一株铜绿假单胞菌及其产生的鼠李糖脂特性研究

Study on Characteristics of a Rhamnolipid Biosurfactant Producing Bacterium and Rhamnolipid Biosurfactant

研究高效代谢表面活性剂的菌种,并对菌种和表面活性剂的理化性质进行分析。采用菌种16S rDNA序列分析对菌种进行鉴定,采用紫外光谱扫描、色谱柱层析、薄层层析、单糖分析对生物表面活性剂的种类进行鉴定,并对表面活性剂的理化性质进行研究。从油田采出水中筛选出一株高效代谢生物表面活性剂的菌株T-1,经16S rDNA鉴定为铜绿假单胞菌属(Pseudomonas aeruginosa)。该菌种以甘油和大豆油为碳源的培养基培养4 d后,其发酵液表面张力30.216 mN/m,EI24为100%。根据表面活性剂的红外光谱分析并结合薄层分析,可确定T-1样品中含有糖脂类物质,进一步的表面活性剂的单糖分析显示水解终产物为单一的鼠李糖,最终确定其产物为鼠李糖脂,苯酚-硫酸法测定其鼠李糖脂产率为5.2 g/L,从发酵液提取的棕黄色生物表面活性剂粗品,其表观临界胶束浓度为45 mg/L,该鼠李糖脂对环境(耐温、耐酸碱、耐盐)具有较强的适应性。该表面活性剂对恶劣环境具有较强的耐受性,可应用于微生物采油等用途。

The aim of this study was to research strains that feature high-producing biosurfactant, and analyze the structure and characteristics of biosurfactant. We used morphological, physiological and biochemical characteristics and 16S rDNA sequences analysis to identify the strain and their characteristics, and used UV Chromatography, TLC, IR to analyze the structure of the biosuractant. Effect of environmental factors including temperature and pH on the biosurfactant were also investigated to confirm the optimum conditions. A strain of bacterium with high capability of de-emulsification was isolated from the co-produced water of oil field. The fermentation supernatant produced by the strain revealed high surface activity, from which a kind of biosurfactant can be acquired by acid precipitation. Its CMC is about 45 mg/L. On the basis of phenotypic and phylogenic analysis, including the phenotype and 16S rRNA, this bacterium was confidently classified to Pseudomonas aeruginosa and named as Pseudomonas aeruginosa T-1. The biosurfactant produced by P. aeruginosa T-I possessed tolerance of pH, inorganic salts and temperature, which showed wide application range. The supernatant of P. aeruginosa T-1 that was precipitated by acetone and crude product of biosurfactant was obtained. Many other proteins were removed after the method of Sevag. The product was purified further by Sephadex G15 column chromatography. The pure product was identified as glycolipid by thin layer chromatography and infrared spectrum analysis. Sugar component of the glycolipid was rhamnose, which was analyzed by paper chromatography. The biosurfactant produed by P. aeruginosa T-1 was rhamnolipid, highest value of which reached to 5.2 g/L at the early stationary phase.