Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent.

AIMS: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. METHODS AND RESULTS: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0.96 h(-1)), whereas Aer. hydrophila concentration decreased by c. 70% in co-culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0.05) and iron (P < 0.01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0.30 g l(-1) h(-1)) and iron (0.60 mg l(-1) h(-1)) uptake rates were also significantly higher (P < 0.01) than the Aer. hydrophila glucose (0.14 g l(-1) h(-1)) and iron (0.43 mg l(-1) h(-1)) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. CONCLUSIONS: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.

Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish.

AIMS: To isolate, select and evaluate Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. METHODS AND RESULTS: Natural isolates obtained from mud sediment and Cyprinus carpio were purified and assessed in vitro for efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions. Based on suitability to predefined characteristics, the isolates B001, B002 and B003 were selected and evaluated in vitro in the presence of Aer. hydrophila and in a preliminary in vivo trial with C. carpio. The inhibitory effect on pathogen growth and the decrease in concentrations of waste ions was demonstrated. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, Bacillus cereus and Bacillus licheniformis, respectively. Isolate B002 did not contain the anthrax virulence plasmids pOX1, pOX2 or the B. cereus enterotoxin. CONCLUSIONS: Selected isolates effected synergistic reduction in pathogen load and the concentrations of waste ions in vitro and in vivo and are safe for use in ornamental aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: A new approach for assessment of biological agents was demonstrated and has yielded putative isolates for development into aquaculture products.

Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3).

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.