Optimization of Sporulation Conditions of Biocontrol Bacteria B579 by Two-step Control Strategy

两步控制策略优化生防菌B579芽孢生成条件(英文)

[Objective] For preparing the biopesticide of Bacillus subtilis B579 with high spore concentration,the sporulation conditions were optimized.[Method] Two-step fermentation control strategy was used,in which,the first phase（0-10 h）was to improve cell growth,and the second phase（10-30 h）was to promote spore formation.Four factors including initial glucose concentration,fermentation pH,temperature（in the second phase）and shaking speed（in the second phase）were optimized using the methods of single factor test and orthogonal experiment.[Result] The initial glucose concentration showed a significant effect on sporulation.The optimal conditions for the spore formation of B.subtilis B579 were as follows：initial glucose concentration 5 g/L,fermentation pH 7.0,the temperature for the first phase 37 ℃,and the shaking speed for the first phase 180 r/min,the temperature for the second phase 40 ℃,and the shaking speed for the second phase 200 r/min;in addition,the first phase was 10 h and the second phase of fermentation was conducted for 30 h.Under such conditions,the spore concentration and spore formation rate could reach 9.43×108 CFU/ml and 90.99%,respectively,which represented 6.70-fold and 2.43-fold increase compared with those before optimization.[Conclusion] The spores concentration of biocontrol agent was improved using two-step control strategy,which provided the basis for biopesticide production in large scale.

[目的]优化生防菌-枯草芽孢杆菌B579芽孢生成的发酵条件,提高芽孢浓度。[方法]采用两步控制策略,即发酵的第1阶段(菌体生长阶段,0～10h)促进细胞生长,发酵的第2阶段(菌体对数生长后期,芽孢生成阶段,10～30h)促进芽孢生成。分别考察了培养基初始葡萄糖浓度、发酵pH值、第2阶段摇床转速、第2阶段发酵温度对芽孢生成的影响。通过单因素试验确定其水平,在单因素实验基础上进行正交试验优化,确定获得高浓度芽孢的最佳发酵条件。[结果]培养基初始葡萄糖浓度是影响芽孢生成的显著因素,芽孢生成的最佳发酵条件是:培养基初始葡萄糖浓度5g/L,发酵pH7.0,第1阶段的发酵温度37℃,摇床转速180r/min,约10h后进入第2阶段,发酵温度提高到40℃,摇床转速调节为200r/min。发酵30h,最终获得的芽孢浓度为9.43×108CFU/ml,芽孢生成率为90.99%,分别是优化前的6.70倍和2.43倍。[结论]利用两步控制发酵策略可提高生防菌B579芽孢浓度,建立的发酵工艺为生防菌剂的大规模生产奠定了基础。