High-density spore production of a B. cereus aquaculture biological agent by nutrient supplementation

Previous studies have demonstrated the efficacy of our Bacillus cereus isolate (NRRL 100132) in reducing concentrations of nitrogenous wastes and inhibiting growth of fish pathogens. In vivo efficacy and tolerance to a range of physiological conditions in systems used to rear Cyprinus carpio make this isolate an excellent candidate for aquaculture applications. Production cost is an important consideration in development of commercially relevant biological products, and this study examines the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modeled to develop a tool for optimization of nutrient concentration in fermentation. An excellent fit of the model was confirmed in laboratory fermentation studies. A cost comparison revealed that production using liquid phytase and ultrafiltered-treated CSL was less expensive than spray-dried CSL and supported cultivation of B. cereus spores at densities higher than 1 x 10(10) CFU ml(-1).