Elucidation of Bacillus subtilis KATMIRA 1933 Potential for Spore Production in Submerged Fermentation of Plant Raw Materials

In this study, the effects of several key factors to increase spore production by Bacillus subtilis subsp. KATMIRA 1933 were evaluated in shake flask experiments. In a synthetic medium, glucose concentration played a crucial role in the expression of bacilli sporulation capacity. In particular, maximum spore yield (2.3 × 10⁹ spores/mL) was achieved at low glucose concentration (2 g/L), and further gradual increase of the carbon source content in the medium caused a decrease in sporulation capacity. Substitution of glucose with several inexpensive lignocellulosic materials was found to be a reasonable way to achieve high cell density and sporulation. Of the materials tested, milled mandarin peels at a concentration of 40 g/L served as the best growth substrate. In these conditions, bacilli secreted sufficient levels of glycosyl hydrolases, providing slow hydrolysis of the mandarin peel's polysaccharides to metabolizable sugars, providing the bacterial culture with an adequate carbon and energy source. Among nitrogen sources tested, peptone was found to favor spore production. Moreover, it was shown that cheese and cottage cheese whey usage, instead of distilled water, significantly increases spore formation. After optimization of the nutrient medium in the shake flask experiments, the technical feasibility of large-scale spore production by B. subtilis KATMIRA 1933 was confirmed in a laboratory fermenter. The spore yield (7 × 10¹⁰ spores/mL) obtained using a bioreactor was higher than those previously reported.