Geert Rombaut

Probiotic Bacteria as Biological Control Agents in Aquaculture

SUMMARY There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.

Advancement of rotifer culture and manipulation techniques in Europe

Abstract Since no artificial feed formulation for first feeding of marine larval fish has been developed yet, live prey feeding remains essential in commercial marine hatchery operations. Because cultured rotifers are relatively poor in eicosapentaenoic acid (EPA: 20:5n-3) and docosahexaenoic acid (DHA: 22:6n-3), it is essential and therefore common practice to enrich these live prey with emulsions of marine oils. The short-term exposure to oil emulsions results in lipid-encapsulated rotifers with high EPA and DHA levels. However, these rotifers are prone to fast losses of their gut content and show a distortion in their protein/lipid balance. Rather than submerging rotifers in oil emulsions, it is often preferred to use formulated culture diets when medium to low enrichment values are needed in live prey. The use of these diets contributes not only to the filling of the gut of the rotifers with nutrients, it generally creates a more stable entire body composition which is important especially when rotifers are not consumed immediately by the larvae. New culture techniques for rotifers, such as closed recirculation systems, are offering new possibilities for continuous supplies of high quality rotifers at 10 times higher densities than in batch cultures. The production increase in these systems is explained by the better water quality obtained by the introduction of protein skimmers, ozone treatment, and biological filtration. Although disinfection of rotifers remains a bottleneck, it has been observed that rotifer populations cultured at high densities are not prone to higher bacterial infestation. Also, the problem of unexplained mortalities in batch cultures seems to be partly solved by the introduction of recirculation systems or by bacterial management (introduction of probionts), which allow more reliable rotifer production.

Selection of bacteria enhancing the growth rate of axenically hatched rotifers (Brachionus plicatilis).

Abstract The effect of bacterial strains on the growth rate of rotifers, Brachionus plicatilis , was determined under monoxenic conditions. The first objective was to obtain sterile rotifer cultures starting from rotifer resting eggs using merthiolate or glutaraldehyde as disinfectant. Sterile rotifer cultures were obtained, without affecting the hatching ability of the resting eggs, when 0.05 μl/l glutaraldehyde was used. This disinfection procedure was used to examine the effect of 20 bacterial strains, isolated from well-performing live-feed production systems, on the population growth rate of rotifers cultured under monoxenic conditions. Five out of the 20 bacterial strains tested were able to improve significantly the asexual reproduction of rotifers. The population growth rate ( μ pop ) of rotifer cultures treated with GR 12 and GR11 (respectively 0.664±0.043 and 0.622±0.062) was significantly higher than the μ pop of the control treatment (0.512±0.101). Overall, the egg ratio after 48 h was significantly higher in the cultures inoculated with the bacterial strains than in the axenic control treatment. The results show that it is possible to control the microbial community in rotifer cultures started from disinfected resting eggs by adding bacterial strains which have a positive effect on the population growth rate.

Monitoring of the evolving diversity of the microbial community present in rotifer cultures

Abstract The genetic fingerprint of the microbiota in the culture water of two different rotifer culture systems (batch versus recirculation) was obtained by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The genetic profile of the bacterial community present in the culture water of a batch culture system changed daily due to shifts occurring at the level of the dominance of bands. By means of statistical tools, it was possible to distinguish three different periods during the development of the microbiota in the recirculation experiment, each corresponding to a typical period during the production of rotifers. Overall, it was obvious that the microbiota in such a recirculation system was less susceptible for variation relative to the microbiota in a batch system. However, some shifts in the genetic profile were observed when technical problems occurred resulting in a reduced water quality or performance of the biofilter. Characterisation of the microbial community present in the recirculation system indicated that the sequences of typical bands showed the highest level of identity to the sequences from the bacterial strain MMB-1 T (representing a species in the genus Marinomonas , Marinomonas mediterranea sp.), M. vaga and Pseudoalteromonas haloplanktis spp. tetraodonis strain IAM 14160 (98%).

Modelling the color development in Biolog microtiter plates by the Gompertz function.

Summary The color development curves obtained from multiple readings of Biolog GN microtiter plates over a long incubation time were fitted to the Gompertz function. This yielded — for each sole-carbon source — the incubation time independent and biologically relevant Gompertz parameters A (maximal extent of color development), μ M (specific color development rate) and λ (lagtime). To evaluate the applicability of the model, the coefficients of determination, the residuals, the biological significance of the parameters, the stability of the parameter estimates and the predicting power of the model were determined. For the pure strains Vibrio alginolyticus and Pseudomonas fluorescens the model passed all the criteria. For the model community consisting of a mixture of 9 strains, all criteria were met for almost all the oxidized carbon sources. However, a high amount of measuring points in the color development curve is necessary (preferably >20) and the reading frequency needs to be the highest in the lagphase and the exponential phase to ensure a high stability and predictability of the estimates of μ M and λ, especially when the color develops very rapidly in the wells. For a few carbon sources the Gompertz model was not appropriate, as the color development curve showed clearly 2 tiers. However, it is substantiated that the second tier may be eliminated from the time series, which allowed the model again to pass all the criteria. Finally, comparison of Biolog fingerprints of environmental samples was made with principal component analysis of the estimated Gompertz parameters for each carbon source. It is shown that this approach is practicable and may yield consistent results for environmental microbial community analysis.