高产脂肽类生物表面活性剂菌株及其驱油机理研究
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摘要
碱-表面活性剂-聚合物三元复合驱(ASP flooding)是一项新型的三次采油技术,但其推广受表面活性剂研究进展的制约。生物表面活性剂具有低毒、无污染、廉价的特点,在各种生物表面活性剂中,脂肽类生物表面活性剂因为它的高表面活性尤其引人关注。本论文主要从脂肽类生物表面活性剂产生菌的筛选方法、菌种鉴定、基因工程菌株的构建、发酵条件的优化、脂肽的理化性质及其驱油机理等方面作了较全面的研究,为其工业化生产和油田现场应用奠定理论基础。主要研究结论如下:
(1)从采自大庆油田的油土样和油水样中,经过富集培养,血平板和油平板分离,并经排油活性法和表面张力法进一步复筛,从中筛选出了8株产生物表面活性剂的优良菌株。经过薄层层析(TLC)和高效液相色谱(HPLC)分析鉴定,其中一株产生脂肽类生物表面活性剂的芽胞杆菌属的ZW-3菌株效果最好,其表面活性剂可以将培养基的表面张力降到31.0mN/m,并具有良好的乳化活性。
(2)对筛选出的ZW-3菌株进行了系统鉴定,通过形态观察、生理生化实验和16S rRNA同源序列比对和进化树分析,发现ZW-3菌株与芽孢杆菌属的枯草芽孢杆菌有98%以上的相似性,所以鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis) ZW-3。
(3)对筛选出的产脂肽类生物表面活性剂的优良菌株ZW-3的摇瓶发酵产脂肽的特性进行了研究。通过单因素实验等方法研究了各种营养和环境因素对菌株ZW-3发酵生产脂肽表面活性剂的影响。结果表明,无机氮源比有机氮源更有利于脂肽表面活性剂的产生;提高培养基的C/N比有利于脂肽的合成和积累。优化后的ZW-3产脂肽的培养基组成(/L)为:可溶性淀粉20.0 g,NH_4NO_3 4.0g,KH_2PO_4 3.0g,Na_2HPO_4 3.0g,酵母粉0.5 g,FeSO_4 6.8μmol,ZnSO_4 0.038mmol,CaCl_2 0.5mmol,MgSO_4 0.2mmol,MnSO_4 0.02mmol,EDTA 50μmol,pH 7.4;最佳培养条件为:以2%接种量将11h种子培养物接入装有100ml发酵培养基(pH 7.4)的500ml摇瓶中,于45℃、250r/min振荡培养53 h。
(4)将血红蛋白基因(vhb)置于RDR细菌启动子驱动下的质粒PSET中,构建PSET-RDR-vhb重组质粒,并通过电激作用转入脂肽代谢菌株-枯草芽孢杆菌株(bacillus subtilis)ZW-3中,重组菌株经酶切和PCR电泳检测鉴定,Southern-blot杂交显示部分转化菌株中外源基因插入基因组DNA,采用一氧化碳差异色谱法测定了血红蛋白的表达量。实验进一步对转化菌株的生长曲线、总蛋白量、过氧化氢酶活性、脂肽的产率进行了测定。结果显示,相比原始菌株,重组菌株数据均有明显提高。
(5)枯草芽孢杆菌ZW-3在可溶性淀粉为碳源的培养基中培养可产生一种脂肽类生物表面活性剂,该生物表面活性剂在100℃、pH 6-10的范围内和100000.0 mg/L的Ca~(2+)/Mg~(2+)离子浓度及12%的高盐浓度下仍维持原有的表面活性。该脂肽表面活性剂在低浓度下就能显著降低水与空气的表面张力从68.92mN/m[0]降低25.19mN/m[0]、原油/水的界面张力从23.53mN/m[0]降低到4.57mN/m[0],其临界胶束浓度(CMC)为33.3mg/L(3.24×10-5mol/L),在此浓度下,当pH 6.0~8.0时表面张力降到最低。更为显著的特点是该脂肽对热相当稳定,在100℃保温2h界面张力不变。该脂肽还有很好的乳化性,稀释10倍后,乳化活性仍为1.36U,仍能达到有关文献报道的生物表面活性剂原液乳化活性水平。ZW-3菌株产生的脂肽增溶与脱附作用显著,可使油水互溶而形成水包油型乳化小滴,镜检时可观察到形成的水包油乳状液,这些特点有利于原油的增采和输送以及环境生物修复等领域的应用,并具有良好的发泡和抗生素作用。该脂肽表面活性剂(0.05%)与低浓度的烷基苯磺酸盐(0.05%)、NaOH溶液(0.6%)进行三元复配,即可达到超低界面张力(10~4mN/m),试推测脂肽类生物表面活性剂和烷基苯磺酸盐复配后,两者之间存在着明显的协同效应,可在不影响界面活性的前提下,降低烷基苯磺酸盐和碱的用量。物理模拟实验结果显示,碱(NaOH,0.6%)-表面活性剂(脂肽,0.05%+烷基苯磺酸盐,0.05%)-聚合物(1000ppm)三元复合驱可提高原油采收率19.6 OOIP。说明脂肽类生物表面活性剂在三元复合驱的研究中具有巨大的应用前景。
The EOR method socalled alkaline–surfactant–polymer (ASP) flooding has proved to be effective in laboratory experiments and field projects .At present restricts to application of this technique is surfactant development. biosurfactants has characteristic of low poisonous, no pollution, inexpensive. Therefore biosurfactants could be used as an economical alternative to chemical surfactants in ASP flooding is feasible. Among the many classes of biosurfactants, lipopeptides are particularly interesting because of their high surface activities and therapeutic potential, which is an inhibitor of fibrin clotting and cyclic AMP phosphodiesterase.
1. Microorganisms capable of producing biosurfactants could be isolated by a series of step including enrichment culture, hemolytic activity assay on blood agar plates and hydrolyzing oil activity assay on oil agar plates etc. Then the fermentation supernatant of first-isolated strains was secondly screened by a drop-collapsing method, oil displacement activity and surface tension method. Eight strains bacteria were isolated with higher surface activity from oil-contaminated soil and water collected from Daqing Oil Field. The results of TLC and HPLC analysis showed that biosurfactants of strain ZW-3 was lipopeptide and it was the best one among them..
The experimental results of the morphological structure, physiological characteristics and the 16S rDNA whole sequence comparison of strain ZW-3 with the strains in Genebank showed that I6S rDNA of stain ZW-3 has more than 98 percent of homogenous with Bacillus subtilis. Therefore strain ZW-3 was identified as Bacillus subtilis ZW-3.
2. The conditions for shaking flask fermentation of strain ZW-3 which has the high productivity of lipopeptide biosurfcant were studied. The influences of the nutrient and environmental factors on the fermentation production are investigated through the complex of orthogonal experiments and one-factor experiments.The optimized composition medium(/L):soluble starch 20.0g, NH_4NO_3 4.0g, Na_2HPO_4·12H2O 3.0g, KH_2PO_4 3.0g, FeSO_4 6.8μmol,ZnSO_4 0.038mmol,CaCl_2 0.5mmol,MgSO_4 0.2mmol,MnSO_4 0.02mmol,EDTA 50μmol, yeast extract 0.5g, initial pH 7.4.The optimal culture condition: 500 mL flask containing 100 mL medium,2.0% seed culture is used, temperature is 40-45℃.On such a condition.
3. Hemoglobin of the gram-negative bacterium Vitreoscilla can bind oxygen strongly and reduce the oxygen requirement of the bacterium. A recombinant plasmid PEST-RDR-vhb was constructed and transferred into strain ZW-3(bacillus subtilis),The results showed that the expression of vhb gene could improve cell growth and enhance the production of surfactin with 31.8% higher than that of control under different dissolved oxygen concentrations. Besides, vhb gene also increased the expression of total protein quantity and CAT.
4. In this study, We investigated changes in surfactin activity under a range of conditions. Experimental variables included NaCl(0-20%), pH(3.0-10.0), Ca~(2+)/Mg~(2+)(0.0-1.0×105mg/L), and temperature (50~120℃). And this surfactin could reduce surface tension of bacterial fermentation culture medium and water/oil-IFT from 68.82 mN/m to 24.88 mN/m and from 23.53 mN/m to 4.57 mN/m , Its critical micelle concentration was tested to be 33.3 mg/L, and it had excellent emulsifying(2.89U) and foaming property.surfactin (concentration 0.066%) and aklyl benzenes sulfonate (concentration 50.0%) were mixed to solution of effective concentration 0.025%～0.4% by allocated proportion 1: 1 in ASP flooding experiment. The result showed the joint of surfactin would reduced the IFT of ASP-system to 10-3mN/m in lower effective concentration (0.025%~0.2%) and lower NaOH concentration(0.4%~0.7%). When NaOH concentration about 0.6% and effective concentration of ASP-system about 0.1% , the IFT reduced to 10-4mN/m. Meanwhile the independent employment of aklyl benzenes sulfonate and NaOH concentration needs 0.1% and 1.0% respectively, only then would gain 10-4mN/m, this indicated surfactin and aklyl benzenes sulfonate has the obvious coordination effect after duplicate match. the results of physical simulation experiment indicated that alkaline–surfactant(surfactin+aklyl benzenes sulfonate)–polymer flooding could enhance oil recovery to 19.6 OOIP.
(1)从采自大庆油田的油土样和油水样中,经过富集培养,血平板和油平板分离,并经排油活性法和表面张力法进一步复筛,从中筛选出了8株产生物表面活性剂的优良菌株。经过薄层层析(TLC)和高效液相色谱(HPLC)分析鉴定,其中一株产生脂肽类生物表面活性剂的芽胞杆菌属的ZW-3菌株效果最好,其表面活性剂可以将培养基的表面张力降到31.0mN/m,并具有良好的乳化活性。
(2)对筛选出的ZW-3菌株进行了系统鉴定,通过形态观察、生理生化实验和16S rRNA同源序列比对和进化树分析,发现ZW-3菌株与芽孢杆菌属的枯草芽孢杆菌有98%以上的相似性,所以鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis) ZW-3。
(3)对筛选出的产脂肽类生物表面活性剂的优良菌株ZW-3的摇瓶发酵产脂肽的特性进行了研究。通过单因素实验等方法研究了各种营养和环境因素对菌株ZW-3发酵生产脂肽表面活性剂的影响。结果表明,无机氮源比有机氮源更有利于脂肽表面活性剂的产生;提高培养基的C/N比有利于脂肽的合成和积累。优化后的ZW-3产脂肽的培养基组成(/L)为:可溶性淀粉20.0 g,NH_4NO_3 4.0g,KH_2PO_4 3.0g,Na_2HPO_4 3.0g,酵母粉0.5 g,FeSO_4 6.8μmol,ZnSO_4 0.038mmol,CaCl_2 0.5mmol,MgSO_4 0.2mmol,MnSO_4 0.02mmol,EDTA 50μmol,pH 7.4;最佳培养条件为:以2%接种量将11h种子培养物接入装有100ml发酵培养基(pH 7.4)的500ml摇瓶中,于45℃、250r/min振荡培养53 h。
(4)将血红蛋白基因(vhb)置于RDR细菌启动子驱动下的质粒PSET中,构建PSET-RDR-vhb重组质粒,并通过电激作用转入脂肽代谢菌株-枯草芽孢杆菌株(bacillus subtilis)ZW-3中,重组菌株经酶切和PCR电泳检测鉴定,Southern-blot杂交显示部分转化菌株中外源基因插入基因组DNA,采用一氧化碳差异色谱法测定了血红蛋白的表达量。实验进一步对转化菌株的生长曲线、总蛋白量、过氧化氢酶活性、脂肽的产率进行了测定。结果显示,相比原始菌株,重组菌株数据均有明显提高。
(5)枯草芽孢杆菌ZW-3在可溶性淀粉为碳源的培养基中培养可产生一种脂肽类生物表面活性剂,该生物表面活性剂在100℃、pH 6-10的范围内和100000.0 mg/L的Ca~(2+)/Mg~(2+)离子浓度及12%的高盐浓度下仍维持原有的表面活性。该脂肽表面活性剂在低浓度下就能显著降低水与空气的表面张力从68.92mN/m[0]降低25.19mN/m[0]、原油/水的界面张力从23.53mN/m[0]降低到4.57mN/m[0],其临界胶束浓度(CMC)为33.3mg/L(3.24×10-5mol/L),在此浓度下,当pH 6.0~8.0时表面张力降到最低。更为显著的特点是该脂肽对热相当稳定,在100℃保温2h界面张力不变。该脂肽还有很好的乳化性,稀释10倍后,乳化活性仍为1.36U,仍能达到有关文献报道的生物表面活性剂原液乳化活性水平。ZW-3菌株产生的脂肽增溶与脱附作用显著,可使油水互溶而形成水包油型乳化小滴,镜检时可观察到形成的水包油乳状液,这些特点有利于原油的增采和输送以及环境生物修复等领域的应用,并具有良好的发泡和抗生素作用。该脂肽表面活性剂(0.05%)与低浓度的烷基苯磺酸盐(0.05%)、NaOH溶液(0.6%)进行三元复配,即可达到超低界面张力(10~4mN/m),试推测脂肽类生物表面活性剂和烷基苯磺酸盐复配后,两者之间存在着明显的协同效应,可在不影响界面活性的前提下,降低烷基苯磺酸盐和碱的用量。物理模拟实验结果显示,碱(NaOH,0.6%)-表面活性剂(脂肽,0.05%+烷基苯磺酸盐,0.05%)-聚合物(1000ppm)三元复合驱可提高原油采收率19.6 OOIP。说明脂肽类生物表面活性剂在三元复合驱的研究中具有巨大的应用前景。
The EOR method socalled alkaline–surfactant–polymer (ASP) flooding has proved to be effective in laboratory experiments and field projects .At present restricts to application of this technique is surfactant development. biosurfactants has characteristic of low poisonous, no pollution, inexpensive. Therefore biosurfactants could be used as an economical alternative to chemical surfactants in ASP flooding is feasible. Among the many classes of biosurfactants, lipopeptides are particularly interesting because of their high surface activities and therapeutic potential, which is an inhibitor of fibrin clotting and cyclic AMP phosphodiesterase.
1. Microorganisms capable of producing biosurfactants could be isolated by a series of step including enrichment culture, hemolytic activity assay on blood agar plates and hydrolyzing oil activity assay on oil agar plates etc. Then the fermentation supernatant of first-isolated strains was secondly screened by a drop-collapsing method, oil displacement activity and surface tension method. Eight strains bacteria were isolated with higher surface activity from oil-contaminated soil and water collected from Daqing Oil Field. The results of TLC and HPLC analysis showed that biosurfactants of strain ZW-3 was lipopeptide and it was the best one among them..
The experimental results of the morphological structure, physiological characteristics and the 16S rDNA whole sequence comparison of strain ZW-3 with the strains in Genebank showed that I6S rDNA of stain ZW-3 has more than 98 percent of homogenous with Bacillus subtilis. Therefore strain ZW-3 was identified as Bacillus subtilis ZW-3.
2. The conditions for shaking flask fermentation of strain ZW-3 which has the high productivity of lipopeptide biosurfcant were studied. The influences of the nutrient and environmental factors on the fermentation production are investigated through the complex of orthogonal experiments and one-factor experiments.The optimized composition medium(/L):soluble starch 20.0g, NH_4NO_3 4.0g, Na_2HPO_4·12H2O 3.0g, KH_2PO_4 3.0g, FeSO_4 6.8μmol,ZnSO_4 0.038mmol,CaCl_2 0.5mmol,MgSO_4 0.2mmol,MnSO_4 0.02mmol,EDTA 50μmol, yeast extract 0.5g, initial pH 7.4.The optimal culture condition: 500 mL flask containing 100 mL medium,2.0% seed culture is used, temperature is 40-45℃.On such a condition.
3. Hemoglobin of the gram-negative bacterium Vitreoscilla can bind oxygen strongly and reduce the oxygen requirement of the bacterium. A recombinant plasmid PEST-RDR-vhb was constructed and transferred into strain ZW-3(bacillus subtilis),The results showed that the expression of vhb gene could improve cell growth and enhance the production of surfactin with 31.8% higher than that of control under different dissolved oxygen concentrations. Besides, vhb gene also increased the expression of total protein quantity and CAT.
4. In this study, We investigated changes in surfactin activity under a range of conditions. Experimental variables included NaCl(0-20%), pH(3.0-10.0), Ca~(2+)/Mg~(2+)(0.0-1.0×105mg/L), and temperature (50~120℃). And this surfactin could reduce surface tension of bacterial fermentation culture medium and water/oil-IFT from 68.82 mN/m to 24.88 mN/m and from 23.53 mN/m to 4.57 mN/m , Its critical micelle concentration was tested to be 33.3 mg/L, and it had excellent emulsifying(2.89U) and foaming property.surfactin (concentration 0.066%) and aklyl benzenes sulfonate (concentration 50.0%) were mixed to solution of effective concentration 0.025%～0.4% by allocated proportion 1: 1 in ASP flooding experiment. The result showed the joint of surfactin would reduced the IFT of ASP-system to 10-3mN/m in lower effective concentration (0.025%~0.2%) and lower NaOH concentration(0.4%~0.7%). When NaOH concentration about 0.6% and effective concentration of ASP-system about 0.1% , the IFT reduced to 10-4mN/m. Meanwhile the independent employment of aklyl benzenes sulfonate and NaOH concentration needs 0.1% and 1.0% respectively, only then would gain 10-4mN/m, this indicated surfactin and aklyl benzenes sulfonate has the obvious coordination effect after duplicate match. the results of physical simulation experiment indicated that alkaline–surfactant(surfactin+aklyl benzenes sulfonate)–polymer flooding could enhance oil recovery to 19.6 OOIP.
引文
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