响应面法优化芽孢杆菌(Bacillussp.)A4的培养参数

Optimization for cultivation parameters of Bacillus sp. A4 using response surface methodology

为了提高芽孢杆菌(Bacillus sp.)A4的菌浓度,采用响应面法对其培养参数进行优化。以单因素实验为基础,筛选适合A4菌生长的碳源和氮源,采用Plackett-Burman实验方法确定影响菌浓度的显著因子;通过最陡爬坡实验逼近菌浓度最大值响应稳定区域;最后根据Box-Behnken实验确定显著因子的最佳水平,建立相关参数的回归方程,得到优化培养参数,并以摇瓶培养实验验证理论参数及方程的科学性。结果显示,最适碳源、氮源分别为玉米浆、大豆蛋白,且它们与温度均为显著影响A4菌浓度的因子,其最适水平分别为20.06 g·L^(-1)、13.29 g·L^(-1)、30.26℃。采用优化后的参数进行摇瓶培养(24 h),A4菌浓度达1.19×109CFU·mL^(-1),与理论计算值(1.24×109CFU·mL^(-1))无显著差异(P>0.05),但显著高于未优化菌株培养浓度(2.69×107CFU·mL^(-1))(P<0.01)。可见,运用响应面法优化芽孢杆菌A4的培养参数,能显著提高A4菌的菌浓度。

In order to increase the concentration of Bacillus sp. Strain A4, we used response surface methodology to optimize the cultivation conditions. We screened the most appropriate carbon source and nitrogen source via single factor experiment. Then, we ob- tained the significant factors for the growth of Strain A4 through Plackett-Burman design experiment. The highest concentration area was determined by the steepest climbing experiment. The optimal cultivation conditions and the correlation equation were analyzed by Box- Behnken central composite design experiment, and were verified by the real cultivation experiment. The results show that corn steep liquor and soybean protein were the optimal carbon and nitrogen sources. The concentration of soybean protein, corn steep liquor and temperature had remarkable effects on the growth of Strain A4, whose optimal values were 20. 06μL-1 , 13.29 g·L-land 30. 26 ~C , respectively. The concentration of Strain A4 reached 1.19 × 109 CFU .mL-1 after being cultured for 24 h, which had no significant difference with the predicted value （1.24 ×109 CFU·mL-1, P 〉 0. 05 ） according to the flask culture experiments, but was higher than that without optimization very significantly （2. 69 ×107CFU·mL-1, P 〈 0. 01 ）. The results indicate that response surface meth- odology can improve concentration of Strain A4.