Res. Microbiol.

151 (2000) 167177

2000 ditions scientifiques et mdicales Elsevier SAS. All rights reserved
S092325080000136-4/REV

Mini-review
Monitoring complex bacterial communities using culture-independent
molecular techniques: application to soil environment
Lionel Ranjard, Franck Poly, Sylvie Nazaret*
Laboratoire dcologie microbienne, UMR CNRS 5557, universit Claude Bernard, Lyon I, 69622 Villeurbanne cedex, France
(Submitted 20 October 1999; accepted 20 December 1999)

Abstract Over the last decade, important advances in molecular biology led to the development of
culture-independent approaches to describing bacterial communities. These new strategies, based on the
analysis of DNA directly extracted from environmental samples, circumvent the steps of isolation and
culturing of bacteria, which are known for their selectivity leading to a non-representative view of the extent
of bacterial diversity. This review provides an overview of the potentials and limitations of some molecular
approaches currently used in microbial ecology. Examples of applications to the study of indigenous soil
microbial community illustrate the feasibility and the power of such approaches. 2000 ditions scientifiques et mdicales Elsevier SAS
bacterial community / soil / molecular biology / DNA / genetic diversity / genetic structure

1. Introduction
Bacteria are an important part of the soil
microflora because of their abundance (up to
109 cells per gram of soil [3]), their species
diversity (a minimum of 4 0007 000 different
bacterial genomes per gram of soil [54]) and the
multiplicity of their metabolic activities. They
play a key role in the biogeochemical cycles of
the main elements (carbon, nitrogen, sulphur,
etc.) and of trace elements (iron, nickel, mercury,
etc.) and are therefore heavily implicated in
energy and nutrient exchanges within the soil.
They also have the potential to reflect the past
history of a given environment. It is therefore
essential to understand the interrelationships

* Correspondence and reprints

Tel.: +33 (0)4 72 44 13 80; fax: +33 (0)4 72 43 12 23;
Nazaret@biomserv.univ-lyon1.fr
Abbreviations: ARDRA, amplified ribosomal DNA restriction analysis; DGGE, denaturing gradient gel electrophoresis; PCR, polymerase chain reaction; RAPD, random
amplified polymorphic DNA; RISA, ribosomal intergenic
spacer analysis; TGGE, temperature gradient gel electrophoresis; T-RFLP, terminal-restriction fragment length polymorphism.

between bacteria and their environment by

studying the structural and functional diversity
of soil bacterial communities and how they
respond to various natural or man-made disturbances.
The diversity of soil bacterial communities
has been investigated for many years using
methods based on isolating and culturing the
micro-organisms. Such techniques are known
for their selectivity and are not considered representative of the extent of the bacterial community. The proportion of cells which can be cultured is estimated to be 0.1% or at most 10% of
the total population [1] and few data are available concerning how closely they reflect the
actual composition of these communities.
Recent advances in the field of molecular biology (extraction of nucleic acids, polymerase
chain reaction (PCR) amplification, DNA cloning, DNA sequencing) have made it possible to
develop techniques which no longer require the
isolation and culture of bacteria and thus reduce
the bias associated with it. These methods
involve i) direct lysis of bacterial cells in soil, ii)
the extraction of the nucleic acids from the
matrix, and iii) the analysis of targeted

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Ranjard et al.

sequences or of the whole body of genetic

information.
Two main types of molecular technique are
available to study bacterial communities using
DNA directly extracted from the soil (figure 1):
molecular approaches which usually
investigate parts of this information by focusing
on genome sequences which are targeted and
amplified by PCR that are called partial community DNA analysis;
molecular approaches which try to investigate all the genetic information in the
extracted DNA and that are called whole community DNA analysis.
The remainder of this mini-review will focus
on various approaches suitable for describing
the genetic diversity and structure of soil bacterial communities, together with the most important original information obtained and the main
technical and conceptual limitations.

2. The partial community DNA analysis

approaches
These approaches consist in the analysis of
PCR-amplified sequences. The most commonly
used target sequences are the genes of the
ribosomal operon, and particularly the rrs gene
(16S rDNA) and the spacer between the rrs and
rrl genes (intergenic spacer (IGS) 16S-23S).
These methods include:
PCR fragment cloning followed by restriction and/or sequencing analysis, which enable
assessment of the diversity of the community in
terms of the number of different species and, to
a lesser extent, the relative abundance of these
species;
genetic fingerprinting, which provides a
global picture of the genetic structure of the
bacterial community.
2.1. PCR fragment cloning and characterization

This approach is used to investigate the

diversity of bacterial communities by producing
a library of clones from rrs sequences obtained
by PCR from DNA extracts. Cloning is used to
separate the sequences so that they can be

characterized individually using PCR/RFLP

(polymerase chain reaction/restriction fragment length polymorphism) and/or by
sequencing. Sequencing allows a fine identification of uncultured bacteria as well as an estimation of their relatedness to known culturable
species. The substantial richness found within
the soil bacterial community and the number of
clones per library (usually about 100 clones)
preclude the application of diversity measurements in terms of species evenness. However,
such a sampling size can be informative for
estimating diversity, both richness and evenness, by considering higher level phylogenetic
groups (i.e. subdivisions within proteobacteria,
high and low G + C firmicutes, Holophaga/
Acidobacterium group, etc.).
This approach was used to compare the
genetic diversity between soils differing in their
geographical location, agricultural practices
and physico-chemical characteristics. Species
common to these different environments were
found and the diversity of some eubacterial and
archaeal taxons was enlarged (for a review
see [23], table I). Other studies have also used
functional genes involved in the nitrogen cycle,
heavy metal resistance or antibiotic metabolism,
as molecular markers for assessing the distribution and the diversity of corresponding functional populations in soil communities (table I).
The main limitations and drawbacks of the
PCR-cloning approach are essentially technical:
it is time-consuming and cumbersome
since it is necessary to sample a large number of
clones in order to obtain a good diversity estimation of the amplified sequences;
it requires very expensive equipment
(sequencer);
there exists bias due to the PCR (i.e. choice
of primers, annealing temperature and numbers
of cycles, inhibition of the enzyme by humic
compounds, formation of chimaeric PCR products) or the cloning strategy used, both of which
have a direct impact on the sampling of the
cloned 16S rDNA sequences and their identification (for a review see [45, 61]). Consequently,
the image of diversity of bacterial communities
may not be totally exact. However, despite the

Monitoring complex bacterial communities

169

Figure 1. Schematic representation of the different molecular approaches for assessing the genetic diversity and structure of soil
bacterial communities. DNAs are directly extracted from soil samples and subsequently analysed either by characterizing particular
sequences targeted and amplified by PCR (approaches called partial community DNA analysis) or by characterizing all the genetic
information (approaches called whole community DNA analysis). PCR products can be analysed by cloning or genetic fingerprint.
Genetic fingerprint consists in a rapid and simple electrophoretic analysis of the PCR products enabling the analysis of the genetic
structure of the community. Characterization of cloned sequences enables assessment of the genetic diversity of a community and can
reveal the phylogenetic affiliation of the community members. Similarly, the sequencing of bands of fingerprint profiles can lead to
identification of particular populations. Whole community DNA analyses such as cross-DNA hybridization and DNA fractionation by
density gradient can help to assess the genetic structure of a community, whereas reassociation kinetics estimates genetic diversity in
terms of the number of different genomes present in a community.

numerous biases that can occur in the PCRcloning method, a recent study by Dunbar et

al. [10] showed that 16S rDNA cloning coupled

to RFLP analysis and partial sequencing was as

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Ranjard et al.

Table I. Overview of the applications of PCR cloning and characterization in soil bacterial community studies.
Applications

References

Characterization of bacterial community, detection of major phylogenetic groups and

identification of genus and species
Diversity investigation of the most active bacterial populations
Identification of novel clusters having no known cultured members
Influence of environmental disturbance on soil genetic diversity
Analysis of the diversity of particular functional groups in soil (i.e. ammonium oxydizers,
methanogenic bacteria, nitrogen-fixing bacteria, heavy metal-resistant bacteria, antibioticproducing bacteria)

valid as plate cultivation for investigating diversity in soil environments.

2.2. Genetic fingerprint techniques

These techniques are also based on PCR

amplification, but do not require a clone library.
They are based on the principle of resolving the
diversity of the amplified sequences simply by
differential electrophoretic migration on agarose or polyacrylamide gels, which depend on
their size (ARDRA, t-RFLP, RISA, RAPD) or
sequence (DGGE, TGGE). The genetic fingerprints provide complex band profiles (i.e. a high
number of different bands per profile) which
yield a representative of the genetic structure of
the community as a whole or of a section of it,
defined by the selected primers. These profiles
are not directly interpretable in terms of richness and evenness, since one band may originate from different species and one cell may be
represented by several bands. This is particularly true with ribosomal sequences, since a
single bacterial species may have several different rRNA sequences [8, 37] and different bacterial species may have closely related rDNA
sequences [18]. Consequently, the genetic fingerprint approaches are especially suitable for
comparing bacterial communities and various
methods have been developed based on this
principle. The profile data can then be analysed
in terms of similarities and relationships can be
represented by a dendrogram of similarities
[49]. Multivariate analyses such as principal
component analysis were also shown to be
useful tools for easily visualizing and comparing changes in data tables derived from modi-

[5, 10, 31, 33, 50, 55, 63]

[13, 14]
[24, 25, 27, 29, 30, 46, 57]
[4, 31, 33, 39, 51]
[7, 17, 48, 51, 59]

fications in the fingerprint profile due to shifts

in genetic structure [60]. Furthermore, this type
of statistical analysis enabled correlation of
these changes with various environmental characteristics or events [43, 44].
2.2.1. Electrophoretic separation using a denaturing
gradient

This method is used to separate fragments of

DNA which have the same size but different
sequences. It was originally developed to detect
specific mutations within the human genome,
and has been adapted to analyse bacterial communities [35]. It involves the separation of
amplicons on polyacrylamide gels containing a
linear gradient of a DNA denaturing agent. This
denaturing agent may be chemical (urea or
formamide), in which case the method is known
as DGGE (denaturing gradient gel electrophoresis) or it may be a physical factor such as the
temperature, in which case the name TGGE
(temperature gradient gel electrophoresis) is
used [13, 35]. The differences between the
sequences of the amplified samples influence
their denaturation. Consequently, amplified
samples with different sequences are separated
during electrophoretic migration yielding complex profiles representing the diversity of the
fragments amplified.
Compared to ARDRA, RISA or RAPD (see
below), DGGE and TGGE require laborious
technical optimization including calibration of
the linear gradient of DNA denaturants (chemical or physical) and improvement of the PCR
primers with the insertion of a GC clamp to
obtain better electrophoretic separation of the
fragments. Nevertheless, DGGE and TGGE of

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171

Table II. Overview of the applications of DGGE and TGGE in soil bacterial community studies.
Applications

References

Study of the genetic structure of the total bacterial community

Study of the genetic structure of a particular phylogenetic group or functional
group
Spatial distribution of bacterial populations
Impact of environmental disturbance on soil community

[13, 14, 40]

[20, 26, 52]

16S rDNA are commonly used to compare the

genetic structure of bacterial communities from
different soil types or submitted to perturbation
(table II). In a restricted case, where the bacterial
diversity is low, DGGE profiles can be used to
define the number of different operational taxonomic units (OTUs) present, enabling determination of the complexity of the community.
DGGE or TGGE of PCR products obtained after
amplification of ribosomal copy DNA of 16S
rRNA (rcDNA) by reverse transcriptase (RT)PCR would indicate the structure or the complexity of the active bacterial populations in a
given soil [13, 14]. Furthermore, individual
bands may be excised, reamplified, and
sequenced, or challenged with a range of probes
for the taxonomic identification of specific
populations within a community.
The specific limitation of this approach lies in
the size of the amplified sequences under investigation since they can no longer be efficiently
separated beyond 500 base pairs. This limit
restricts the precision of the subsequent identification of the bands based on sequence comparison and the choice of the regions of the rrs
gene to define the PCR primers or probes for
further identification. In addition, the resolution
of the profiles obtained, in terms of band numbers, is not always sufficient to illustrate the
considerable bacterial diversity in indigenous
soil communities, and some studies have shown
that fragments of different sequences might
migrate at the same position [56].

[13]
[11, 12, 41]

2.2.2. ARDRA (amplified ribosomal DNA restriction

analysis) and T-RFLP (terminal-RFLP).

ARDRA involves digesting the 16S rDNA

sequences obtained by PCR using restriction
enzymes and separating the restricted fragments on agarose or polyacrylamide gels. The
main advantage of this method is its convenience, because it does not require any particular development. This technique has been used
to demonstrate changes in the genetic structure
of bacterial communities following changes in
environmental conditions or exposure to exogenous toxic compounds (table III). Like DGGE, it
can be used for the taxonomic identification of
bacterial populations by hybridization of the
profile using specific probes.
The main limitation of this method lies in the
choice of restriction enzymes, which is crucial
for obtaining optimum resolution. Preliminary
tests for the enzyme choice must be performed
to ensure the highest resolution in detecting
changes in communities. In this method, the
complexity of the profile is great and no information can be deduced in detecting and quantifying a specific ribosomal pattern. An alternative has recently been found using
fluorochrome-labelled primers. This makes it
possible to detect only the terminal fragments
and it is therefore known as T-RFLP or
terminal-RFLP. The profiles obtained are simpler in terms of the numbers of bands. A data
bank has been produced of the sizes of the 16S
rDNA terminal restriction fragment for a given

Table III. Overview of the applications of ARDRA in soil bacterial community studies.
Applications

References

Analysis of genetic similarities between different soils

Study of the genetic structure of a particular phylogenetic group or functional group
Impact of environmental disturbance on soil community

[58]
[6, 42, 58]
[9, 49, 53]

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Ranjard et al.

Table IV. Overview of the applications of RISA in soil bacterial community studies.
Applications

References

Comparative study of the genetic structure from various soil microenvironments

Impact of environmental perturbation on genetic structure of soil community

[43]
[4, 44, 47]

enzyme on a large set of bacterial species and

this makes it possible to identify the bands
present in a community by a profile to profile
comparison [32]. Running such profiles on acrylamide gel with laser detection (sequencing system) increases the resolution and sensitivity
power and enables the interpretation of peak
height and area which can be further interpreted in terms of number and abundance of
OTUs. Such an approach has also been successfully adapted to functional genes such as the
mercury resistance determinants (mer genes [6])
to assess the complexity and the distribution of
functional groups in soil matrix.
2.2.3. RISA (ribosomal intergenic spacer analysis)

RISA involves the analysis of the length polymorphism of the spacer between the rrs and rrl
genes (IGS) whose size varies from 50 bp to
more than 1.5 kb depending on the species. The
primers target regions within the adjacent genes
and can be defined so that part of the rrs gene is
co-amplified. The subsequent sequencing of certain bands can then allow taxonomic identification of specific populations within a community. The various amplified IGSs are directly
separated on polyacrylamide gels on the basis
of size [4]. As mentioned for T-RFLP, fluorescent
primers can be used to further run RISA profiles
on sequencing gel systems and then to increase
the resolution power. The high size variability
of this spacer can then allow detection of slight
changes in the genetic structure of the community, and the high sequence variability [36] may
provide a finer taxonomic identification of
bands than can be obtained using 16S rDNA
sequences (as in DGGE or ARDRA). However,
to date, the IGS sequence databank is not large
enough to permit such efficient identification (in
September 1999, 26 191 16S rDNA sequences
and 561 IGS sequences were available in the
data bank).

Although RISA is easy to use and does not

require any particular development, it has not
been used to any significant extent at the community level (table IV). Several works have
demonstrated the reproducibility and the
capacity of this approach to detect modifications in community structure between different
soil types and even between closely related
communities such as those associated with various microenvironments of a soil [43, 44].
2.2.4. RAPD (random amplified polymorphic DNA)

The RAPD technique uses short random

primers (about 10 bp) which anneal at different
places on the genomic DNA, generating PCR
products of various lengths further resolved on
agarose or acrylamide gel. This technique was
demonstrated to be rapid and sensitive for
revealing differences between similar complex
prokaryotic genomes [19]. Though some works
have evidenced the lack of reproducibility of
this technique owing to its great sensitivity to
PCR artefacts [19], recent studies conducted at
the community level demonstrated its reproducibility in comparing the genetic structure of
complex bacterial communities [60]. The main
limitation is that it cannot provide phylogenetic
information about the bacterial composition of
the community.
The only study conducted on the soil bacterial community using this method was developed by Xia et al. [62]. The authors assessed the
molecular genetic response of different soil
communities to the application of 2,4-D and
demonstrated the absence of a pesticide effect.
2.2.5. Limitations of genetic fingerprint techniques

Initially, genetic fingerprints were developed

to discriminate and to identify pure bacterial
strains. When cultured populations were found
to be poorly representative of the community, in
qualitative and quantitative terms [1], these

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173

Table V. Overview of the applications of cross-DNA hybridization in soil bacterial community studies.
Applications
Analysis of genetic similarities between different soils
Impact of chemical pollutants on soil genetic diversity

methods were then adapted for whole communities which are made up of a complex mixture
of target sequences. Compared to the PCRcloning/RFLP/sequencing strategies, genetic
fingerprint methods are easier to perform and
less time-consuming allowing any investigations which involve a large number of differently treated samples. However, PCR is also
involved in these approaches and may lead to a
distorted view of bacterial community structure
owing to the previously mentioned biases (formation of chimaeric sequences, differential
PCR, etc.; see part 2.1. and [61]). Furthermore,
technical problems inherent in electrophoresis
still limit these approaches. In some cases,
DGGE has been reported not to be able to
separate amplicons harbouring different
sequences [56]. Similarly, resolution of differentsize bands in ARDRA and RISA profiles may
not always be possible especially for high
molecular weight fragments. Another limitation
is the complexity of such genetic profiles, which
leads to difficulties in data analysis since they
exhibit several hundred bands of different
intensities.
To date, only a few works have compared the
efficiency of different fingerprinting approaches
for analysing bacterial community structure.
The works of Ranjard et al. [43] demonstrated
that ARDRA and RISA techniques gave the
same clustering for the bacterial communities
associated with the various microenvironments
of a soil. Recently, Moeseneder et al. [34], by
comparing the complexity of different bacterial
communities in terms of OTUs with T-RFLP
and DGGE of 16S rDNA, demonstrated the
higher sensitivity of the former. To date, it is
difficult to conclude as to the better resolution
or sensitivity of a fingerprinting technique since
it seems to be dependent on the type of bacterial
community studied and the type of molecular
markers (taxonomic, functional or random). The

References
[15, 62]
[62]

choice of a technique for a given community

had to be deduced from a testing procedure of
several approaches.

3. The whole genomic community DNA

analysis approaches
3.1. Total genomic cross-DNA hybridization

The technique involves DNA extraction and

purification from environmental samples and
cross-hybridization between DNA from one
sample with that from the other [28]. The DNA
from one sample is radioactively labelled and is
used as a template. The extent to which the
radiolabelled probe anneals to the filter-bound
target DNA reflects the similarity of probe and
target and consequently the extent to which the
population structure of bacterial communities is
similar. Applications to the communities of soil
differing in texture, land use and location and to
the response of communities exposed to pesticides are reported (table V).
This technique might not be appropriate for
detecting differences between communities
with relatively high DNA sequence similarity,
since a high degree of variability of crosshybridization has sometimes been observed
[62].
3.2. Thermal denaturation and reassociation of
whole extracted DNA

This technique involves the heat denaturation

of DNA followed by reassociation of the
homologous single strands. The proportion of
DNA renatured is generally expressed as the
function of the product (Cot) of the concentration of nucleotides (moles/L) and the reaction
time. Under specified conditions, the Cot1/2
value, corresponding to one half of the reassociation reaction, is directly proportional to the
size of the genome or the complexity of the

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Ranjard et al.

Table VI. Overview of the applications of reassociation kinetics in soil bacterial community studies.
Applications

References

Estimation of species numbers in a complex community

Analysis of genetic similarities between different soils
Comparative study of genetic diversity between culturable and nonculturable bacteria
Impact of chemical pollutants on soil genetic diversity
Impact of root exudates on soil genetic diversity

DNA. The Cot1/2 value is used as an index of

the genetic diversity in a bacterial community
based on DNA extracted from the soil [54].
Though this technique makes it possible to
estimate the genetic diversity in terms of the
number of different genomes (richness), it does
not provide any details about the relative abundance of individual genomes (evenness).
Using this method, Torsvik and collaborators
have estimated that the number of different
bacterial genomes present per gram of soil
ranged from 350 to 10 000 according to soil
types [40, 54]. They have also suggested that
most of this diversity is present in the fraction of
bacteria which cannot be cultured. This technique can also be used to evaluate the impact of
exogenous compounds on a soil community.
Atlas et al. [2] demonstrated that soil amendment with a pesticide (2,4,5-T) decreases the
genetic diversity of the bacterial community
estimated by thermal denaturation and reassociation kinetics of the extract DNA. The overview of the applications using this approach to
study soil bacterial community are listed in
table VI.
3.3. Extracted whole DNA fractionation
by base composition using density gradient

This method is based on the fact that prokaryotic DNAs vary in G + C content from ca. 24 to
76% [21]. It uses an intercalating agent, bisbenzimidazole, which is preferentially bound to A +
T base pairs, thus amplifying the differences in
the gravity of the DNA according to the specific
G + C content. The extracted DNA is centrifuged through a caesium chloride density gradient in the presence of bisbenzimidazole. This
yields a DNA community profile of the distribution of %G + C values providing a molecular

[54]
[40]
[40, 54]
[2, 41]
[16]

fingerprint of the overall community structure.

Furthermore, since %G + C of chromosomal
DNA is generally characteristic for phylogenetic
groups of bacteria at the genus level, the relative
abundance of DNA at each %G + C is an
indication of the relative abundance of phylogenetic groups in the bacterial community. Holben and Harris [21] showed that the majority of
DNA extracted from cultivated soil corresponds
to the %G + C of range 5573 which includes
bacterial genera known to be abundant in soil
communities under field conditions, e.g. Agrobacterium (G + C = 5964%), Alcaligenes (G + C =
5561%), Arthrobacter (G + C = 6369%), and
Pseudomonas (G + C = 5866%).
This method provides a low level of taxon
resolution, since several taxonomic groups can
share the same G + C range. Nevertheless, it led
to detection of modifications in the composition
of the soil community due to the influence of
carbon amendment or vegetation cover [21, 39].
An overview of applications using this
approach for the study of soil bacterial communities is given in table VII. This method can be
extended by using analyses providing a more
accurate identification of some bacterial populations by hybridization with specific probes. In
situations where the diversity of the community
is considered too complex to be tackled as a
whole, this method can be used to select a part
of the community based on its %G + C. This
sample can then be subjected to more detailed
molecular analysis (PCR-cloning-sequencing) to
identify its constituent populations [38, 39].
The three previously described methods
exhibit common disadvantages:
the need for a large amount of DNA (
50 g);

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175

Table VII. Overview of the applications of DNA fractionation by base composition in soil bacterial community studies.
Applications

References

DNA composition (phyla, genus) of soil bacterial community

Impact study of environmental changes on indigenous community structure
Localization of functional gene within a community by hybridization to the profile

the need for high DNA quality in terms of

purity and size integrity;
the co-extraction of DNA from live bacterial cells and of contaminant DNA (i.e. extracellular DNA, DNA from dead cells, DNA originating from fungi or other soil eukaryotes) that
can bias the analysis;
expensive equipment (ultracentrifuge for
DNA fractionation technique).

4. Concluding remarks
These molecular ecology methods have
enabled us to extend our understanding of
microbial ecology and will continue to do so.
However, due to the various biases or limitations for each of them, they are not a substitute
for more conventional methods (cultured population studies, measurement of activity, etc.) but
they must be viewed as complementary methods for use in investigating the ecology of
bacteria in their natural habitats. Along this line,
future prospects in microbial ecology must be of
a polyphasic nature, combining and selecting a
range of molecular biological and microbiological techniques in order to understand the relationship between microorganisms and their
environment.
These methods have primarily been used to
assess the composition of microbial communities (identification of genus, species, or phylogenetic groups) and to monitor, over space and
time, changes due to environmental disturbances (figure 1) using as a target ribosomal
genes and/or function-specific genes. Data
focussing on the activity of microbial populations and on gene expression and regulation in
situ are still scarce, especially in a soil environment. Development of tools for addressing
these points will be an important task in the
coming years.

[21, 38]
[21, 39]
[22]

Acknowledgments
The authors thanks Benoit Cournoyer, Philippe Normand, and Jacques Balandreau for
their comments on the manuscript.

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