利用表型芯片技术筛选利于枯草芽胞杆菌Bacillus subtilis BAB-1生长与芽胞形成的营养物质

为获得枯草芽胞杆菌Bacillus subtilis BAB-1菌株的芽胞高效率生产技术,采用Biolog微生物细胞表型芯片(phenotype microarray,PM)技术分析了适合该菌株细胞生长的营养物质、渗透压和pH等生长环境条件;通过单因素试验分析了5种碳源及5种氮源对该菌株生长及芽胞形成的影响。结果表明,BAB-1菌株能够利用114种碳源、228种氮源、29种磷源及29种硫源作为其营养物质,其中52种碳源、66种氮源、3种硫源及1种磷源能够明显促进该菌株的细胞生长;BAB-1菌株对渗透压的忍受能力较强,在1%～10%氯化钠、3%～6%氯化钾、2%～5%硫酸钠、5%～20%乙二醇、1%～6%甲酸钠、2%～7%尿素、1%～12%乳酸钠、20～200 mmol/L磷酸钠、10～100 mmol/L硝酸钠、10～100 mmol/L硫酸铵中均能较好的生长;BAB-1菌株对pH的耐受范围较广,在4.5≤pH≤10.0条件下均能够生长。葡萄糖与氯化铵分别是最适宜BAB-1菌株生长的碳源与氮源,在不同培养时间其菌体总量均最高,48 h时该菌株的菌体总量分别达到6.90×10~8CFU/mL与6.87×10~8CFU/mL;培养12 h时,D-木糖、D-核糖、L-阿拉伯糖及熊果苷能够显著提高BAB-1菌株的芽胞形成率,其中L-阿拉伯糖与熊果苷的芽胞形成率较高,分别为61.94%与66.92%;培养12～36 h,L-脯氨酸、L-谷氨酰胺及L-天冬酰胺能够显著提高BAB-1菌株的芽胞形成率,芽胞形成率介于27.88%～82.37%之间。表明表型芯片技术可以应用于枯草芽胞杆菌生长及芽胞形成营养物质的高通量分析及培养工艺的优化中。

Using phenotype microarray to screen the nutrient factors contributing to the growth and sporulation in Bacillus subtilis BAB-1

To obtain high sporulation efficiency of Bacillus subtilis strain BAB-1, the suitable culture conditions including nutrient, osmotic pressure and pH value were analyzed by using biolog phenotype microarray (PM) technology. The effects of five carbon and five nitrogen substrates on growth and sporulation of strain BAB-1 were evaluated with single factor test. The results showed that strain BAB-1 was able to metabolize 114 carbon substrates, 228 nitrogen substrates, 29 phosphorus substrates and 29 sulfur substrates.Amongthem,52carbon,66nitrogen,three sulfur and one phosphorus sources could obviously promote the growth of strain BAB-1. It had strong tolerance to osmolytes with up to 1%-10% sodium chloride, 3%-6% potassium chloride, 2%-5% sodium sulphate, 5%-20% ethylene glycol, 1%-6% sodium formate,2%-7%urea,1%-12%sodiumlactate,20-200mmol/Lsodiumphosphate,10-100mmol/L sodium nitrate and 10-100 mmol/L ammonium sulfate. Strain BAB-1 could grow well under pH between 4.5 and 10.0. Glucose and ammonium chloride were the optimum carbon and nitrogen sources for the growth of strain BAB-1, respectively, and the number of viable bacteria was significantly higher than those of other substrates at different culture durations. At 48 h, the number of viable bacteria reached 6.90×10⁸ CFU/mL and 6.87×10⁸ CFU/mL respectively. D-xylose, D-ribose, L-arabinose and arbutin could significantly enhance sporulation rates of strain BAB-1 at 12 h, and the sporulation rate of L-arabinose and arbutin were 61.94% and 66.92% respectively; L-proline, L-cysteine, L-glutamine and L-asparagine could prominently improve the sporulation rate of strain BAB-1 at 12 h to 36 h, and the sporulation rate reached from 27.88% to 82.37%. The results indicated that PM technology could apply to high-throughput analysis of the nutrients conducive to the growth and sporulation of B.subtilis and optimization of cultivation process.