregates
(220 m) [25]. Such microhabitats offer the most favorable
conditions for microbial growth with respect to water and
substrate availability, gas diffusion and protection against
predation. Particle size had a higher impact on microbial
diversity and community structure than did factors like bulk
pH and the type and amount of organic compound input
[26]. Results showed that the microbial diversity in fractions
with small soil particles was higher than that in fractions
with large soil particles, and that most of the soil microbial
community was particle-specific. A high diversity of bacteria
belonging to the Holophaga/Acidobacterium division and
Prosthecobacter were present in small particles (silt and clay).
Large particles (sand) harbored few members of the
Holophaga/Acidobacterium division, and were dominated by
bacteria belonging to the -proteobacteria. Other investiga-
tions indicate that the type and amount of available organic
substrates strongly influence the abundance of microbial
groups and their functional diversity in soil ecosystems
[27,28]. Smit et al. [29

] used their own data and data from

recent literature on the distribution of 16S rDNA sequences
among five main bacterial divisions to search for relation-
ships between the abundance of microbial groups and soil
nutritional status. Their results suggested that soil with a
high content of readily available nutrients showed positive
selection for - and -proteobacteria, this being indicative of
r-selection, which is selection for bacteria with potentially
high growth rates. In low-nutrient soil or soil with a high
content of recalcitrant substrates, the percentage of
Acidobacteriumincreased, this being indicative of k-selection,
which is selection for bacteria with lower growth potential
but higher capability to compete for substrates. The ratio
between the number of proteobacteria and Acidobacterium
was suggested to be indicative of the nutritional status of
Microbial diversity and function in soil: from genes to ecosystems Torsvik and vres 243
soils. The ratio was low in oligotrophic soil, medium in
agricultural soil with low organic input, and high in
agricultural soil with high organic input [29

,30].
Competitive interactions are thought to be a key factor
controlling microbial community structure and diversity
[11

]. Soil structure and water regime influence

competitive interactions by causing spatial isolation within
communities. Soil with high spatial isolation showed high
microbial diversity, whereas soil with lower spatial isolation
showed much lower diversity and was dominated by a few
microorganisms. The high diversity in soil with high spatial
isolation may also have been caused by a higher hetero-
geneity of carbon resources in this soil and, consequently,
a higher niche variation. Soil bacteria are subjected to
considerable seasonal fluctuations in environmental
conditions such as temperature, water content and
nutrient availability. An important issue to elucidate is how
environmental changes and seasonal variations influence
qualitative variation in community composition. Smit et al.
[29

] found that bacterial biomass did not change signifi-

cantly during the seasons, but that both culturing and
molecular fingerprinting techniques demonstrate signifi-
cant variations in community composition. Culturing
techniques show that the proportion of fast-growing
bacteria was lowest in winter and highest in summer, and
that the highest species richness was found in spring and
autumn. This seemed to correlate with enhanced micro-
bial activities and nutrient input from fertilization (spring)
and plant debris after harvest (autumn). Molecular finger-
printing indicated that the community, to a large extent,
consisted of stable dominant populations, but that less-
abundant populations, as revealed by low-intensity bands
on a DGGE gel, showed distinct seasonal differences.
Culture-dependent methods and molecular methods
reveal strikingly different microbial populations in soil. It
has been demonstrated that Gram-positive bacteria with
high GC content [29

] and Gram-positive bacteria with low

GC content [31] were abundant among isolates, but had
very low abundance or were nearly absent among clones.
The microbial diversity of soil and the interactions
between different trophic levels were elucidated in a simple
ecosystem model in which primary producers (plants) and
decomposers (microbes) were linked through cycling of a
limiting nutrient factor [32] for the primary producers. The
model shows that the efficiency of nutrient recycling from
organic compounds to decomposers is a key parameter that
controls ecosystem processes (productivity and biomass of
the functional groups). The model predicts that microbial
diversity has a positive effect on nutrient cycling efficiency,
and contributes to increased ecosystem processes. One
major effect that microbial diversity can have on ecosystem
processes is to ensure that all organic compounds are
recycled. Organic compound diversity may have a negative
or neutral effect on a stable ecosystem. Most soils are
exposed to fluctuating environmental conditions and, in
fluctuating ecosystems and a long-term perspective, the
high diversity of organic substrate is likely to have a
positive effect on the function. The relationship between
microbial diversity and soil processes may not be linear
because many processes are carried out by a consortium of
microorganisms. In interacting consortia, small linear
changes in microbial diversity may result in non-linear
changes in process.
Functional diversity of soil microorganisms
Functional diversity is an aspect of the overall microbial
diversity in soil, and encompasses a range of activities. The
relationship between microbial diversity and function in soil
is largely unknown, but biodiversity has been assumed to
influence ecosystem stability, productivity and resilience
towards stress and disturbance. The catabolic response
profile (CRP) [33], which is a measure of short-term sub-
strate-induced respiration, has been used to calculate the
diversity (range and evenness) of catabolic functions
expressed in situ. Catabolic diversity has been used to inves-
tigate the effect of stress and disturbance on the diversity
and resilience of soil microbial communities. When match-
ing soils from different long-term-managed environments
(crop and pasture) were subjected to stress and disturbance,
it was demonstrated that microbial communities with low
catabolic evenness (crop fields) were less resistant to stress
and disturbance than were microbial communities with high
catabolic evenness (pasture). Other soil properties might
also have contributed to the resistance. After a major
disturbance (such as landslips, volcanic eruptions or retreat
of glaciers), marked changes in functional diversity
(catabolic evenness) in developing soil ecosystems have been
demonstrated [34]. The functional diversity was initially low
in non-vegetated (underdeveloped) sites, but as vegetation
was established, the diversity rapidly increased and, in older
successions, the catabolic evenness declined. These diversity
patterns broadly paralleled patterns in plant and fungal
diversity. The relationships between microbial community
composition and physiological capacity [35] were elucidated
using PLFA profiles and two functional measures, namely
substrate utilization capacities (BIOLOG) and enzyme
activities. Broad-scale community composition (PFLA
profiles) correlated well with specific enzyme activities,
especially enzymes responsible for initial degradation of
macromolecules such as lignocellulose. The lack of correla-
tion between BIOLOG and PLFA assays may be due to
the fact that BIOLOG selects for a minor part of the micro-
bial community utilizing readily available simple substrates,
whereas PLFA includes the total community. Macro-
molecule degradation and demineralization enzyme activities
may measure functions that immediately respond to litter
input and are more clearly related to changes in the active
part of microbial communities.
Conclusions and future directions
Recent advances in soil community analysis using molecular
methods agree with the earlier data on total genetic
diversity by indicating an enormous microbial diversity in
soil. Soil diversity exceeds that of aquatic environments,
244 Ecology and industrial microbiology
and is a great resource for biotechnological exploration of
novel organisms, products and processes. Novel methods
and approaches enable us to explore this vast diversity.
Studies of sequence information from organisms in soil
microhabitats and their gene expression under different
conditions will provide guidelines for designing new and
improved culturing methods that resemble their natural
niches. New tools in bioinformatics and statistical analysis
enable us to handle the huge amount of data obtained
through multidimensional studies that combine growth-
independent molecular analyses with analyses of microbial
growth, activity and physiology, and integrate measures
of environmental parameters. Such polyphasic studies
integrate different aspects of microbial diversity and
provide a more complete picture of microbial diversity and
a deeper understanding of the interactions in soil microbial
ecosystems. Studies of microbial sequences, comparative
genomics and microarray technology will improve our
understanding of the structure/function relationships and
the effects of abiotic and biotic factors on soil microbial
communities. It is conceivable that the research field
dealing with the interaction of genomes with the environ-
ment will be an important topic in the future.
Acknowledgements
This work was supported by grant from the Norwegian Research Council
(NFR), project number 140658/720, and the Nordic Academy for Advanced
Study (NorFA).
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