Spores of Bacillus subtilis are being used as probiotics and competitive exclusion agents for animal consumption. Commercial production media often include relatively expensive components of animal origin that are a potential source for the presence of adventious agents, therefore undesirable for use in production scale. In this study a new animal-free component, chemically defined medium, was tested for B. subtilis spore production. Medium composition was optimized with respect to vitamin composition, carbon, nitrogen and calcium concentrations. A fed-batch bioprocess was developed, being the effect on sporulation of the carbon to nitrogen ratio at the end of the exponential growth phase studied. The developed strategy consisted of an initial and a final batch phase and an intermediate fed-batch phase with the addition of a feeding solution containing glucose and calcium and the addition of a feeding solution of ammonium sulphate, using an exponential and a constant feeding profile, respectively. Using the fed-batch strategy, it was possible to achieve a maximum spore production of 3.6 × 1010 spores/mL, corresponding to a 5 folds increase when compared to the preliminary batch experiments.
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