Optimization Of Bacillus Sonorensis BP-2 Fermentation Processes For The Production Of Activity Component Against Blue Mould Disease Citrus

In this paper, the fermentation processes of Bacillus sonorensis BP-2 were optimized in shake-flask and 15-L fermenter, for producing the activity component against blue mould disease citrus.Fisrstly, the fermentation conditions, seed medium and fermentation were optimized in shak-flask. The results in shake-flask fermentation revealed that 30 mL of a medium volume in 250 mL shake-flask,240 rpm of revolution,6.0-8.0 of an initial medium pH and 5% of an inoculum volume were the optimal fermentation conditions for Bacillus sonorensis BP-2. According to the orthogonal design of L9(43), the optimal seed medium for Bacillus sonorensis BP-2 was composed of (g/L):glucose,50; peptone,15; (NH4)2SO4, 13; KH2PO4,2.0; MgSO4,3.0; MnCl2,1.0. Based on the central composite design and response surface analysis, the final composition of the optimal medium for Bacillus sonorensis BP-2 fermentation was as follow (g/L):glucose,50; peptone,21.29; (NH4)2SO4, 8.7; KH2PO4,2.0; MgSO4,3.0; MnCl2,1.0; NaCl 5.0.Secondly, the mode of shake-flask fed-bach fermentation was investigated in detail. The feeding strategy of carbon source showed:when 2.0-2.5 g/l00mL of total sugar was maintained during 48-96 h of fermentation, the relative titer of activity component was 783.03 mg/L, which was 64.34% higher than that (476.48 mg/L) obtained under shake-flask bach fermentation. Based on this fact, the feeding strategy of nitrogen source was implemented, and it was discovered that NH4+ feeding could not only satisfy the demand for cell growth but also avoid its inhibition on the biosynthesis of activity component. When 80 mmol/L of NH4+ was contained in fermentation medium, and the additional 80 mmol/L of NH4+ was fed at 24,28,32 and 36 h, the final cell growth (OD600) and relative titer of activity component were 32.39 and 980.2 mg/L, respectively.Finally, the fermentation control strategies in 15-L fermenter were performed. The results of pH control strategy during the fed-batch fermentation showed that the optimum pH was 6.80-7.00, and the final cell growth (DCW) and relative titer of activity component were increased from 18.07 g/L and 517.36 mg/L (obtained in batch fermentation in 15-L fermenter) to 31.85 g/L and 2142.18 mg/L, respectively. The DO control strategy was then invesigated, and the results showed that two-stage DO control (0-60:20% of DO; 60-end: 10% of DO) was superior to one-stage of 10% or 20% of DO control. The final relative titer of activity component was 2357.72 mg/L, which was 10.06% higher than that (2142.18 mg/L) obtained under one-stage of 10% of DO control. The feeding strategy of was also studied in 15-L fermenter. When the NH4+ concentration in fermentation medium was decreased from 140 mmol/L to 80 mmol/L, and per 10 mmol/L of NR4+ was fed at 18,24,30,36,42,48,54 and 60 h, the final relative titer of activity component was achieved 2502.18 mg/L.