17.1 Introduction
This chapter presents the main methods for the study of
microorganisms in the environment. This study passes first
through an adapted sampling for the different environments
explored. The various components of the microbial community can then be characterized from the point of view of their
biomass, their activity, and their diversity. To respond to
these various issues, techniques as varied as those of flow
cytometry, molecular biology, biochemistry, molecular isotopic tools, or electrochemistry are implemented. These
different methods are described with their advantages and
disadvantages for different types of biotopes (water, soil,
sediment, biofilms). A final important point discussed in
this chapter concerns the isolation of microorganisms from
the environment and their culture in the laboratory. Without
being exhaustive, this chapter emphasizes the importance
of using appropriate and efficient methodological tools
to properly explore the still mysterious compartment of
microorganisms in the environment.
17.2 Sampling Techniques in Microbial
Ecology
17.2.1 Soils Sampling Techniques
Sampling of soil for microbial ecology studies can be achieved
with a shovel, but more usually using a hand auger (diameter
between 1 and 10 cm) or vehicle-mounted hydraulic auger,
which allows to collect at a constant depth. The depth is
important because it influences many parameters (oxygen,
temperature, water content, concentration of organic matter,
etc.). The depth studied is variable in function of the questions.
It can reach several meters (Box 17.1), but more often it is
the layer between 0 and 30 cm that is studied. Techniques
described above (use of a shovel or an auger) concern masses
of soil from few hundred grams to several kilograms.
A major element to be taken into account during the sampling is the quantity and the quality of the organic materials
that will in part determine the functioning of heterotrophic
microflora representing the majority of the soil microorganisms. For this reason, many studies take into account the
organization of the soil horizon. The superficial horizons
(0–30 cm) are the most studied, because the most intense
microbial activity is observed in these horizons. For the
same reason, the rhizospheric soil (under the influence of
the roots) is often the object of particular attention. Sampling rhizospheric soil is done by shaking the roots to
remove not adhering soil, the soil still adhering constituting
rhizospheric soil.
Box 17.1: Sampling the Deep Biosphere
Michel Magot
The results of a study published in 1998 sounded like a
thunderclap in the sky of microbial ecology. The
authors suggested that the amount of biomass hidden
in the deep Earth subsurface, down to about 4,000 m
deep, would be equivalent or higher to that of all living
organisms on its surface (Whitman et al. 1998)! The
total population of prokaryotes, only denizens of the
deep, was estimated between 25 and 250 Â 10
28 cells,
while about 15 years ago most microbiologists
believed that the Earth subsurface was sterile beyond
a few tens meters deep. Why did it take more than a
century of research in microbiology for the estimation
of such a great unknown biodiversity? The main reason
undoubtedly lies in the difficulty of obtaining uncontaminated geological samples or deep fluids. Three
main ways to collect microbiologically representative
deep subsurface samples will be briefly described
below.
Collecting Fluid Samples from Pre-existing Wells
This is a priori the simplest and cheapest method.
This is what the American microbiologist Edson
Bastin did during the 1920s to study sulfate-reducing
bacteria from American oilfield waters, suggesting
for the first time that bacteria could live in the deep
subsurface. Whether collecting petroleum fluids or
water samples from deep aquifers, the technique is
the same: in principle, it just needs a production or
monitoring well drilled in the geological formation
of interest to collect liquid samples by following
some basic rules to maintain aseptic conditions and
avoid contact with oxygen. Studies based on such
samples, either by conventional microbiology or
molecular methods, show that things are really not
that easy! Multiple sources of contamination can
lead to erroneous conclusions. The two main ones
result from (i) the drilling fluids used during the implementation of the well, which will be discussed below,
and (ii) the colonization of the entire inner part of the
well (the tubing) by a biofilm, which most often has
nothing to do with the indigenous subsurface
microorganisms (Magot 2005). A solution is nevertheless available to collect representative subsurface
M. Magot
Institut des Sciences Analytiques et de Physico-chimie pour
l’Environnement et les Mate ´riaux (IPREM), UMR CNRS 5254,
Universite ´ de Pau et des Pays de l’Adour, B.P. 1155, 64013 Pau
Cedex, France
(continued)
758
F. Joux et al.
This chapter presents the main methods for the study of
microorganisms in the environment. This study passes first
through an adapted sampling for the different environments
explored. The various components of the microbial community can then be characterized from the point of view of their
biomass, their activity, and their diversity. To respond to
these various issues, techniques as varied as those of flow
cytometry, molecular biology, biochemistry, molecular isotopic tools, or electrochemistry are implemented. These
different methods are described with their advantages and
disadvantages for different types of biotopes (water, soil,
sediment, biofilms). A final important point discussed in
this chapter concerns the isolation of microorganisms from
the environment and their culture in the laboratory. Without
being exhaustive, this chapter emphasizes the importance
of using appropriate and efficient methodological tools
to properly explore the still mysterious compartment of
microorganisms in the environment.
17.2 Sampling Techniques in Microbial
Ecology
17.2.1 Soils Sampling Techniques
Sampling of soil for microbial ecology studies can be achieved
with a shovel, but more usually using a hand auger (diameter
between 1 and 10 cm) or vehicle-mounted hydraulic auger,
which allows to collect at a constant depth. The depth is
important because it influences many parameters (oxygen,
temperature, water content, concentration of organic matter,
etc.). The depth studied is variable in function of the questions.
It can reach several meters (Box 17.1), but more often it is
the layer between 0 and 30 cm that is studied. Techniques
described above (use of a shovel or an auger) concern masses
of soil from few hundred grams to several kilograms.
A major element to be taken into account during the sampling is the quantity and the quality of the organic materials
that will in part determine the functioning of heterotrophic
microflora representing the majority of the soil microorganisms. For this reason, many studies take into account the
organization of the soil horizon. The superficial horizons
(0–30 cm) are the most studied, because the most intense
microbial activity is observed in these horizons. For the
same reason, the rhizospheric soil (under the influence of
the roots) is often the object of particular attention. Sampling rhizospheric soil is done by shaking the roots to
remove not adhering soil, the soil still adhering constituting
rhizospheric soil.
Box 17.1: Sampling the Deep Biosphere
Michel Magot
The results of a study published in 1998 sounded like a
thunderclap in the sky of microbial ecology. The
authors suggested that the amount of biomass hidden
in the deep Earth subsurface, down to about 4,000 m
deep, would be equivalent or higher to that of all living
organisms on its surface (Whitman et al. 1998)! The
total population of prokaryotes, only denizens of the
deep, was estimated between 25 and 250 Â 10
28 cells,
while about 15 years ago most microbiologists
believed that the Earth subsurface was sterile beyond
a few tens meters deep. Why did it take more than a
century of research in microbiology for the estimation
of such a great unknown biodiversity? The main reason
undoubtedly lies in the difficulty of obtaining uncontaminated geological samples or deep fluids. Three
main ways to collect microbiologically representative
deep subsurface samples will be briefly described
below.
Collecting Fluid Samples from Pre-existing Wells
This is a priori the simplest and cheapest method.
This is what the American microbiologist Edson
Bastin did during the 1920s to study sulfate-reducing
bacteria from American oilfield waters, suggesting
for the first time that bacteria could live in the deep
subsurface. Whether collecting petroleum fluids or
water samples from deep aquifers, the technique is
the same: in principle, it just needs a production or
monitoring well drilled in the geological formation
of interest to collect liquid samples by following
some basic rules to maintain aseptic conditions and
avoid contact with oxygen. Studies based on such
samples, either by conventional microbiology or
molecular methods, show that things are really not
that easy! Multiple sources of contamination can
lead to erroneous conclusions. The two main ones
result from (i) the drilling fluids used during the implementation of the well, which will be discussed below,
and (ii) the colonization of the entire inner part of the
well (the tubing) by a biofilm, which most often has
nothing to do with the indigenous subsurface
microorganisms (Magot 2005). A solution is nevertheless available to collect representative subsurface
M. Magot
Institut des Sciences Analytiques et de Physico-chimie pour
l’Environnement et les Mate ´riaux (IPREM), UMR CNRS 5254,
Universite ´ de Pau et des Pays de l’Adour, B.P. 1155, 64013 Pau
Cedex, France
(continued)
758
F. Joux et al.