生物表面活性剂产生菌的分离鉴定及培养条件优化
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摘要
作为天然表面活性剂的一种,生物表面活性剂(Biosurfactant)主要是指微生物在一定培养条件下产生的一些代谢产物,如糖脂类、脂多糖、脂肽、脂蛋白以及中性类脂衍生物等。和常见的化学表面活性剂相比,它们除具有降低表面张力、稳定乳化液等相同特性外,还具有无毒、可自然生物降解、生态安全等化学表面活性剂所不具备的特性及高表面活性和生理活性等优点,因此近年来备受国内外研究学者的关注。综合生物表面活性剂的特点及其广泛应用领域,筛选出能够代谢廉价底物且性能稳定的高产菌株、设计经济有效的产物分离纯化方法、通过优化培养条件等方法提高生物表面活性剂的发酵产率和提取效率,是进一步推广生物表面活性剂走向市场,逐渐取代传统化学表面活性剂的关键。
本文通过富集培养及蓝色凝胶平板筛选,从炼油厂内长期石油污染土中分离出9株高效产生物表面活性剂的土著微生物。对其发酵液进行初步和精确性能验证后,挑选出一株性能稳定且表面张力良好的菌株,命名为S2。提取了菌株的DNA基因组,通过PCR扩增得到长约1.2Kb的16SrDNA片段,结合细菌生理生化试验,将该菌株鉴定为铜绿假单胞菌(Pseudomonas aeruginosa).根据GenBank数据库中与菌株同源性最近的菌株的分类地位及序列信息对菌株进行了系统进化分析,并在核酸数据库GenBank上申请登录号：EU590647。
进一步对菌株S2在发酵培养条件下所产生物表面活性剂进行了初步提取和精确提取,将精提后的产物用高效液相色谱和薄层层析等方法进行分析鉴定,最终确定发酵液中生物表面活性剂为鼠李糖脂。进一步测定了发酵过程中发酵液表面张力和菌体生长量的变化曲线,结果表明,菌株S2在12h内便可将发酵培养液的表面张力从最初的65.9mN/m降低至36.8mN／m。并利用最终的纯化产物测定出铜绿假单胞菌S2所产鼠李糖脂的临界胶束浓度值(CMC)为250mg／L。比较了生物表面活性剂和几种常用的化学表面活性剂的乳化性能。试验结果表明,菌株S2发酵液能维持菜油与水乳化液体积在83%左右且长达150h以上,其乳化性能优于化学表面活性剂。
为保证后续试验及发酵过程中始终保持最适培养条件,以发酵液中生物量及鼠李糖含量作为试验指标,对菌株S2的培养基配方进行了碳源及氮源的影响试验,最终选定碳源为菜油,氮源为硝酸钠。以光密度(OD610)值和发酵液中的鼠李糖含量为主要指标,比较碳源、氮源、初始pH和发酵时间等4个因素对发酵液中鼠李糖产量的影响。每个因素取3个水平,进行正交试验,最终确定发酵培养的最优条件为：菜油25 g/L, NaNO3 15 g/L, KCl1.1 g/L, NaCl1.1 g/L, KH2PO43.4 g/L, K2HPO44.4 g/L MgSO40.5 g/L,酵母膏0.5 g／L,微量元素溶液5.0ml,培养基初始pH值7.5,发酵时间96h。在该培养条件下,生物表面活性剂最高产率可达到4.9g／L。
Biosurfactants are high surface-active agents synthesized by microorganisms that exhibite pronounced surface activity. They mainly include glycolipid, lipopolysaccharide, lipopeptid, and lipoprotein, etc. Biosurfactants have not only physical and chemical characteristics similiar to chemical surfactants, but biosurfactants have much lower toxicity to ecological system and easily biodegradable. Therefore, biosurfactants are studied widely in recent years worldwide. The objectives of the present study are to (1) isolate the biosurfactant-producing microorganisms with high yield and surface activity, (2) purify and analyze the products of strain S2, (3) optimum the isolate's growth condition.
In this study, the microorganisms capable of producing biosurfactants were isolated from oil sludge samples collected from Yangzi Refinery, Jiangsu Province, China by enrichment culture and blue agar plate in term of the surface tension of fermentation liquor. Nine strains with high surface activity were isolated. It was found that one of these ioslates (hereinafter named by S2) was of the highest surfact activity. Total DNA of the strain S2 was extracted and amplified for 16S rDNA gene sequences. The purified PCR product was sequenced and deposited in the GenBank database with accession number EU590647. Phylogenesis of strain S2 was built in term of the closest related bacterias found in GenBank database. Its morphology and physiological characterics were also analyzed. Combining physiological, biochemical and molechular test results, strain S2 was identified as Pseudomonas aeruginosa.
The biosurfactant components of metabolite it produced were also analyzed. The products of strain S2 (hereinafter called PAS2) in fermentation liquor consisted mainly of rhamnolipid through purification of products and anlysised by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). The critical micelle concentration (CMC) of PAS2 is 250mg/L, which is much lower than normal chemical surfactants.
The emulsification activity of fermentation liquor and common chemical surfactants were also tested. The results showed the emulsification ability of PAS2 was better than Sodium dodecyl sulfate (SDS) and Cetyltrimethylammonium bromide (CTAB), as indicating that PAS2 could maintain the emulsified solution volume at the ratio of 83% for more than 150 hours. Using biomass and rhamnolipid content as two main test indexes, the optimal medium and fermentation conditions of biosurfactant by strain S2 were studied using single factor and orthogonal experiment. It was found that the optimum carbon and nitrogen sources are vegetable oil and NaNO3, respectively. Under this condition, the maximal rhamnolipid concentration can be reached up to 4.9g/L.
It was concluded that biosurfactant PAS2 has a promising application in environmental remediation and other fields.
本文通过富集培养及蓝色凝胶平板筛选,从炼油厂内长期石油污染土中分离出9株高效产生物表面活性剂的土著微生物。对其发酵液进行初步和精确性能验证后,挑选出一株性能稳定且表面张力良好的菌株,命名为S2。提取了菌株的DNA基因组,通过PCR扩增得到长约1.2Kb的16SrDNA片段,结合细菌生理生化试验,将该菌株鉴定为铜绿假单胞菌(Pseudomonas aeruginosa).根据GenBank数据库中与菌株同源性最近的菌株的分类地位及序列信息对菌株进行了系统进化分析,并在核酸数据库GenBank上申请登录号：EU590647。
进一步对菌株S2在发酵培养条件下所产生物表面活性剂进行了初步提取和精确提取,将精提后的产物用高效液相色谱和薄层层析等方法进行分析鉴定,最终确定发酵液中生物表面活性剂为鼠李糖脂。进一步测定了发酵过程中发酵液表面张力和菌体生长量的变化曲线,结果表明,菌株S2在12h内便可将发酵培养液的表面张力从最初的65.9mN/m降低至36.8mN／m。并利用最终的纯化产物测定出铜绿假单胞菌S2所产鼠李糖脂的临界胶束浓度值(CMC)为250mg／L。比较了生物表面活性剂和几种常用的化学表面活性剂的乳化性能。试验结果表明,菌株S2发酵液能维持菜油与水乳化液体积在83%左右且长达150h以上,其乳化性能优于化学表面活性剂。
为保证后续试验及发酵过程中始终保持最适培养条件,以发酵液中生物量及鼠李糖含量作为试验指标,对菌株S2的培养基配方进行了碳源及氮源的影响试验,最终选定碳源为菜油,氮源为硝酸钠。以光密度(OD610)值和发酵液中的鼠李糖含量为主要指标,比较碳源、氮源、初始pH和发酵时间等4个因素对发酵液中鼠李糖产量的影响。每个因素取3个水平,进行正交试验,最终确定发酵培养的最优条件为：菜油25 g/L, NaNO3 15 g/L, KCl1.1 g/L, NaCl1.1 g/L, KH2PO43.4 g/L, K2HPO44.4 g/L MgSO40.5 g/L,酵母膏0.5 g／L,微量元素溶液5.0ml,培养基初始pH值7.5,发酵时间96h。在该培养条件下,生物表面活性剂最高产率可达到4.9g／L。
Biosurfactants are high surface-active agents synthesized by microorganisms that exhibite pronounced surface activity. They mainly include glycolipid, lipopolysaccharide, lipopeptid, and lipoprotein, etc. Biosurfactants have not only physical and chemical characteristics similiar to chemical surfactants, but biosurfactants have much lower toxicity to ecological system and easily biodegradable. Therefore, biosurfactants are studied widely in recent years worldwide. The objectives of the present study are to (1) isolate the biosurfactant-producing microorganisms with high yield and surface activity, (2) purify and analyze the products of strain S2, (3) optimum the isolate's growth condition.
In this study, the microorganisms capable of producing biosurfactants were isolated from oil sludge samples collected from Yangzi Refinery, Jiangsu Province, China by enrichment culture and blue agar plate in term of the surface tension of fermentation liquor. Nine strains with high surface activity were isolated. It was found that one of these ioslates (hereinafter named by S2) was of the highest surfact activity. Total DNA of the strain S2 was extracted and amplified for 16S rDNA gene sequences. The purified PCR product was sequenced and deposited in the GenBank database with accession number EU590647. Phylogenesis of strain S2 was built in term of the closest related bacterias found in GenBank database. Its morphology and physiological characterics were also analyzed. Combining physiological, biochemical and molechular test results, strain S2 was identified as Pseudomonas aeruginosa.
The biosurfactant components of metabolite it produced were also analyzed. The products of strain S2 (hereinafter called PAS2) in fermentation liquor consisted mainly of rhamnolipid through purification of products and anlysised by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). The critical micelle concentration (CMC) of PAS2 is 250mg/L, which is much lower than normal chemical surfactants.
The emulsification activity of fermentation liquor and common chemical surfactants were also tested. The results showed the emulsification ability of PAS2 was better than Sodium dodecyl sulfate (SDS) and Cetyltrimethylammonium bromide (CTAB), as indicating that PAS2 could maintain the emulsified solution volume at the ratio of 83% for more than 150 hours. Using biomass and rhamnolipid content as two main test indexes, the optimal medium and fermentation conditions of biosurfactant by strain S2 were studied using single factor and orthogonal experiment. It was found that the optimum carbon and nitrogen sources are vegetable oil and NaNO3, respectively. Under this condition, the maximal rhamnolipid concentration can be reached up to 4.9g/L.
It was concluded that biosurfactant PAS2 has a promising application in environmental remediation and other fields.
引文
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