一株产生脂肽的枯草芽孢杆菌的分离鉴定及脂肽对原油的作用

Isolation and identification of surfactin producing Bacillus Subtilis strain and its effect of surfactin on crude oil

摘要:

从大庆油田地层水中分离到一组能高效产生生物表面活性剂的菌株, 采用sfp基因PCR鉴定的方法从中分离到一株芽孢杆菌ZW-3, 该菌株能够产生大量表面活性物质, 采用细菌生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株为枯草芽孢杆菌(Bacillus subtilis), 通过薄层层析色谱(TLC)、高效液相色谱(HPLC)分析其代谢产物, 初步鉴定为脂肽(Lipopeptide); 该脂肽生物表面活性剂理化性质显示它能使培养基的表面张力从68.92mN/m降低25.19mN/m、原油/水的界面张力从23.53mN/m降低到4.57mN/m, 与1.8%的NaOH溶液复配可以将油水界面张力降低到1.2×10-3mN/m, 其临界胶束浓度为33.3mg/L(3.24×10-5mol/L), 并具有较好的乳化活性和发泡性能, 说明该菌株代谢的脂肽生物表面活性剂在提高石油采收率中具有广泛的应用前景。

关键词:枯草芽孢杆菌; 多相分类; 脂肽; 生物表面活性剂; 理化性质; 原油

Abstract:

Strains producing biological surfactants were isolated from formation water of Daqing oil field, Heilongjiang Province, China. From which a lipopeptide producing strain ZW-3 was screened out by PCR of the sfp gene. The mor-phology ,cultural characteristics ,physiological , biochemical properties and chemotaxonomy of strain ZW-3 were studied. The strain is rod shaped (0.7～0.8 mm×2~3 mm) , gram-positive, spore-forming and aerobic bacteria. Its (G+C) content was determined to be 42.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to the genus Bacillus subtilis, and the metabolites of strain ZW-3 was analyzed by thin layer chromatog-raphy and high performance liquid chromatography, the result indicated that the biosurfactant strain ZW-3 produced was surfactin. It could reduce surface tension of bacterial fermentation culture medium and water/oil- interfacial tension from 68.82 mN/m to 24.88 mN/m and from 23.53 mN/m to 4.57 mN/m ,respectively, and its mixture with 1.8%NaOH could reduce water/oil- interfacial tension to an ultra low level (1.2×10-3mN/m), Its critical micelle concentration (CMC) was tested to be 33.3 mg/L(3.24×10-5 mol/L)at 25℃, and it had excellent emulsifying(2.89U) and foaming property. All these results showed that this biosurfactant had great potential in pharmaceutics, environmental protection, cosmetic, oil recov-ery and many other application fields.

Key words:Bacillus subtilis; polyphasic taxonomy; lipopeptide; biosurfactant; physico-chemical properties; crude oil