响应面法优化蓝藻溶藻菌CZBC1发酵培养工艺

Fermentation culture technique of cyanobacteria algae-lysing bacteria CZBC1 optimized by response surface method

【目的】优化蓝藻溶藻菌——蜡样芽孢杆菌CZBC1的发酵培养工艺,为蓝藻溶藻菌制剂的工业化发酵生产提供技术支持。【方法】通过单因素试验筛选适合蜡样芽孢杆菌CZBC1生长的最适碳源和氮源,在单因素试验的基础上,采用中心组合试验设计(CCD)确定关键因子的最佳数量水平,并以Desig-Expert 8.0.5进行回归分析,通过响应面分析获得CZBC1的最佳发酵培养工艺参数。【结果】蜡样芽孢杆菌CZBC1的最佳碳源、氮源分别为麸皮+糖蜜(1∶1)和酵母膏。以麸皮+糖蜜用量(A)、酵母膏用量(B)、pH(C)、培养时间(D)为因素变量,CZBC1芽孢数(lgN)为响应值,拟合得到二次多元回归方程为lgN=8.5019-0.2222A+0.0998B-0.1292C+0.3801D+0.2194AB-0.1133AC+0.1350AD+0.2049BC-0.0651BD+0.0638CD-0.1260A^2-0.3152B^2-0.0380C^2-0.2533D^2,其中,碳源(麸皮+糖蜜)用量与氮源(酵母膏)用量的交互作用、碳源用量与pH的交互作用、碳源用量与培养时间的交互作用、氮源用量与pH的交互作用对CZBC1芽孢数的影响均达极显著水平(P<0.01,下同)。响应面分析优化得到的蜡样芽孢杆菌CZBC1最佳发酵培养参数:麸皮+糖蜜6.84 g/L,酵母膏3.36 g/L,pH 6.50,培养时间42 h。此条件下,CZBC1芽孢数的实际值为5.75×10~8CFU/mL,与理论值(5.90×10~8CFU/mL)间无显著差异(P>0.05),但极显著高于优化前采用营养肉汤发酵培养的芽孢数(1.28×10~7CFU/mL)。【结论】采用响应面法优化得出蜡样芽孢杆菌CZBC1最佳发酵培养工艺能有效提高菌株的芽孢数量,且模型拟合效果较好,可用于指导蓝藻溶藻菌制剂的工业化发酵生产。

【Objective】The fermentation cultivation technique of cyanobacteria algae-lysing bacteria-Bacillus cereus CZBC1were optimized in order to provide technical support for the industrial fermentation production of algae-lysing bacteria agent.【Method】Based on single-factor experiment,the optimal carbon source and nitrogen source for the growth of B.cereus CZBC1were screened.Then,on this basis,the optimal value of key factors was determined by using central composite design(CCD).Regression analysis was conducted by Desig-Expert8.0.5,and the optimum fermentation cultivation parameters of CZBC1were obtained by response surface analysis.【Result】The results showed that the best carbon source and nitrogen source for B.cereus CZBC1were bran+molasses(1∶1)and yeast extract respectively.Amount of bran+molasses(A),amount of yeast extract(B),pH(C)and culture time(D)were set as factor variables,and spore Number of strain CZBC1(lgN)as response value,based on which the quadratic multiple regression equation was fitted as follows:lgN=8.5019-0.2222A+0.0998B-0.1292C+0.3801D+0.2194AB-0.1133AC+0.1350AD+0.2049BC-0.06513BD+0.06380CD-0.1260A2-0.3152B2-0.0380C2-0.2533D2.Among them,interaction between amounts of carbon source(bran+molasses)and nitrogen source(yeast extract),interaction between carbon source and pH,interaction between carbon source and culture time,and interaction between nitrogen source and pH had extremely significant effects on CZBC1spore number(P<0.01,the same below).The optimum fermentation culture parameters obtained by response surface analysis for B.cereus CZBC1were as follows:bran+molasses6.84g/L(mL/L),yeast extract3.36g/L,pH6.50,and42h culture time.Under such conditions,the actual spore number of CZBC1was5.75×108CFU/mL,which had no significant difference with the theoretical value(5.90×108CFU/mL)(P>0.05),but it was significantly higher than the spore number cultured by nutrient broth before optimization(1.28×107CFU/mL).【Conclusion】The optimum fermentation culture technique for B.cereus CZBC1optimized by response surface method can effectively improve the spore number.The model established shows sound fitting effects,and can be applied to guide the industrial fermentation production of cyanobacteria algae-lysing bacteria agent.