et al. 2011). Several of these biomolecules, present only
in viable cells, can be used as markers to identify major
taxonomic and functional groups and even some bacterial taxonomic groups (cf. Sect. 17.6.1). They have been
used for the study of soil microbial communities (Ritchie
et al. 2000), deep-sea ecosystems (Fang et al. 2003;
Schrenk et al. 2003), marine ecosystems contaminated
by petroleum hydrocarbons (Mazzella et al. 2005), or
different stages of composting (Eiland et al. 2001).
(ii) Hopanes (pentacyclic triterpenoids to 30 carbon
atoms), whose complex chemical structure has to withstand the processes of diagenesis and catagenesis*, are
biomarkers that are used to identify the location and
condition of synthesis of petrol oil and its level of
maturity. The hopanes and steranes are also used in
environmental geochemistry. In effect, each oil provenance has a specific chemical composition, a true
“molecular fingerprint” that can pinpoint the source of
an oil spill (cf. Chap. 16).
(iii) Glycerol ethers that are specific to Archaea and which
have demonstrated the involvement of these
microorganisms in the anaerobic biodegradation of
methane (cf. Box 14.9).
(iv) Quinones which have been used to characterize the
evolution of microbial communities (bacteria and
fungi) during the thermophilic phase during a
composting process (Tang et al. 2007).
Pigments
Some microorganisms contain pigments such as
chlorophylls, bacteriochlorophylls, carotenoids, and
phycobiliproteins, with characteristics allowing their use as
taxonomic markers:
(i) They are present in photosynthetic microorganisms that
can be distinguished from other members of the microbial community.
(ii) Certain pigments are limited to certain microbial classes or genera, allowing characterization of the taxonomic composition of photosynthetic microbial
communities at the class or genus levels.
(iii) They are highly colored, and considering the
chlorophylls and phycobiliproteins, they are fluorescent
at wavelengths in the visible spectrum, allowing their
detection at very low sensitivity levels.
(iv) They are labile and rapidly degraded after cell death,
allowing to target mainly living cells.
The main drawbacks to the use of pigments as taxonomic
markers are:
(i) Since they are labile, they must be protected from light,
oxygen, acidity, and alkali during sampling and analysis.
In solution they form isomers spontaneously.
(ii) Their distribution is complex, some pigments being
species specific and others covering many different
microbial classes.
(iii) Their expression is variable, and the pigment level per
cell varies depending on environmental parameters
such as irradiation or nutrients.
Despite these drawbacks, pigment analysis is a method of
choice to monitor the abundance and composition of photosynthetic microorganisms. It has been used to analyze the
diversity of picophytoplankton in the Pacific Ocean by semiautomatic methods such as flow cytometry (Blanchot and
Rodier 1996) of cyanobacteria in microbial mats by confocal
microscopy (Fourcans et al. 2004) or of microbial mats after
extraction and HPLC analysis (Nubel et al. 1999). Another
interesting example is the HPLC analysis of pigments from
microbial mats growing in arctic ecosystems (Vincent et al.
2004), which highlighted the presence of cyanobacteria in
cryo-ecosystems.
8.4.1.3 Analysis of Metabolic Capacities
of Communities
BIOLOG plates that permit to establish patterns of individual
carbon substrate utilization were originally designed for the
identification of microbial isolates. Ease of use and the possibility afforded to obtain rapid results resulted in their utilization for the analysis of metabolic capacities of microbial
communities for the first time by Garland and Mills (1991),
despite the often-heard criticism of lack of reproducibility: the
latter would be due to the nonlinearity of substrate utilization
over time and the difficulties of standardizing the inoculum
(Haack et al. 1995). In particular, the inoculum density and
incubation time are critical, and the interpretation of results
has to be done with caution. Moreover, BIOLOG analysis is a
method based on cells expressing metabolic activities and
each metabolic profile observed therefore reflects only partly
the nature and structure of the targeted microbial community
derived from an environmental sample.
BIOLOG-ECO plates were developed to include 31
substrates among the most used in many natural
environments. These 31 carbon sources are repeated three
times, thus providing data replication. Metabolic fingerprints
of microbial communities can then be processed by multivariate statistical analyses.
Gamo and Shoji (1999) have developed a method using
BIOLOG-MPN plates to enumerate bacteria by the MPN
method on a range of substrates. This method has been used
to characterize the temporal changes of functional diversity
for bacterioplankton communities (Matsui et al. 2001).
BIOLOG plates have been widely used in soil microbiology
(Garland and Mills 1991). They helped to highlight the
resistance of bacterial communities of forest soils during
application of glyphosate at concentrations used
268
P. Normand et al.
in viable cells, can be used as markers to identify major
taxonomic and functional groups and even some bacterial taxonomic groups (cf. Sect. 17.6.1). They have been
used for the study of soil microbial communities (Ritchie
et al. 2000), deep-sea ecosystems (Fang et al. 2003;
Schrenk et al. 2003), marine ecosystems contaminated
by petroleum hydrocarbons (Mazzella et al. 2005), or
different stages of composting (Eiland et al. 2001).
(ii) Hopanes (pentacyclic triterpenoids to 30 carbon
atoms), whose complex chemical structure has to withstand the processes of diagenesis and catagenesis*, are
biomarkers that are used to identify the location and
condition of synthesis of petrol oil and its level of
maturity. The hopanes and steranes are also used in
environmental geochemistry. In effect, each oil provenance has a specific chemical composition, a true
“molecular fingerprint” that can pinpoint the source of
an oil spill (cf. Chap. 16).
(iii) Glycerol ethers that are specific to Archaea and which
have demonstrated the involvement of these
microorganisms in the anaerobic biodegradation of
methane (cf. Box 14.9).
(iv) Quinones which have been used to characterize the
evolution of microbial communities (bacteria and
fungi) during the thermophilic phase during a
composting process (Tang et al. 2007).
Pigments
Some microorganisms contain pigments such as
chlorophylls, bacteriochlorophylls, carotenoids, and
phycobiliproteins, with characteristics allowing their use as
taxonomic markers:
(i) They are present in photosynthetic microorganisms that
can be distinguished from other members of the microbial community.
(ii) Certain pigments are limited to certain microbial classes or genera, allowing characterization of the taxonomic composition of photosynthetic microbial
communities at the class or genus levels.
(iii) They are highly colored, and considering the
chlorophylls and phycobiliproteins, they are fluorescent
at wavelengths in the visible spectrum, allowing their
detection at very low sensitivity levels.
(iv) They are labile and rapidly degraded after cell death,
allowing to target mainly living cells.
The main drawbacks to the use of pigments as taxonomic
markers are:
(i) Since they are labile, they must be protected from light,
oxygen, acidity, and alkali during sampling and analysis.
In solution they form isomers spontaneously.
(ii) Their distribution is complex, some pigments being
species specific and others covering many different
microbial classes.
(iii) Their expression is variable, and the pigment level per
cell varies depending on environmental parameters
such as irradiation or nutrients.
Despite these drawbacks, pigment analysis is a method of
choice to monitor the abundance and composition of photosynthetic microorganisms. It has been used to analyze the
diversity of picophytoplankton in the Pacific Ocean by semiautomatic methods such as flow cytometry (Blanchot and
Rodier 1996) of cyanobacteria in microbial mats by confocal
microscopy (Fourcans et al. 2004) or of microbial mats after
extraction and HPLC analysis (Nubel et al. 1999). Another
interesting example is the HPLC analysis of pigments from
microbial mats growing in arctic ecosystems (Vincent et al.
2004), which highlighted the presence of cyanobacteria in
cryo-ecosystems.
8.4.1.3 Analysis of Metabolic Capacities
of Communities
BIOLOG plates that permit to establish patterns of individual
carbon substrate utilization were originally designed for the
identification of microbial isolates. Ease of use and the possibility afforded to obtain rapid results resulted in their utilization for the analysis of metabolic capacities of microbial
communities for the first time by Garland and Mills (1991),
despite the often-heard criticism of lack of reproducibility: the
latter would be due to the nonlinearity of substrate utilization
over time and the difficulties of standardizing the inoculum
(Haack et al. 1995). In particular, the inoculum density and
incubation time are critical, and the interpretation of results
has to be done with caution. Moreover, BIOLOG analysis is a
method based on cells expressing metabolic activities and
each metabolic profile observed therefore reflects only partly
the nature and structure of the targeted microbial community
derived from an environmental sample.
BIOLOG-ECO plates were developed to include 31
substrates among the most used in many natural
environments. These 31 carbon sources are repeated three
times, thus providing data replication. Metabolic fingerprints
of microbial communities can then be processed by multivariate statistical analyses.
Gamo and Shoji (1999) have developed a method using
BIOLOG-MPN plates to enumerate bacteria by the MPN
method on a range of substrates. This method has been used
to characterize the temporal changes of functional diversity
for bacterioplankton communities (Matsui et al. 2001).
BIOLOG plates have been widely used in soil microbiology
(Garland and Mills 1991). They helped to highlight the
resistance of bacterial communities of forest soils during
application of glyphosate at concentrations used
268
P. Normand et al.