Fed-batch cultivation was performed at 37 °C for 24 h in a 3-L bioreactor (Infors HT, Bottmingen, Switzerland) [30 (link)] containing 1.2 L of optimized medium. Seed culture (100 mL LB medium containing 30 μg/mL kanamycin) was cultivated in 500-mL shake flasks and grown at 37 °C for 8 h with shaking at 200 rpm before inoculation into the bioreactor.
Dissolved oxygen (DO) and temperature were controlled at 30% and 33 °C, respectively, and phosphoric acid or ammonia solution was added, as needed, to maintain the pH at 7.0. DO, pH, temperature, and flow rates (200–700 rpm) were regulated automatically by the Infors fermentation device (Infors HT). Upon reduction of the carbon source and increase in DO in the primary medium, the feed medium was added to maintain the B. subtilis growth rate, thereby initiating the exponential-feeding phase of fed-batch cultivation. After fermentation the quantity of SPase was determined by scanning the area of each band on reduced SDS-PAGE gels, and then calculating with Image-Master TotalLab software (Amersham Biosciences) using purified SPase as a reference.
Dissolved oxygen (DO) and temperature were controlled at 30% and 33 °C, respectively, and phosphoric acid or ammonia solution was added, as needed, to maintain the pH at 7.0. DO, pH, temperature, and flow rates (200–700 rpm) were regulated automatically by the Infors fermentation device (Infors HT). Upon reduction of the carbon source and increase in DO in the primary medium, the feed medium was added to maintain the B. subtilis growth rate, thereby initiating the exponential-feeding phase of fed-batch cultivation. After fermentation the quantity of SPase was determined by scanning the area of each band on reduced SDS-PAGE gels, and then calculating with Image-Master TotalLab software (Amersham Biosciences) using purified SPase as a reference.