CONTROL OF THE MICROBIAL COMMUNITY IN ROTIFER
CULTURES (BRACHIONUS PLICATILIS)
PhD Thesis by Geert Rombaut,
Faculty of Agricultural and Applied Biological
Sciences, Ghent University, Belgium, 2001, 194 pp.
Summary:
The necessity, and the general
aspects of rotifers (Brachionus plicatilis) and rotifer cultures in
larviculture are demonstrated and described in Chapter 1 (Situation of the
necessity of live food in larviculture) and 2 (General aspects of rotifers).
The problems related to the often poor reproducible results in terms of
quantitative and qualitative aspects of a rotifer culture can be defined
partly to the negative interactions between rotifers (Brachionus plicatilis) and
the associated bacteria. The present study examined and characterised the
developing microbial community which is associated with routine rotifer
batch cultures and with a newly-developed recirculation culture system. It
described a methodology to evaluate the effect of individual bacterial
strains on the population growth rate of rotifers with the selection of
probiotic bacterial strains applicable in rotifer cultures in view. The
application of these selected bacterial strains was set as an objective to
find a way out for the inconsistent results.
In Chapter 3 (Probiotics in
aquaculture) and 4 (Microbial ecology of a rotifer culture), literature on
the possible modes of action and the use of probiotic bacteria as biological
control agents in aquaculture and in rotifer cultures has been reviewed.
In Chapter 5 (Monitoring of the
evolving diversity of the microbial community present in rotifer cultures),
the microbial community present in two different rotifer culture systems was
monitored by means of a molecular fingerprinting based method, i.e.
denaturing gradient gel electrophoresis (DGGE). It was demonstrated that the
microbiota present in a batch system was more sensitive for daily shifts in
the genetic fingerprints compared to the microbiota associated with a
rotifer recirculation culture system. Moreover, further identification of
the different bands in the genetic profiles of the bacterial community
present in the recirculation system demonstrated that two different
bacterial genera, i.e. Marinomonas
and Pseudoalteromonas, dominated the system.
In Chapter 6 (Selection of
bacteria enhancing the growth rate of axenically hatched rotifers (Brachionus
plicatilis),
a new methodology
was
described to disinfect mictic eggs of rotifers by means of glutaraldehyde.
By this method, it was possible to set up axenic batch cultures that could
be used as a basic tool to screen bacterial strains, acquired from different
sources, for their effect on the rotifer population growth rate. Five
bacterial strains were selected based on their positive effects on the
asexual reproduction of rotifers, while other strains did not or negatively
affect the asexual reproduction. Overall, the egg ratio after 48 h was
significantly higher in the rotifer cultures inoculated with the bacterial
strains than in the axenic control cultures. These results suggested that it
would be worthwhile to scrutinise the possible nutritional role of bacteria
in the observed positive effect.
Attempts made to elucidate this
mode of action are presented in Chapter 7 (Feasibility to detect enzymatic
activity in the intestinal tract of rotifers Brachionus plicatilis after
visualisation of bacterial grazing by means of microscopic analysis of
stained rotifers). It was demonstrated that the bacterial mixture, provided
as only food source, could not sustain a normal rotifer population growth.
Nevertheless, as it can be assumed that no other microorganisms could
influence the effect of the inoculated bacterial strain in the monoxenic
cultures, the nutritional effect of the bacteria was thought to be at level
of the produced metabolites and enzymes. Microscopic pictures of stained
rotifers, cultured under these monoxenic conditions, indicated that the
corona and the intestinal tract of the rotifers was colonised with active
bacterial cells. Therefore, this observation can support the idea that the
enzymatic activity or the production of limiting substances could be at the
basis of the observed positive effect. By means of a biochemical test
(APIZYM), the enzymatic profile of the bacterial strains was documented.
These tests revealed that the bacterial strain which exhibit a positive
effect on the asexual reproduction of the rotifers, showed an increased and
more diverse enzymatic profile compared tot the other bacterial isolates.
Some of the bacterial exoenzymes, such as glucosamindase and glucosidase
were produced in higher amounts by these putative probiotic strains.
Chapter 8 (The use of a
multispecies probiotic mixture in a rotifer culture system) describes the in
vivo evaluation of the probiotic effect of the five selected bacterial
strains which was done by pre-emptively colonising the culture water of the
rotifers and the feed solution with selected bacterial strains. Because of
the complicated nature of microbial management, all the in vivo
experiments were performed with a mixture of five selected bacterial
strains. All the rotifer cultures inoculated with the probiotic solution
yielded a higher or an equal rotifer population growth. Yet, the beneficial
properties of the bacterial mixture were not exhibited consistently.
Different formulation of the
probiotic mixture were considered as an application for rotifer batch
cultures to produce rotifers of a much more consistent quality and quantity.
The immobilisation of the different bacterial strains in alginate beads and
Lentikats did not have the same effect as the fresh bacterial cultures. Yet,
higher rotifer densities were obtained when alginate beads with entrapped
bacteria were used compared to alginate beads without entrapped bacteria.
These finding endorse the hypothesis that the observed probiotic effect is
caused by the production of exoenzymes and/or extracellular products.
In Chapter 9 (A nitrifying culture
(ABIL) used as probiotic supplement in rotifer batch cultures and as starter
for marine nitrifying biofilters) a nitrifying inoculum (ABIL) was used in
batch cultures as probiotic mixture. This bacterial mixture was able to
improve the culture conditions consistently, resulting in significant higher
rotifer densities (50-150 higher, p<0.05). For
similar reasons, the nitrifiers were subsequently examined for their
capacity to enhance the start-up of biofilters, commonly installed in
aquaculture rearing tanks. Of the
different carrier materials used in these biofilters, i.e. CaCO3,
gravel and a PVC matrix (Bionet), CaCO3 gave by far the best
results. It was furthermore demonstrated that the biofilter removed part of
the ammonium by means of stripping of ammonia and part by nitrification. In
a third set of experiments, effectively nitrifying biofilter systems were
connected to rotifer culture tanks and thus operated over a period of up to
ten days. It was demonstrated that the ABIL started CaCO3- based
biofilters allowed excellent rotifer growth reaching rotifer densities up to
5500 rotifers per mL. Moreover, a new system in which the ABIL culture
together with hollow fibres was used to remove ammonium was developed and
demonstrated to be effective for supporting rotifer growth. Interestingly,
this approach yielded, in contrast to the conventional biofilter systems,
nitrate as endproduct
of the nitrification. Overall, the use of the dense nitrifying culture
either to seed batch cultures, conventional biofilters or hollow fibre
bioreactor systems in support of rotifer cultures were demonstrated to be
effective for improving the water quality.
It has been shown in this research that the bacterial
populations have an influence on the output of rotifer cultures. Moreover,
the use of probiotics as microbial control agents in rotifer cultures could
give rise to better and more
consist rotifer culture performances.