原油污染土壤中生物表面活性剂菌株的筛选和产物鉴定

Screening and product identification of biosurfactant-producing strains in crude oil-contaminated soil

摘要:

【目的】从原油污染的土壤中分离出产表面活性剂的枯草芽孢杆菌（Bacillus subtilis） SX-20，并对其产物进行提取及结构分析。【方法】采用氯化十六烷基吡啶和溴百里酚蓝混合溶液（cetylpyridinium chloride-bromothymol blue，CPC-BTB）显色反应结合血琼脂平板简单高效的筛选得到产脂肽的枯草芽孢杆菌。通过酸沉淀、甲醇萃取和旋转蒸发提取发酵所产生的粗产物，该产物对痤疮丙酸杆菌（Propionibacterium acnes）具有良好的抑制作用。运用傅里叶红光变换光谱（Fourier transform infrared spectroscopy，FTIR）、氨基酸分析和液相质谱联用（liquid chromatography-mass spectrometry，LC-MS）对粗产物的成分进行分析。【结果】筛选所得的菌株所产物质是含C15脂肪酸链和7个氨基酸形成的环状的脂肽表面活性剂。【结论】本研究为筛选脂肽类生物表面活性剂提供了一定的理论基础和技术路线，有利于后续获得高产的低成本的生物表面活性剂。

关键词:原油污染土壤;枯草芽孢杆菌;表面活性剂;脂肽;筛选;鉴定

Abstract:

[Objective] To screen out a surfactant-producing microbial strain Bacillus subtilis SX-20 from the soil contaminated by crude oil for a long time and then extract and analyze the products of the strain. [Methods] We used cetylpyridinium chloride-brominated thymol blue combined with blood agar plate for the screening and obtained a strain capable of producing lipopeptide. The crude fermentation product of the strain was extracted by acid precipitation, methanol extraction, and rotary evaporation, and it showed good inhibitory effect on Propionibacterium acnes. Its composition was analyzed by Fourier transform infrared spectroscopy (FTIR), amino acid analysis, and liquid chromatography-mass spectrometry (LC-MS). [Results] The surfactant-producing strain screened out was identified as Bacillus subtilis and named SX-20. The product of the screened strain was a cyclic lipopeptide containing a C15 fatty acid chain and 7 amino acid residues. [Conclusion] This study provided a theoretical basis and technical route for the screening of biosurfactant-producing strains, which is conducive to the subsequent acquisition of high-yield and low-cost biosurfactants.

Key words:crude oil-contaminated soil;Bacillus subtilis;surfactant;lipopeptide;screening;identification