一株高产表面活性剂的南极土地杆菌的分离及其特性

Isolation and characterization of an Antarctic strain of Pedobacter sp. producing surfactant

摘要:

【背景】生物表面活性剂具有毒性低、生物兼容性好和可降解等优点，是化学表面活性剂的优良替代物。目前产生物表面活性剂的微生物多为常温菌，从低温环境中挖掘高产新型生物表面活性剂的生产菌株具有重要的意义。【目的】从南极土壤中筛选产表面活性剂的低温微生物，对其表面活性剂进行纯化和结构解析并评估其性能。【方法】采用排油圈法对分离自南极菲尔德斯半岛土壤样品中的细菌菌株进行筛选，获得一株在菌苔表面产白色固体颗粒的菌株，对菌株进行形态观察和16S rRNA基因序列分析以确定该菌株系统发育地位。利用高效液相色谱法(high performance liquid chromatography, HPLC)分离产物，并用核磁共振波谱(nuclear magnetic resonance, NMR)技术对产物的化学结构进行鉴定。采用单因素试验和响应面设计方法对发酵培养基进行优化。此外，对产物的乳化性能及其对柴油的降解能力进行评估。【结果】得到了一株高产生物表面活性剂的耐冷土地杆菌属(Pedobacter) GW9-17，其最优发酵培养基(g/L)组成为：可溶性淀粉18.0、胰蛋白胨9.0、C₃H₃NaO₃4.4、K₂HPO₄3.6、MgSO₄1.2，pH 7.0±0.2。在此条件下，按10% (体积分数)接种量、28 ℃、180 r/min培养7 d后表面活性剂的浓度可达到(3.0±0.5) g/L。利用HPLC和NMR技术发现菌株GW9-17表面活性剂主要成分为flavolipid-9U,9U，表面活性剂粗提物的甲醇-水溶液(体积比1:1)对液体石蜡油有良好的乳化能力，菌株GW9-17在柴油含量为5%时，在4 ℃和28 ℃条件下对柴油的降解率分别达到40.1%和57.3%。【结论】从南极土壤中获得了一株高产低分子量表面活性剂的低温Pedobacter sp. GW9-17，其产物flavolipids具有对烷烃类污染物质增溶分解的潜力，对石油污染的低温生态修复有重要的利用潜能。

关键词:生物表面活性剂;flavolipids;土地杆菌;柴油降解

Abstract:

[Background] With low toxicity, good biocompatibility, and biodegradability, biosurfactants are excellent substitutes for chemical surfactants. Most of the available biosurfactant-producing bacteria are mesophilic and it is of great significance to mine efficient biosurfactant-producing strains from cryogenic environments such as the Antarctic. [Objective] To screen psychrotolerant bacteria producing surfactants from the Antarctic soil samples and analyze the chemical structures and properties of the purified surfactants. [Methods] The biosurfactant-producing bacteria from the soil samples in Fildes Peninsula, Antarctic were screened by the oil spreading method. A strain with white solid particles on the colony surface was obtained and identified based on the morphology and the phylogenetic analysis of the 16S rRNA gene sequence. The components in the product were separated by high performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR). The fermentation medium of the strain was optimized by single factor experiment and response surface design. The emulsifying ability of the product and the diesel oil-degrading ability of the strain were evaluated. [Results] Pedobacter sp. GW9-17 with high surfactant production was screened out. The optimal fermentation medium contained (g/L): soluble starch 18.0, tryptone 9.0, C₃H₃NaO₃ 4.4, K₂HPO₄ 3.6 and MgSO₄ 1.2, with pH value 7.0±0.2. The concentration of the product reached (3.0±0.5) g/L after the strain was incubated in the optimal medium at 28 ℃ and 180 r/min for 7 days with an inoculum amount of 10% (volume fraction). The main component of the surfactant was flavolipid-9U,9U, and the methanol-aqueous solution (the volume ratio is 1:1) of the surfactant crude extract had good emulsifying ability for liquid paraffin oil. In the medium with 5% diesel oil, the strain GW9-17 showed the diesel oil-degrading rates of 40.1% and 57.3% at 4 ℃ and 28 ℃, respectively. [Conclusion] A psychrotolerant strain, Pedobacter sp. GW9-17, efficiently producing the surfactant with a low molecular weight was isolated from the Antarctic soil. The product demonstrated the potential to degrade alkane pollutants and remediate the low-temperature environment of oil pollution.

Key words:biosurfactants;flavolipids;Pedobacter;diesel oil degradation