Marine Microbial Bioprospecting 11.3 Methods for Microbial Bioprospecting in Marine Environments 309
Part B | 11.3
There are certain phylogenetic groups which per se
constitute interesting targets for bioprospection. Actinomycetes (within the phylum Actinobacteria) are widely
known for their capabilities of producing metabolites,
which include antibiotics, antitumor and immunosuppressive agents and enzymes, among others [11.20].
Novel compounds with biological activities have already been isolated from marine actinomycetes [11.6].
Culture-independent studies have shown that the majority of actinomycetes from marine environments are not
recovered by cultivation-based methods, and that marine actinomycetes are very different phylogenetically
from their terrestrial counterparts [11.21, 22]. Deep-sea
sediments and marine flora and fauna allow the access
of true marine actinomycetes, away from the influence
of land wash-offs [11.6]. Given the fact that 45% of
all microbial bioactive secondary metabolites currently
derive from actinomycetes and that only 10% of these
metabolites is estimated to have been discovered so far,
it becomes evident that advances in the ability to access
this yet unexploited diversity will provide a new source
for the discovery of secondary metabolites [11.23].
11.3 Methods for Microbial Bioprospecting in Marine Environments
Both culture-dependent and independent methods have
uncovered an incredible diversity of microorganisms
whose metabolisms largely have yet to be characterized [11.24, 25]. These methods have been further
empowered by genomic-level information, which in
turn is supported by sequencing technologies and bioinformatics [11.26, 27]. In the next sections, traditional,
state-of-the-art and emerging approaches used for bioprospecting marine microorganisms with biotechnological potential will be reviewed.
11.3.1 Culturing Techniques
Microbial bioprospection and biodiscovery is currently severely limited by the lack of laboratory
cultures [11.28]. Although culture-independent approaches have revolutionized environmental microbiology, the development of biotechnological applications
from the genetic potential of microbial communities as
well as fundamental environmental research must be
anchored by the corresponding study of pure cultures.
Furthermore, this novel diversity needs to be deeply
characterized and adequately preserved in order to guarantee its future availability [11.29]. Novel cultivation
methods, fortunately, continue to emerge as alternatives to overcome culture limitations [11.30]. These
methods rely on advances in basic biological and ecological knowledge in order to best simulate the natural
environment, as well as on the development of new
technologies for more efficient screenings (Table 11.1).
With the aid of sophisticated high-throughput cultivation techniques, the proportion of microorganisms
from marine environments represented in culture has
increased significantly over the last years [11.31].
High-throughput dilution-to-extinction culture is
one of the most powerful and sensitive approaches
for the culture of marine microorganisms such as bacterioplankton. This technique led to the cultivation
of the first member of the widespread but yet uncultured marine SAR11 clade [11.40]. This method
consists in dilution of bacteria up to 110 cells per
well in microtiter plates, using low-nutrient filtered
seawater. High-throughput screening based on fluorescence microscopy clearly improved the technique over
conventional methods, allowing rapid and sensitive detection of growing cells [11.35]. In later studies, this
approach was coupled to long-term incubation at low
temperatures to allow the recovery of new microbial
variants [11.39].
The diffusion chamber [11.32] is a device in which
microbial cells are inoculated in an agar matrix separated from the source environment by membranes,
isolating the cells but allowing nutrients and growth
factors to pass through. The use of this device greatly
improved the proportion of culturable bacteria from
marine sediments [11.32]. Another version of this approach is the microbial trap, which selectively enriches
for filamentous bacteria (e.g. actinomycetes) by allowing the filament colonization of the sterile agar through
membranes with 0:2 m pores [11.34]. Microdroplet
encapsulation in an agarose matrix, combined with
growth detection by flow cytometry, led to the recovery
of new clades from the marine environment [11.36, 37].
This approach is similar to the diffusion chamber in the
sense that the agarose is porous, and nutrients and signaling molecules can diffuse into the growing colony
and waste metabolites can diffuse out. Another advantage of the approach is that the microdroplets are physically separated and, because they are much larger than
bacterial cells, they can be manipulated [11.28, 36].
Currently, second-generation high-throughput automated methods are being developed from these environ-
Part B | 11.3
There are certain phylogenetic groups which per se
constitute interesting targets for bioprospection. Actinomycetes (within the phylum Actinobacteria) are widely
known for their capabilities of producing metabolites,
which include antibiotics, antitumor and immunosuppressive agents and enzymes, among others [11.20].
Novel compounds with biological activities have already been isolated from marine actinomycetes [11.6].
Culture-independent studies have shown that the majority of actinomycetes from marine environments are not
recovered by cultivation-based methods, and that marine actinomycetes are very different phylogenetically
from their terrestrial counterparts [11.21, 22]. Deep-sea
sediments and marine flora and fauna allow the access
of true marine actinomycetes, away from the influence
of land wash-offs [11.6]. Given the fact that 45% of
all microbial bioactive secondary metabolites currently
derive from actinomycetes and that only 10% of these
metabolites is estimated to have been discovered so far,
it becomes evident that advances in the ability to access
this yet unexploited diversity will provide a new source
for the discovery of secondary metabolites [11.23].
11.3 Methods for Microbial Bioprospecting in Marine Environments
Both culture-dependent and independent methods have
uncovered an incredible diversity of microorganisms
whose metabolisms largely have yet to be characterized [11.24, 25]. These methods have been further
empowered by genomic-level information, which in
turn is supported by sequencing technologies and bioinformatics [11.26, 27]. In the next sections, traditional,
state-of-the-art and emerging approaches used for bioprospecting marine microorganisms with biotechnological potential will be reviewed.
11.3.1 Culturing Techniques
Microbial bioprospection and biodiscovery is currently severely limited by the lack of laboratory
cultures [11.28]. Although culture-independent approaches have revolutionized environmental microbiology, the development of biotechnological applications
from the genetic potential of microbial communities as
well as fundamental environmental research must be
anchored by the corresponding study of pure cultures.
Furthermore, this novel diversity needs to be deeply
characterized and adequately preserved in order to guarantee its future availability [11.29]. Novel cultivation
methods, fortunately, continue to emerge as alternatives to overcome culture limitations [11.30]. These
methods rely on advances in basic biological and ecological knowledge in order to best simulate the natural
environment, as well as on the development of new
technologies for more efficient screenings (Table 11.1).
With the aid of sophisticated high-throughput cultivation techniques, the proportion of microorganisms
from marine environments represented in culture has
increased significantly over the last years [11.31].
High-throughput dilution-to-extinction culture is
one of the most powerful and sensitive approaches
for the culture of marine microorganisms such as bacterioplankton. This technique led to the cultivation
of the first member of the widespread but yet uncultured marine SAR11 clade [11.40]. This method
consists in dilution of bacteria up to 110 cells per
well in microtiter plates, using low-nutrient filtered
seawater. High-throughput screening based on fluorescence microscopy clearly improved the technique over
conventional methods, allowing rapid and sensitive detection of growing cells [11.35]. In later studies, this
approach was coupled to long-term incubation at low
temperatures to allow the recovery of new microbial
variants [11.39].
The diffusion chamber [11.32] is a device in which
microbial cells are inoculated in an agar matrix separated from the source environment by membranes,
isolating the cells but allowing nutrients and growth
factors to pass through. The use of this device greatly
improved the proportion of culturable bacteria from
marine sediments [11.32]. Another version of this approach is the microbial trap, which selectively enriches
for filamentous bacteria (e.g. actinomycetes) by allowing the filament colonization of the sterile agar through
membranes with 0:2 m pores [11.34]. Microdroplet
encapsulation in an agarose matrix, combined with
growth detection by flow cytometry, led to the recovery
of new clades from the marine environment [11.36, 37].
This approach is similar to the diffusion chamber in the
sense that the agarose is porous, and nutrients and signaling molecules can diffuse into the growing colony
and waste metabolites can diffuse out. Another advantage of the approach is that the microdroplets are physically separated and, because they are much larger than
bacterial cells, they can be manipulated [11.28, 36].
Currently, second-generation high-throughput automated methods are being developed from these environ-