地衣芽孢杆菌BF-002高产芽孢的碳源/氮源浓度协同优化控制

Synergistic Optimization Control of Carbon Source/Nitrogen Source Concentration for High-yield Spores of Bacillus licheniformis BF-002

目的:探讨碳源和氮源浓度对地衣芽孢杆菌BF-002细胞生长和芽孢生成的协同调控作用,提高发酵液中的活细胞数量和芽孢数量。方法:在5 L发酵罐规模下,考察氮源浓度变化、碳源浓度变化对细胞生长和芽孢生成的影响规律,建立通过调节碳源浓度启动芽孢生成、促进营养体细胞向芽孢持续高效转化的方法,最终建立碳源/氮源协同流加控制策略,提高发酵结束时刻的活细胞和芽孢产量。结果表明:碳源充足、氮源浓度降低至临界水平(约0.5 g/L)或氮源浓度充足、碳源浓度降低至临界水平(约2.0 g/L),均可启动芽孢生成。协同调控碳源和氮源浓度,培养前24 h将葡萄糖和氨基氮浓度分别控制在10 g/L和1.5 g/L,24 h后将葡萄糖和氨基氮的浓度维持在2 g/L和0.5 g/L,持续至48 h,活细胞和芽孢数量分别达到2.88×1010 CFU/mL和2.56×1010 CFU/mL。与单独控制葡萄糖浓度的最优批次相比,芽孢数量提高2.91倍。结论:将对数生长期的碳源和氮源浓度同时控制在适宜水平,可以得到较高的活细胞量。之后,降低碳源和氮源浓度,将其维持在各自的临界水平,可以促进营养体细胞向芽孢持续高效转化。

Objective:To investigate the synergistic regulation of carbon and nitrogen sources on the cell growth and spore generation of Bacillus licheniformis BF-002,so as to increase the number of living cells and spores in fermentation broth.Methods:Under the scale of 5 L fermentor,the effects of nitrogen source concentration and carbon source concentration on cell growth and spore formation were investigated,and the method of starting spore formation and promoting the continuous and efficient transformation of vegetative cells into spores by adjusting carbon source concentration was established.Finally,the carbon source/nitrogen source synergistic feeding control strategy was established to improve the production of living cells and spores at the end of fermentation.The results showed that spore generation could be initiated when the concentration of carbon source decreased to the critical level(about 0.5 g/L)or when the concentration of carbon source decreased to the critical level(about 2.0 g/L).The concentrations of glucose and amino nitrogen were controlled at 10 g/L and 1.5 g/L,respectively before 24 h,and maintained at 2 g/L and 0.5 g/L after 24 h for 48 h.The number of living cells and spores reached 2.88×10^(10) CFU/mL and 2.56×10^(10) CFU/mL,respectively.Compared with the optimal batch with glucose concentration controlled alone,the number of spores increased 2.91 times.Conclusion:When the concentration of carbon source and nitrogen source in logarithmic growth period is controlled at an appropriate level,a higher amount of living cells can be obtained.Subsequently,by reducing the concentration of carbon source and nitrogen source and keeping them at their respective critical levels,we can promote the continuous and efficient transformation of vegetative cells into spores.