In Stirred Tank Reactor

The medium chosen for the stirred tank reactor experiments was the Landy medium at a pH value of 6.5. A slight modification was done by reducing the concentration of glutamic acid from 5 to 2 g⋅L^–1. A cheaper nitrogen source, ammonium sulfate, was added to balance correctly the medium without impact on mycosubtilin production. The Landy medium composition was modified as follows: glucose, 20 g⋅L^–1; glutamic acid, 2 g⋅L^–1, (NH₄)₂SO₄, 2.3 g⋅L^–1; yeast extract, 1 g⋅L^–1; K₂HPO₄, 1 g⋅L^–1; MgSO₄ 0.5 g⋅L^–1; KCl, 0.5 g⋅L^–1; CuSO₄, 1.6 mg⋅L^–1; Fe₂(SO₄)₃, 1.2 mg⋅L^–1; and MnSO₄, 0.4 mg⋅L^–1. For each inoculum and culture in a bioreactor, B. subtilis BBG116 and BBG125 cells were taken from a −80°C frozen stock, and two precultures were done in a modified Landy medium. Stirred tank reactors used in this study were Bioflo 3000 (New Brunswick, NJ, United States) and EZ-Control (Applikon Biotechnology B.V., Netherlands) with both a nominal maximal working volume of 5 L. The bioreactor, containing 3 L of culture medium, was inoculated with early logarithmic grown cells and prewashed with one volume of sterile physiological water after a 5-min and 3,000-g centrifugation. The initial value of OD at 600 nm comprised between 0.1 and 0.2 u⋅OD. The temperature was 30°C, and the pH was controlled at the value of 6.5 by adding KOH (3 M) or H₂SO₄ (3 M) solutions. In order to have a precise regulation and avoid the degradation of the foam by titrants solutions, these additions were directly carried out on the surface of the culture medium thanks to a flush tube. The same technique was used for the feeding of the fresh substrate, thus limiting the impact of the foam on the dispersion of the substrate. The dissolved oxygen (DO) concentration was controlled above 15% of the saturation thanks to the adaptive stirrer speed. Aeration of the broth was ensured with insufflating 0.22-μm sterilized air at 0.25 VVM. The purity of the culture was tested on Mossel agar plates (Serva, Heidelberg, Germany). Culture medium, waste, and collected foam samples were taken under sterile working conditions.