Marine Enzymes – Production and Applications 14.2 Cultivation Techniques of Marine Bacteria 415
Part B | 14.2
Seawater contains approximately 10
4
10
6
cells ml
1 , whereas on surfaces exposed to seawater much higher cell densities, of six orders of
magnitude, can be detected [14.6]. Thus, the applied
nutrient media should reflect the nutrient conditions
of the natural habitat. However, commonly used
nutrient media feature a high carbon content, which is
contradictory for the fermentation of marine strains.
A special cultivation technique applicable for those
strains that are classified as non-reproducible under
laboratory conditions is the so-called dilution technique [14.7], whereas the populations in a sample are
measured, diluted to a small and known number of
cells, inoculated into unamended sterilized seawater,
and afterwards examined for a defined period of time.
Another nutrient component which is often available to a less degree in seawater and in artificial nutrient
broth is iron; therefore growth may be limited with respect to iron deficiency [14.8, 9]. Because of the limited
bioavailability of iron, several organisms have adapted
themselves and are able to release particular iron-complexing agents, so-called siderophores, enabling the
organism to increase iron uptake [14.10].
Although marine microorganisms have specific nutrient requirements, many complex media have been
developed for the cultivation of marine microorganisms under lab-scale conditions. The most prominent is
the so-called Marine Broth 2216, which was developed
in its primary formulation by Zobell in 1941 [14.11].
This complex medium can be regarded as the main reference medium in the field of marine microbiology.
To reflect the special nutrient needs of distinct marine
microorganisms it is sometimes utilized in diluted concentrations. Moreover, to satisfy the need for iron, it
contains a well-accessible iron salt (0:1% iron-citrate).
Besides the cultivation of freely suspended cells,
enrichment of organisms deriving from surfaces exposed to seawater or from tissues of higher marine
organisms can be a challenging task. The organisms
commonly grow as conglomerates of bacteria, fungi, algae, and protozoans and represent a biofilm. Within the
biofilm the organisms benefit from the accompanying
organisms in the extracellular matrix (commensalism
and mutualism). Furthermore, the organisms are sheltered within the matrix thus they are often more tolerant towards unphysiological conditions than their suspended counterparts, e.g., towards high/low pH, pressure, and antibiotics. Microorganisms are exposed to
varying conditions in their natural habitats with regard
to pH, temperature, osmolality, or nutrients and they
have evolved specific adaptation systems. Thus, they
are able to respond to such fluctuations by activation
or repression of gene expression [14.12]. Apart from
these mechanisms alternative stimuli–response regulation patterns can be found in bacterial cell–cell communication by means of small signal molecules, called
autoinducers. So far, a multitude of signal molecules
and the induced signal cascades have been identified;
this form of molecular regulation of signal transduction was defined as quorum sensing (QS). This term
was given according to its special mechanism and was
inaugurated by Fuqua et al. [14.13]. Hence, many interactions within such a microenvironment are mediated
via different (chemical) QS communication patterns.
During the phase of cell growth a specific autoinducer molecule is produced as a function of increasing
cell-population density. The bacteria are able to detect
and subsequently respond to the accumulation of these
compounds, thus, they are capable of distinguishing between high and low cell population density and control
gene expression in variation of cell population. By using such QS circuits a population of microorganisms
can control the gene expression of the entire community [14.14]. Such a cell–cell communication was first
described by Hastings and Nealson [14.15, 16], who
elucidated the cell-density depending expression of luciferase of Vibrio fischeri. The microorganism emits
light when growing inside the specialized light organs
of the host organism but not when living freely in
the ocean. So far, many communication strategies have
been discovered, whereas distinct signal compounds
have been found, depending on the species. Whereas
Gram-positive bacteria commonly utilize amino acids
or small peptides for QS [14.17, 18], derivatives of
fatty acids (acyl-homoserine lactones) serve as autoinducers for Gram-negative bacteria [14.19]. Alternative
communication strategies like hierarchical systems for
cross-species communication have been summarized by
Whitehead et al. and can be found elsewhere [14.19].
The discovery of the QS communication system is also
of interest for the cultivation processes of marine microorganisms. For example, Yan et al. used a biofilm
approach, where two epiphytic isolates were cultivated
in a modified roller-bottle bioreactor to produce antimicrobial compounds [14.20]. Due to mimicking of
the intertidal environment by application of this type
of bioreactor, an enhanced production of antimicrobial compounds was found. When the organisms were
cultivated in a planktonic state no antimicrobial activity was observed in the supernatant. Since QS is
facilitated in the biofilm, it is assumed that production of the compounds is related to the communi-
Part B | 14.2
Seawater contains approximately 10
4
10
6
cells ml
1 , whereas on surfaces exposed to seawater much higher cell densities, of six orders of
magnitude, can be detected [14.6]. Thus, the applied
nutrient media should reflect the nutrient conditions
of the natural habitat. However, commonly used
nutrient media feature a high carbon content, which is
contradictory for the fermentation of marine strains.
A special cultivation technique applicable for those
strains that are classified as non-reproducible under
laboratory conditions is the so-called dilution technique [14.7], whereas the populations in a sample are
measured, diluted to a small and known number of
cells, inoculated into unamended sterilized seawater,
and afterwards examined for a defined period of time.
Another nutrient component which is often available to a less degree in seawater and in artificial nutrient
broth is iron; therefore growth may be limited with respect to iron deficiency [14.8, 9]. Because of the limited
bioavailability of iron, several organisms have adapted
themselves and are able to release particular iron-complexing agents, so-called siderophores, enabling the
organism to increase iron uptake [14.10].
Although marine microorganisms have specific nutrient requirements, many complex media have been
developed for the cultivation of marine microorganisms under lab-scale conditions. The most prominent is
the so-called Marine Broth 2216, which was developed
in its primary formulation by Zobell in 1941 [14.11].
This complex medium can be regarded as the main reference medium in the field of marine microbiology.
To reflect the special nutrient needs of distinct marine
microorganisms it is sometimes utilized in diluted concentrations. Moreover, to satisfy the need for iron, it
contains a well-accessible iron salt (0:1% iron-citrate).
Besides the cultivation of freely suspended cells,
enrichment of organisms deriving from surfaces exposed to seawater or from tissues of higher marine
organisms can be a challenging task. The organisms
commonly grow as conglomerates of bacteria, fungi, algae, and protozoans and represent a biofilm. Within the
biofilm the organisms benefit from the accompanying
organisms in the extracellular matrix (commensalism
and mutualism). Furthermore, the organisms are sheltered within the matrix thus they are often more tolerant towards unphysiological conditions than their suspended counterparts, e.g., towards high/low pH, pressure, and antibiotics. Microorganisms are exposed to
varying conditions in their natural habitats with regard
to pH, temperature, osmolality, or nutrients and they
have evolved specific adaptation systems. Thus, they
are able to respond to such fluctuations by activation
or repression of gene expression [14.12]. Apart from
these mechanisms alternative stimuli–response regulation patterns can be found in bacterial cell–cell communication by means of small signal molecules, called
autoinducers. So far, a multitude of signal molecules
and the induced signal cascades have been identified;
this form of molecular regulation of signal transduction was defined as quorum sensing (QS). This term
was given according to its special mechanism and was
inaugurated by Fuqua et al. [14.13]. Hence, many interactions within such a microenvironment are mediated
via different (chemical) QS communication patterns.
During the phase of cell growth a specific autoinducer molecule is produced as a function of increasing
cell-population density. The bacteria are able to detect
and subsequently respond to the accumulation of these
compounds, thus, they are capable of distinguishing between high and low cell population density and control
gene expression in variation of cell population. By using such QS circuits a population of microorganisms
can control the gene expression of the entire community [14.14]. Such a cell–cell communication was first
described by Hastings and Nealson [14.15, 16], who
elucidated the cell-density depending expression of luciferase of Vibrio fischeri. The microorganism emits
light when growing inside the specialized light organs
of the host organism but not when living freely in
the ocean. So far, many communication strategies have
been discovered, whereas distinct signal compounds
have been found, depending on the species. Whereas
Gram-positive bacteria commonly utilize amino acids
or small peptides for QS [14.17, 18], derivatives of
fatty acids (acyl-homoserine lactones) serve as autoinducers for Gram-negative bacteria [14.19]. Alternative
communication strategies like hierarchical systems for
cross-species communication have been summarized by
Whitehead et al. and can be found elsewhere [14.19].
The discovery of the QS communication system is also
of interest for the cultivation processes of marine microorganisms. For example, Yan et al. used a biofilm
approach, where two epiphytic isolates were cultivated
in a modified roller-bottle bioreactor to produce antimicrobial compounds [14.20]. Due to mimicking of
the intertidal environment by application of this type
of bioreactor, an enhanced production of antimicrobial compounds was found. When the organisms were
cultivated in a planktonic state no antimicrobial activity was observed in the supernatant. Since QS is
facilitated in the biofilm, it is assumed that production of the compounds is related to the communi-