棉花黄萎病生防芽孢杆菌S12产芽孢培养基的响应面优化

Optimization of Culture Medium for Spore Production from a Biocontrol Spore Bacterium S12 against Cotton Verticillium Wilt By Using Response Surface Methodology

枯草芽孢杆菌(Bacillus subtilis)S12是分离自新疆棉田根际土壤的一株广谱抑菌菌株,尤其对棉花黄萎病大丽轮枝菌(Verticillium dahliae Kleb.)孢子萌发和菌丝生长具有较强的抑制作用。为了提高菌株S12发酵液中芽孢产量,对其发酵培养基组分及配比进行优化。首先通过单因素试验分别分析6种不同碳源、氮源和无机盐对菌株S12芽孢产量的影响,随后采用Plackett-Burman试验设计确定影响芽孢产量的主要因子,并利用Box-Behnken试验设计及响应面分析法优化确定主要影响因子的最佳配比。最终获得菌株S12产芽孢培养基的优化配比组合为:玉米粉1.30%、大豆蛋白胨1.51%、CaCO30.08%、NaH2PO4·2H2O0.2%、Na2HPO4·2H2O 0.4%。优化后芽孢产量达到7.46×108 mL-1,与初始培养基相比提高了387.58%。可见,通过优化培养基组分,提高发酵液中芽孢产量,降低生产原料成本,为其进一步生防菌剂的工业化生产奠定基础。

Bacillus subtilis S12 with a broad-spectrum antifungal activity was isolated from cotton rhizosphere soil of Xinjiang.The strain S12 had strong inhibition effect on mycelium growth and spore germination of Verticillium dahliae Kleb.In order to improve the spores yield of the strain S12,the medium composition was optimized.The effects of six kinds of carbon sources,nitrogen sources and inorganic salts on the yield of spores produced by strain S12 were studied by single factor analysis in order to gain the optimum carbon,nitrogen and inorganic salt.Plackett-Burman experimental design was used to determine the main factors which affected spore production.And Box-Behnken design and response surface analysis were used to further investigate the mutual interaction between the variables,and to identify optimal values that bring maximum spore yield.The final optimal culture medium was corn meal 1.30%,soybean tryptone 1.51%,CaCO3 0.08%,NaH2PO4·2H 2O 0.2%and Na2HPO4·2H2O 0.4%.Under this optimum condition,the spore quantity of strain S12 reached 7.46×10^8 mL^-1.And the yield of spores increased 387.58%,compared with its original medium.The spore number in the fermented liquid was improved by optimization of culture medium.At the same time,the material cost was reduced.This study laid the foundation for the industrialization of Bacillus subtilis S12 as a kind of biocontrol agent.