防治蓟马的球孢白僵菌SDDZ-9菌株液体发酵工艺优化

【目的】球孢白僵菌(Beauveria bassiana)作为一种重要的虫生真菌,已广泛应用于害虫生物防治,但其不成熟的规模化生产技术一直是限制球孢白僵菌广泛应用的主要问题之一。开展球孢白僵菌液体发酵工艺研究,提高液体发酵生物量,从而为固体发酵阶段提供优质的种子液,进而提高固体发酵的产孢量,为球孢白僵菌规模化生产提供参考。【方法】影响球孢白僵菌液体发酵的培养条件有温度、装样量、摇床转速、接种浓度和p H等,首先采用Plackett-Burman试验设计,筛选出影响球孢白僵菌菌株SDDZ-9的液体发酵的主要因子,然后运用最陡爬坡路径法,以试验值变化的梯度方向和各因素效应值的大小分别确定爬坡方向和变化步长,确定响应面的中心点,逼近最佳值区域,最后采用central composite design(CCD)响应曲面法,建立多元二次回归方程来拟合各因素与效应值之间的关系,并对影响发酵生物量的各因素及其交互作用进行响应面分析和评价,确定对西花蓟马(Frankliniella occidentalis)毒力较高的球孢白僵菌菌株SDDZ-9液体发酵的最优培养条件。【结果】培养温度、装样量、转速和浓度是影响发酵生物量的主要因子,一定范围内的p H变化对此菌株的液体发酵影响较小。在自然p H下,当培养温度27.08℃、装样49.72 m L(250 m L三角瓶)、摇床转速205.45 r/min、接种浓度1.122×107孢子/m L时,发酵48 h产生的生物量最大。在优化发酵条件下进行液固双相发酵试验,得到的球孢白僵菌液体发酵生物量为14.769 g·L-1,与模型预测生物量相符,固体发酵后产孢量为20.16 g·kg-1,含孢量1.84×1011孢子/g。【结论】在优化发酵工艺条件下进行发酵,可显著提高球孢白僵菌的产孢量,此液体发酵工艺为球孢白僵菌的规模化生产奠定了基础。

Optimization of Fermentation Process of Beauveria bassiana of SDDZ-9 Against Frankliniella occidentalis

【Objective】Entomopathogenic fungus Beauveria bassiana has been widely applied as an important biocontrol agent for controlling many insect pests, while the poorly large-scale production technology of B. bassiana is one of the key issues which may consequently restrict its wide application. Study on the liquid fermentation process research of B. bassiana can improve the liquid fermentation of biomass, thereby providing high-quality fermentation broth for solid liquid fermentation stage and improving spore production of solid fermentation. The study may serve as a basis for the large-scale production technology of B. bassiana. 【Method】The liquid fermentation of B. bassiana is affected by several factors, such as temperature, volume of liquid, rotate speed, initial conidia concentration, pH and so on. Firstly, Plackett-Burman (PB) design was used to screen the main factors which affected the liquid fermentation. Secondly, the method of the steepest ascent path was applied to identify the central point of response surface and approach the optimal region. The climbing direction and the change range were determined by gradient direction of experimental value changes and the effect value of each factor, respectively. Lastly, the central composite design (CCD) was adopted to identify the optimal liquid fermentation concentration by setting up a multiple quadratic regression equation, analyzing and evaluating the correlations among these factors which affect the mycelium mass of B. bassiana SDDZ-9, one of the most virulent strains against western flower thrips (Frankliniella occidentalis. 【Result】Temperature, volume of liquid, rotate speed and initial conidia concentration were the main factors on mycelium mass. The change of pH within certain limits had less influence on this strain of liquid fermentation. Without considering pH, when the fermentation conditions of temperature, volume of liquid, rotate speed and initial concentration were 27.08℃, 49.72 mL/250 mL of triangular flask, 205.45 r/min, and 1.122×10⁷ conidia/mL, the maximum mycelium mass was produced after 48 h of fermentation. Using the culture conditions to produce conidia, the mycelium mass obtained was about 14.769 g·L^-1, which was consistent with the predicted biomass. After the solid state fermentation, the yield achieved about 20.16 g·kg^-1 and 1.84×10¹¹ conidia/g.【Conclusion】The spore production of B. bassiana could be significantly improved under optimized fermentation conditions. The results may provide reference for large-scale liquid fermentation of B. bassiana SDDZ-9.