中国生物工程杂志

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 CO₂ 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 / OD₆₀₀ by fed-batch on tail gas CO₂ concentration feedback, and the number of spores can reach 4.25×10 ⁹ cfu/ mL. The number of spores can get 1.87×10¹⁰ 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.