Optimizing submerged cultivation of Bacillus velezensis for endospore production and formulation

Considering the high efficacy of Bacillus velezensis AP-3 strain in controlling plant diseases, optimizing large-scale and cost-effective endospore production is crucial for providing accessible bioproducts to the agricultural sector. This study aimed to optimize the physicochemical and nutritional parameters of B. velezensis AP-3 submerged liquid fermentation using cost-effective media suitable for high-yield endospore production. A series of experiments was conducted to evaluate the effects of temperature, ultrasonic wave exposure, inoculum volume, aeration, and culture medium composition on sporulation. Additionally, wettable powder formulations were developed using corn-starch and potato-starch as carriers, supplemented with soybean lecithin. These formulations were assessed for suspensibility and shelf life at room temperature and at 40 °C. Among the physical variables, temperature had the strongest impact on fermentation performance, with endospore production ranging from 4.17 × 10⁷ CFU mL⁻¹ at 22 °C to 7.30 × 10⁸ CFU mL⁻¹ at 34 °C in chemically defined culture medium (T1). Culture medium composition and nutrient sources significantly influenced sporulation. The highest yields were observed in the following media: molasses+cottonseed flour (T8), autoclaved chemically defined culture medium (T1) modified by Vilanova (T6), non-autoclaved chemically defined culture medium (T1) modified by Vilanova (T7), T1 plus sucrose+protein hydrolysate+salts (T11), molasses+protein hydrolysate (T12), molasses+isolated soy protein+yeast extract (T13), and T1 medium modified by Vilanova supplemented with protein hydrolysate (T14). After 72 h of fermentation, these media resulted in yields of 2.32, 2.28, 2.03, 1.17, 1.01, 1.15, and 1.08 × 10⁹ CFU mL⁻¹, respectively. The bio-formulations exhibited a shelf life exceeding 3 years at room temperature.