Marine Microbial Bioprospecting 11.3 Methods for Microbial Bioprospecting in Marine Environments 311
Part B | 11.3
cubation (i. e., the same amount of effort focused either
on many short-term or fewer long-term cultivation experiments) [11.41]. This theory could explain previous
findings, such as why, for example, representatives of
the phylum Verrucomicrobia that had been considered
unculturable for many years, were eventually cultivated
using rather conventional techniques. Dilution cultures
were used to separate the very fast growers, but long
incubation times were not necessary [11.41, 53].
11.3.2 Culture Independent Gene-Targeted
Methods
In spite of the recent advances in microbial culturing,
the majority of environmental microorganisms are still
unculturable. Out of the more than 100 bacterial divisions that have been proposed to date, only 30 possess
a cultivated representative [11.29]. Moreover, marine
microbes are at the top of the list of those unculturable
by conventional methods [11.26]. Since the landmark
studies of Woese and Pace [11.54, 55], which stated
the basis for molecular phylogeny, culture-independent
methods have revolutionized our understanding of microbial communities [11.56] and currently stand on
their own as a valid alternative for bioprospection.
These methods are based on the information provided
by biomolecules, mainly deoxyribonucleic acid (DNA),
bypassing the need of cultivation by extracting these
biomolecules directly from the environmental sample.
Although they are not exempt from biases [11.57], they
are still the best way to gain access to the overwhelming
biodiversity of environmental microbes.
Culture-Independent Phylogenetic
Approaches
Among culture-independent methods, the approach
based on the molecular phylogeny of rRNA (ribosomal
ribonucleic acid), particularly the small subunit (16S
rRNA for archaea and bacteria), continues to be one
of the most widely used. This gene has two properties
that have positioned it as a building block for a universal molecular phylogenetic framework: its presence in
all forms of life and a domain structure with variable
evolutionary rates, which enables phylogenetic reconstruction at various levels. Fingerprinting techniques,
polymerase chain reaction (PCR) clone libraries, and
microscopy-based techniques like fluorescence in situ
hybridization (FISH) have been routinely utilized over
the last decades to describe and compare the structure
and composition of microbial communities [11.58].
More recently, large-scale sequencing of 16S rRNA
gene hypervariable regions has brought new strength to
the classical phylogenetic approaches, which suffered
a number of limitations associated with low coverage
and cloning biases [11.59]. These approaches can be
used as a guide for phylogenetically-driven biodiscovery. For example, analysis of community 16S rRNA
by sequencing or fingerprinting can be used to select the most diverse sampling sites, thus maximizing
novel taxa recovery in culture (Fig. 11.2, [11.23]). This
approach is based upon the premise that taxonomic diversity is coupled to chemical diversity due to the role
that secondary metabolism plays in speciation [11.23].
Functional Gene-Based Approaches
Approaches based on functional genes, which focus on
the potential of the community to perform an activity of interest, can give a complementary view to the
phylogenetic approach. Gene coding for key enzymes
participating in different environmental processes, such
as sulfate reduction [11.60], denitrification [11.61], nitrogen fixation [11.62], ammonia oxidation [11.63],
hydrocarbon biodegradation [11.64, 65] among others,
have been studied in the marine environment. Targets
include not only bacterial but also archaeal populations
and subgroups within these, by means of the use of
primers with different specificities. Due to its highly
focused nature, this approach is very powerful. However, one of its major drawbacks is the relative lack
of database sequence information for functional genes,
with respect to the 16S RNA gene. Another shortcoming is the lack of accuracy in taxonomic assignment due
to lateral gene transfer [11.66].
Functional genes have the potential to be used
as biomarkers in assays developed for the environmental biotechnology field, including wastewater treatment [11.67], and environmental remediation
(Fig. 11.2, [11.68]). These molecular biological tools
have been applied in the marine environment, for example, for the study of hydrocarbon degrading bacterial
populations [11.64, 65]. In particular, quantitative polymerase chain reaction (qPCR) is a promising technique
due to its quantitative nature, high sensitivity and the
possibility of high throughput analysis [11.68]. However, this tool is still in its infancy for field applications
in marine environments.
Linking Phylogeny and Function: Labeled
Isotopic-Based Approaches
One of the long-standing goals of environmental microbiology is the possibility to link the phylogenetic identity of an uncultured microorganism with its function in
Part B | 11.3
cubation (i. e., the same amount of effort focused either
on many short-term or fewer long-term cultivation experiments) [11.41]. This theory could explain previous
findings, such as why, for example, representatives of
the phylum Verrucomicrobia that had been considered
unculturable for many years, were eventually cultivated
using rather conventional techniques. Dilution cultures
were used to separate the very fast growers, but long
incubation times were not necessary [11.41, 53].
11.3.2 Culture Independent Gene-Targeted
Methods
In spite of the recent advances in microbial culturing,
the majority of environmental microorganisms are still
unculturable. Out of the more than 100 bacterial divisions that have been proposed to date, only 30 possess
a cultivated representative [11.29]. Moreover, marine
microbes are at the top of the list of those unculturable
by conventional methods [11.26]. Since the landmark
studies of Woese and Pace [11.54, 55], which stated
the basis for molecular phylogeny, culture-independent
methods have revolutionized our understanding of microbial communities [11.56] and currently stand on
their own as a valid alternative for bioprospection.
These methods are based on the information provided
by biomolecules, mainly deoxyribonucleic acid (DNA),
bypassing the need of cultivation by extracting these
biomolecules directly from the environmental sample.
Although they are not exempt from biases [11.57], they
are still the best way to gain access to the overwhelming
biodiversity of environmental microbes.
Culture-Independent Phylogenetic
Approaches
Among culture-independent methods, the approach
based on the molecular phylogeny of rRNA (ribosomal
ribonucleic acid), particularly the small subunit (16S
rRNA for archaea and bacteria), continues to be one
of the most widely used. This gene has two properties
that have positioned it as a building block for a universal molecular phylogenetic framework: its presence in
all forms of life and a domain structure with variable
evolutionary rates, which enables phylogenetic reconstruction at various levels. Fingerprinting techniques,
polymerase chain reaction (PCR) clone libraries, and
microscopy-based techniques like fluorescence in situ
hybridization (FISH) have been routinely utilized over
the last decades to describe and compare the structure
and composition of microbial communities [11.58].
More recently, large-scale sequencing of 16S rRNA
gene hypervariable regions has brought new strength to
the classical phylogenetic approaches, which suffered
a number of limitations associated with low coverage
and cloning biases [11.59]. These approaches can be
used as a guide for phylogenetically-driven biodiscovery. For example, analysis of community 16S rRNA
by sequencing or fingerprinting can be used to select the most diverse sampling sites, thus maximizing
novel taxa recovery in culture (Fig. 11.2, [11.23]). This
approach is based upon the premise that taxonomic diversity is coupled to chemical diversity due to the role
that secondary metabolism plays in speciation [11.23].
Functional Gene-Based Approaches
Approaches based on functional genes, which focus on
the potential of the community to perform an activity of interest, can give a complementary view to the
phylogenetic approach. Gene coding for key enzymes
participating in different environmental processes, such
as sulfate reduction [11.60], denitrification [11.61], nitrogen fixation [11.62], ammonia oxidation [11.63],
hydrocarbon biodegradation [11.64, 65] among others,
have been studied in the marine environment. Targets
include not only bacterial but also archaeal populations
and subgroups within these, by means of the use of
primers with different specificities. Due to its highly
focused nature, this approach is very powerful. However, one of its major drawbacks is the relative lack
of database sequence information for functional genes,
with respect to the 16S RNA gene. Another shortcoming is the lack of accuracy in taxonomic assignment due
to lateral gene transfer [11.66].
Functional genes have the potential to be used
as biomarkers in assays developed for the environmental biotechnology field, including wastewater treatment [11.67], and environmental remediation
(Fig. 11.2, [11.68]). These molecular biological tools
have been applied in the marine environment, for example, for the study of hydrocarbon degrading bacterial
populations [11.64, 65]. In particular, quantitative polymerase chain reaction (qPCR) is a promising technique
due to its quantitative nature, high sensitivity and the
possibility of high throughput analysis [11.68]. However, this tool is still in its infancy for field applications
in marine environments.
Linking Phylogeny and Function: Labeled
Isotopic-Based Approaches
One of the long-standing goals of environmental microbiology is the possibility to link the phylogenetic identity of an uncultured microorganism with its function in