绿僵菌IMI330189液体发酵动力学研究
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摘要
金龟子绿僵菌(Metarhizium anisopliae) IMI330189菌株对蝗虫具有很好的防治效果，已经在南非沙漠蝗虫和我国草原蝗虫的防治上得到了推广应用。本论文主要以189菌株为研究对象，对其液体发酵条件和发酵培养基进行了优化，并进行了液体发酵动力学研究。研究结果如下：
装液量200mL/1000mL，转速200r/min，27℃条件下，一级种菌龄72h，发酵培养85h，初始pH为6.5，接种量2.5%为最优摇瓶发酵条件。
应用响应面（RSM）法对189菌株液体发酵培养基进行了优化。Plackett-Burman设计从6个因素中筛选出了对菌丝体生物量有显著影响的3个因素：蔗糖、酵母浸粉和FeCl3·6H2O；最陡爬坡试验确定了中心组合试验的最大响应区域；通过中心组合设计和响应面分析确定最佳浓度。优化培养基组分为：蔗糖44.394g/L，酵母浸粉17.104g/L，氯化铁0.206g/L，磷酸氢二钾6g/L，硫酸镁1.5g/L，氯化钙0.4g/L。在此培养基中，生物量干重可达到25.028g/L，比优化前提高了63%。
建立了189菌株的菌体生长、糖消耗和氮消耗动力学模型，并运用Oringn7.5软件拟合求解出各模型参数。结果表明，模型拟合和实验数据具有良好的适应性，拟合的模型能较好的反映189菌株的液体发酵过程。分析了发酵过程中的比速率变化规律，总糖比消耗速率在发酵9.6h达到最大值0.246h-1；总氮的最大比消耗速率为0.007h-1，出现在发酵10.3h时；而菌丝体比生长速率在发酵培养22.8h才达到最大值，为0.084h-1。总糖的得率系数是随着发酵时间的延长先逐渐升高再降低，在发酵培养39.8h达到最高值0.861g/g。
利用优化培养基进行液体发酵培养，采用不同发酵时间的菌液对固体发酵载体接种进行固体发酵，将液体发酵与固体发酵产孢相结合，分析固体发酵产孢量与液体发酵终点时菌体及发酵液的状态之间的关系，确定189菌株液体发酵终点的参数为：培养时间51~75h；菌体处于指数增长中后期，生物量达到15~20g/L；发酵液为淡褐色，粘稠；pH值为5左右，处于稳定期而没有开始上升。
Metarhizium anisopliae strain IMI330189is of high efficiency in locust control and has been usedin locust control in South Africa and China's grassland. In this paper, the fermentation medium and theliquid fermentation conditions of Metarhizium anisopliae strain IMI330189were optimized, and theliquid fermentation kinetics were studied. The results are as follows:
Under the condition of liquid volume200mL/1000mL flask,200r/min,27°C, the optimized shakeflask fermentation conditions are as follows: the fermentation time of seed is72h, fermentation time is85h, the initial pH is6~6.5, and the inoculum is2.5%.
Liquid fermentation medium for189strain was optimized by response surface method (RSM).Firstly, three factors which have a significant impact on the biomass had been screened out from sixmedium composition by the use of Plackett-Burman design: sugar, yeast extract and FeCl36H2O;secondly, the response region of central composite design was determined by the steepest ascent test;Finally, the optimal concentration of the three significant factors were determined by the use of centralcomposite design and response surface analysis. The optimized medium composition is: sucrose44.394g/L, yeast extract17.104g/L, FeCl3·6H2O0.206g/L, K2HPO46g/L, MgSO4·7H2O1.5g/L, andCaCl20.4g/L, respectively. In the optimized medium, the biomass of Metarhizium anisopliae reached25.028g/L,63%higher than that in the initial medium.
Kinetic models of biomass growth, suger consumption and nitrogen consumption were established,and the parameters of the models were determined by Origin7.5software. The results showed that themodel simulation matched well with the experimental observations, the fitted models basically reflectthe liquid fermentation process of189strain. The change of specific rate during the fermentation processwas analyzed. The maximum specific growth rate was found to be0.084h-1at22.8h, specific total sugarconsumption rate0.246h-1at9.6h and specific total nitrogen consumption rate0.007h-1at10.3h. Themaximum yield coefficient of biomass from total sugar was0.861g/g at39.8h.
Finally, the Metarhizium anisopliae was cultivated using the optimized liquid fermentation medium,then the solid fermentation carrier were inoculated with the broth in different fermentation age for solidfermentation, linked liquid fermentation and solid fermentation of Metarhizium anisopliae. Therelationship between solid state fermentation sporulation and the state of fermentation broth at the end ofliquid fermentation was analyzed, and draw the conclution that the following parameters should beconsidered to deside the end of liquid fermentation: culture time51~75h; cell in the middle and lateperiod of exponential growth and the biomass have reach15~20g/L; the fermentation broth is lightbrown and sticky; pH value is about5and stable and has not start to rise.
装液量200mL/1000mL，转速200r/min，27℃条件下，一级种菌龄72h，发酵培养85h，初始pH为6.5，接种量2.5%为最优摇瓶发酵条件。
应用响应面（RSM）法对189菌株液体发酵培养基进行了优化。Plackett-Burman设计从6个因素中筛选出了对菌丝体生物量有显著影响的3个因素：蔗糖、酵母浸粉和FeCl3·6H2O；最陡爬坡试验确定了中心组合试验的最大响应区域；通过中心组合设计和响应面分析确定最佳浓度。优化培养基组分为：蔗糖44.394g/L，酵母浸粉17.104g/L，氯化铁0.206g/L，磷酸氢二钾6g/L，硫酸镁1.5g/L，氯化钙0.4g/L。在此培养基中，生物量干重可达到25.028g/L，比优化前提高了63%。
建立了189菌株的菌体生长、糖消耗和氮消耗动力学模型，并运用Oringn7.5软件拟合求解出各模型参数。结果表明，模型拟合和实验数据具有良好的适应性，拟合的模型能较好的反映189菌株的液体发酵过程。分析了发酵过程中的比速率变化规律，总糖比消耗速率在发酵9.6h达到最大值0.246h-1；总氮的最大比消耗速率为0.007h-1，出现在发酵10.3h时；而菌丝体比生长速率在发酵培养22.8h才达到最大值，为0.084h-1。总糖的得率系数是随着发酵时间的延长先逐渐升高再降低，在发酵培养39.8h达到最高值0.861g/g。
利用优化培养基进行液体发酵培养，采用不同发酵时间的菌液对固体发酵载体接种进行固体发酵，将液体发酵与固体发酵产孢相结合，分析固体发酵产孢量与液体发酵终点时菌体及发酵液的状态之间的关系，确定189菌株液体发酵终点的参数为：培养时间51~75h；菌体处于指数增长中后期，生物量达到15~20g/L；发酵液为淡褐色，粘稠；pH值为5左右，处于稳定期而没有开始上升。
Metarhizium anisopliae strain IMI330189is of high efficiency in locust control and has been usedin locust control in South Africa and China's grassland. In this paper, the fermentation medium and theliquid fermentation conditions of Metarhizium anisopliae strain IMI330189were optimized, and theliquid fermentation kinetics were studied. The results are as follows:
Under the condition of liquid volume200mL/1000mL flask,200r/min,27°C, the optimized shakeflask fermentation conditions are as follows: the fermentation time of seed is72h, fermentation time is85h, the initial pH is6~6.5, and the inoculum is2.5%.
Liquid fermentation medium for189strain was optimized by response surface method (RSM).Firstly, three factors which have a significant impact on the biomass had been screened out from sixmedium composition by the use of Plackett-Burman design: sugar, yeast extract and FeCl36H2O;secondly, the response region of central composite design was determined by the steepest ascent test;Finally, the optimal concentration of the three significant factors were determined by the use of centralcomposite design and response surface analysis. The optimized medium composition is: sucrose44.394g/L, yeast extract17.104g/L, FeCl3·6H2O0.206g/L, K2HPO46g/L, MgSO4·7H2O1.5g/L, andCaCl20.4g/L, respectively. In the optimized medium, the biomass of Metarhizium anisopliae reached25.028g/L,63%higher than that in the initial medium.
Kinetic models of biomass growth, suger consumption and nitrogen consumption were established,and the parameters of the models were determined by Origin7.5software. The results showed that themodel simulation matched well with the experimental observations, the fitted models basically reflectthe liquid fermentation process of189strain. The change of specific rate during the fermentation processwas analyzed. The maximum specific growth rate was found to be0.084h-1at22.8h, specific total sugarconsumption rate0.246h-1at9.6h and specific total nitrogen consumption rate0.007h-1at10.3h. Themaximum yield coefficient of biomass from total sugar was0.861g/g at39.8h.
Finally, the Metarhizium anisopliae was cultivated using the optimized liquid fermentation medium,then the solid fermentation carrier were inoculated with the broth in different fermentation age for solidfermentation, linked liquid fermentation and solid fermentation of Metarhizium anisopliae. Therelationship between solid state fermentation sporulation and the state of fermentation broth at the end ofliquid fermentation was analyzed, and draw the conclution that the following parameters should beconsidered to deside the end of liquid fermentation: culture time51~75h; cell in the middle and lateperiod of exponential growth and the biomass have reach15~20g/L; the fermentation broth is lightbrown and sticky; pH value is about5and stable and has not start to rise.
引文
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