Marine Microbial Bioprospecting 11.3 Methods for Microbial Bioprospecting in Marine Environments 313
Part B | 11.3
The most widely used method is DNA-SIP, in which
DNA is separated in caesium chloride gradients and
further purified and analyzed by cloning and sequencing [11.70]. The RNA-based SIP approach maintains
the sequence-based phylogenetic resolution of DNASIP, but focuses directly on the RNA molecule itself
rather than its gene, with the advantage of a high copy
number and a turnover that is independent of cell replication [11.71]. Marine environments studied by this
method include marine and estuarine sediments [11.72–
74] and seawater samples [11.75]. Biotechnological
applications of SIP have mainly addressed issues related to environmental biotechnology [11.76].
SIP depends upon the availability of stable isotopes (
13 C,
15 N,
18 O) and of substituted substrate compounds [11.69]. However, they have the advantage of
generating de novo information about the identity of the
populations associated with a certain metabolic process.
Interestingly, different incubation times can be used to
follow the carbon flow in the different members of the
community. The results can then be further tested experimentally, for example, by means of imaging techniques
or targeted culture (Fig. 11.2).
Microscopy provides information about spatial
arrangement and physical interactions of cells, which
is applicable to spatially complex environments such
as biofilms, consortia and symbiotic assemblages
(Fig. 11.2). The development of fluorescence insitu hybridization (FISH) enabled the detection and
identification of single microbial cells in environmental samples by means of rRNA-targeted gene
probes [11.77]. Microscope-based enumeration of
cells makes this method an excellent approach for
quantitative estimations, which is more accurate than
conventional PCR. Furthermore, the technique is suitable for the use of multiple hierarchical probes in the
same sample, which reduces the possibility of false
positives. This powerful method has been coupled with
the microautoradiography technique (FISH-MAR),
which offers the possibility to directly observe the
incorporation of substrates labeled with a radioactive
isotope into single microbial cells [11.78]. As in SIP,
the main limitation of this technique is the availability of
radiolabeled substrates, with the additional concern of
safety issues. In addition, some environmental samples
bearing cells with low ribosome content (e.g., marine
oligotrophic environments) can have detection problems with FISH. Horseradish peroxidase (HRP)-labeled
oligonucleotide probes and tyramide can be used to
enhance the signal intensities of hybridized cells. This
approach is sometimes called catalyzed reporter deposition FISH (CARD-FISH, [11.79]), which can also be
coupled to microautoradiography, further increasing
its potential [11.80]. Raman microspectroscopy and
nanometer-scale secondary-ion mass spectrometry
(nanoSIMS [11.81, 82]), are other techniques that are
currently under development and may potentially be
useful in the future for bioprospecting.
11.3.3 Omics and Meta-Omics Approaches
The large-scale study of genes (genomics), transcripts
(transcriptomics), proteins (proteomics), metabolites
(metabolomics), lipids (lipidomics), and interactions
(interactomics) are globally defined as omics in the
study of individual species and are often referred to
as meta-omics approaches when microbial communities
are analyzed [11.83]. These are rapidly evolving fields,
which are highly dependent on the development and improvement of technologies and analysis tools. Besides
their critical role for understanding the structure and
function of microbial communities, they represent powerful approaches for the bioprospection of biological
products and activities with biotechnological potential
in marine environments.
Genomics
Since the publication of the genome of the bacterium Haemophilus influenzae Rd in 1995 [11.84],
the number of sequenced genomes has expanded
quickly [11.85]. By the end of 2012, the Genomes Online Database (GOLD, Table 11.2) listed more than
4000 completed genome projects, 90% of them belonging to bacteria. Approximately 60% of these genomes
where finished, that is, all segments obtained after the
assembly were ordered, all gaps were closed, and any
ambiguities or discrepancies were resolved after a series of rigorous quality-control steps [11.86, 87]. As the
finishing step increases the cost and time required to sequence a genome, often the final goal is to obtain a draft
genome, represented by a number of contigs or scaffolds [11.87]. Although there are limitations for the use
of draft sequences in some applications [11.88], draft
assemblies are a powerful resource for bioprospecting,
as the majority of the genes of an organism are usually
represented in its draft genome [11.86].
Not only the number of genomes sequenced so far
represents a minimal proportion of the microbial diversity present in our planet [11.89], but also some
phylogenetic groups of microorganisms (such as members of the Proteobacteria and Firmicutes) are greatly
over-represented, while other groups have no represen-
Part B | 11.3
The most widely used method is DNA-SIP, in which
DNA is separated in caesium chloride gradients and
further purified and analyzed by cloning and sequencing [11.70]. The RNA-based SIP approach maintains
the sequence-based phylogenetic resolution of DNASIP, but focuses directly on the RNA molecule itself
rather than its gene, with the advantage of a high copy
number and a turnover that is independent of cell replication [11.71]. Marine environments studied by this
method include marine and estuarine sediments [11.72–
74] and seawater samples [11.75]. Biotechnological
applications of SIP have mainly addressed issues related to environmental biotechnology [11.76].
SIP depends upon the availability of stable isotopes (
13 C,
15 N,
18 O) and of substituted substrate compounds [11.69]. However, they have the advantage of
generating de novo information about the identity of the
populations associated with a certain metabolic process.
Interestingly, different incubation times can be used to
follow the carbon flow in the different members of the
community. The results can then be further tested experimentally, for example, by means of imaging techniques
or targeted culture (Fig. 11.2).
Microscopy provides information about spatial
arrangement and physical interactions of cells, which
is applicable to spatially complex environments such
as biofilms, consortia and symbiotic assemblages
(Fig. 11.2). The development of fluorescence insitu hybridization (FISH) enabled the detection and
identification of single microbial cells in environmental samples by means of rRNA-targeted gene
probes [11.77]. Microscope-based enumeration of
cells makes this method an excellent approach for
quantitative estimations, which is more accurate than
conventional PCR. Furthermore, the technique is suitable for the use of multiple hierarchical probes in the
same sample, which reduces the possibility of false
positives. This powerful method has been coupled with
the microautoradiography technique (FISH-MAR),
which offers the possibility to directly observe the
incorporation of substrates labeled with a radioactive
isotope into single microbial cells [11.78]. As in SIP,
the main limitation of this technique is the availability of
radiolabeled substrates, with the additional concern of
safety issues. In addition, some environmental samples
bearing cells with low ribosome content (e.g., marine
oligotrophic environments) can have detection problems with FISH. Horseradish peroxidase (HRP)-labeled
oligonucleotide probes and tyramide can be used to
enhance the signal intensities of hybridized cells. This
approach is sometimes called catalyzed reporter deposition FISH (CARD-FISH, [11.79]), which can also be
coupled to microautoradiography, further increasing
its potential [11.80]. Raman microspectroscopy and
nanometer-scale secondary-ion mass spectrometry
(nanoSIMS [11.81, 82]), are other techniques that are
currently under development and may potentially be
useful in the future for bioprospecting.
11.3.3 Omics and Meta-Omics Approaches
The large-scale study of genes (genomics), transcripts
(transcriptomics), proteins (proteomics), metabolites
(metabolomics), lipids (lipidomics), and interactions
(interactomics) are globally defined as omics in the
study of individual species and are often referred to
as meta-omics approaches when microbial communities
are analyzed [11.83]. These are rapidly evolving fields,
which are highly dependent on the development and improvement of technologies and analysis tools. Besides
their critical role for understanding the structure and
function of microbial communities, they represent powerful approaches for the bioprospection of biological
products and activities with biotechnological potential
in marine environments.
Genomics
Since the publication of the genome of the bacterium Haemophilus influenzae Rd in 1995 [11.84],
the number of sequenced genomes has expanded
quickly [11.85]. By the end of 2012, the Genomes Online Database (GOLD, Table 11.2) listed more than
4000 completed genome projects, 90% of them belonging to bacteria. Approximately 60% of these genomes
where finished, that is, all segments obtained after the
assembly were ordered, all gaps were closed, and any
ambiguities or discrepancies were resolved after a series of rigorous quality-control steps [11.86, 87]. As the
finishing step increases the cost and time required to sequence a genome, often the final goal is to obtain a draft
genome, represented by a number of contigs or scaffolds [11.87]. Although there are limitations for the use
of draft sequences in some applications [11.88], draft
assemblies are a powerful resource for bioprospecting,
as the majority of the genes of an organism are usually
represented in its draft genome [11.86].
Not only the number of genomes sequenced so far
represents a minimal proportion of the microbial diversity present in our planet [11.89], but also some
phylogenetic groups of microorganisms (such as members of the Proteobacteria and Firmicutes) are greatly
over-represented, while other groups have no represen-