A 100 mL shake flask containing 20 mL of complete chemically defined medium was inoculated with one cryovial of B. subtilis from the frozen stock. The seeded culture was incubated at 37˚C and 150 rpm on a rotary shaker (Innova 4330, New Brunswick Scientific Co. Inc., USA) for 28 hours to a final optical density (595 nm) of approximately 8.0. The cells were then used to inoculate the 2-L bioreactor at an inoculum size of 1% (v/v).