地衣芽孢杆菌BF-002高产芽孢的氮源流加工艺研究

Research on a Fed-batch of Nitrogen Source Fermentation Process to Improve the Spores of Bacillus Licheniformis BF-002

目的:提高地衣芽孢杆菌BF-002的芽孢产量,实现氮源流加过程的自动化控制,降低生产成本,为其他芽孢杆菌提高芽孢产率的研究提供一种思路。方法:通过摇瓶做单因素实验,筛选最佳温度和碳氮源,在此基础上进行5 L发酵罐实验。初始添加不同浓度的氮源,探索芽孢形成与氮源的关系。提出相对氨基氮的概念,通过恒速补料、间歇补料和基于尾气CO_(2)浓度反馈流加三个策略控制相对氨基氮浓度水平。采用Python语言编写计算机控制程序,实现基于尾气CO_(2)浓度反馈流加策略的自动化控制。结果:摇瓶筛选最佳温度及碳氮源分别为:37℃、葡萄糖、鱼粉蛋白胨、豆粕。上罐结果表明,相对氨基氮浓度越低芽孢率越高,采用基于尾气CO_(2)浓度反馈流加能将相对氨基氮控制在8.42 mg/OD_(600)水平,芽孢量可达4.25×10^(9) cfu/mL。利用计算机程序自动控制低价氮源氯化铵的流加,可以使芽孢量达到1.87×10^(10) cfu/mL,是前期最优批次的4.4倍,同时降低原料成本。结论:将相对氨基氮浓度控制在适宜水平可以得到芽孢量较高的培养液,自动流加氯化铵策略能降低生产成本并实现自动化控制,为研究芽孢杆菌产孢提供一种思路。

Objective:Base on spore products of Bacillus licheniformis can be produced comparatively easily and incorporated into everyday foods/feeds with high stability retaining their viability.The spore production with a good sporulation efficiency is a key step in bio-products development.It is important to study a production method that can reduce manufacturing costs and realize automatic control to enhance the productivity of Bacillus licheniformis and provide a way for other bacilli to heighten the productivity of spores.Methods:The optimal culture temperature,carbon sources and nitrogen sources of Bacillus licheniformis BF-002 were determined by the single factor experiment in shake flasks.On this basis,the experiment of the 5 L fermentor was carried out.It explored the relationship between spore formation and nitrogen sources that different concentrations of nitrogen sources were added initially.It controlled relative amino nitrogen level by fed-batch,constant speed of fed-batch and fed-batch on tail gas CO_(2) concentration feedback.The computer control program was written in Python language to realize automatic control.Results:The optimal culture temperature,carbon source and nitrogen source determined by the single factor experiment were:37℃,glucose and peptone from fish:soybean meal=1∶1.The results showed that the lower the relative amino nitrogen was,the higher the spore rate was.The relative amino nitrogen was controlled at the level of 8.42 mg/OD_(600) by fed-batch on tail gas CO_(2) concentration feedback,and the number of spores can reach 4.25×10^(9) cfu/mL.The number of spores can get 1.87×10^(10) cfu/mL by using an automatic fed-batch of ammonium chloride,4.4 times the optimal batch in the early stage.Conclusion:There was a correlation between spore rate and relative amino nitrogen.The strategy of using an automatic fed-batch of ammonium chloride can reduce the production cost and realize automatic control,which provides an idea for the study of Bacillus.