脂肽-糖脂混合生物表面活性剂产生菌筛选和优化培养

Identification of Bacillus subtilis THY-7 and high titer optimization for the blend-biosurfactant of lipopeptide and glycolipid

摘要:

结合脂肽和糖脂的性能优势，致力于产脂肽-鼠李糖脂混合型生物表面活性剂的新菌株选育和培养条件优化。采用血平板溶血圈法初筛菌株、改进排油圈法快速检测产量以及飞行时间质谱鉴定产物结构。对优选菌株的碳源、氮源和磷酸盐缓冲液、重要金属离子浓度等进行了单因子和正交试验，优化了培养基和培养条件。采用高压液相色谱和蒽酮比色法定量分析了产物组成。筛选获得了同时积累糖脂和脂肽的新菌株，鉴定命名为芽胞杆菌Bacillus subtilis THY-7。摇瓶分批培养48 h，细胞OD600为37.0，产物浓度2.4 g/L，分别是优化前的3.4倍和3.1倍。发酵罐补料分批培养，泡沫中产物浓度达到4.5 g/L，且74％为表面活性素，22％为鼠李糖脂。B. subtilis THY-7是具有脂肽-鼠李糖脂高产潜力的优选菌株。

Abstract:

Biosurfactants (BSs) are highlighted owing to their multiple advantages in diverse applications. To screen a superior strain that producing a blend-biosurfactant of lipopeptide and glycolipid, the hemolytic activity assay on blood agar plates, the modified oil-red spreading test and MALDI-TOF Mass Spectrometry identification of the purified products was carried out. Bacillus subtilis THY-7 was selected and its principal products were surfactin and dirhamnolipid. The medium component and culture conditions of THY-7 were optimized by both single factor and orthogonal experiments. After 48 h optimal batch culture in flask, the cell density (OD600) was 37.0 and the product titer was 2.4 g/L, which was 3.4 folds and 3.1 folds of that under original condition, respectively. A fed-batch culture in a 5 L fermentor was further performed coupling with in situ recovery of foam, in which the titer of blend-BS increased to 4.5 g/L at 25 h. Quantification by HPLC and anthrone colorimetry revealed that surfactin and dirhamnolipid accounted for 74% and 22% of the blend-BS, respectively.