一株具有噬菌体抗性的芽孢杆菌BS-2的鉴定及葡萄糖流加工艺优化

Identification of a Bacillus Strain BS-2 with Anti-phages and Optimization of Glucose Feeding Strategy

筛选噬菌体抗性菌株以解决枯草芽孢杆菌(Bacillus subtilis)生产过程中噬菌体污染问题并提高抗性菌株的发酵水平。采用双层平板法从异常发酵液中分离并纯化以枯草芽孢杆菌为宿主的噬菌体,利用纯化得到的噬菌体筛选实验室芽孢杆菌库以得到抗性菌株,结合16S rDNA和gyrA基因序列进行系统发育分析确定其分类地位,以分批发酵的生长曲线及葡萄糖含量曲线作为基础,对发酵培养基的初糖浓度及葡萄糖的流加方式进行优化提高筛选到的噬菌体抗性菌株的发酵水平。从异常发酵液中分离到噬菌体S01及S02并在实验室芽孢杆菌库中筛选到一株噬菌体无法侵染的芽孢杆菌BS-2,结合16S rDNA及gyrA基因序列的系统发育分析将BS-2鉴定为枯草芽孢杆菌,按照葡萄糖初始浓度15 g/L,葡萄糖浓度低于5 g/L时以2 g/L·h的速度流加葡萄糖,补加量10 g/L的流加补料方法,可以将发酵水平提高至2.43×1010 CFU/mL。枯草芽孢杆菌BS-2具有较好的噬菌体抗性且经过工艺优化可以达到较高发酵水平,有较强工业化应用潜力。

This work aims to screen anti-phage strain for solving the problem of phage infection in Bacillus subtilis production and increase fermentation of anti-phage strain.Double-layer-agar plating method was used to screen and purify phages with Bacillus subtilis as host from abnormal fermentation broth,and anti-phage strains were obtained by screening the Bacillus library in our laboratory using the purified anti-phages.Phylogenetic trees were constructed by 16 S rDNA and gyrA gene sequences analysis to identify the anti-phages strain.Based on glucose content curve and growth curve of anti-phages strain batch fermentation,initial glucose concentration in the medium and glucose feeding strategy were optimized for improving the fermentation of the screened anti-phage strains.As results,phages S01 and S02 were screened from the abnormal fermentation broth,and a strain Bacillus sp.BS-2,which was not infected by the phages,was screened from the Bacillus library in our laboratory.According to the 16 S rDNA and the phylogenetic trees of gene gyrA,the strain BS-2 was characterized as Bacillus subtilis.The biomass was up to 2.43×1010 CFU/mL,while suitable initial concentration of glucose was 15 g/L,and glucose was fed into the broth at the speed of 2 g/L·h when the glucose concentration in the broth was lower than 5 g/L,and the total supplying quantity was 10 g/L.B.subtilis BS-2 shows strong ability of anti-phages and high titer under optimized fermentation conditions,which has promising potential for largescale industrialization.