【作者】 郝东辉；

【导师】 林建强；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 世界范围内原油贮量的日益减少带来了严重的能源危机,而以常规技术,一般只能开采出30%的原油,大量石油尤其是高粘油滞留在储油层。微生物提高原油采收率（MEOR）是目前公认的开采油藏中剩余油和枯竭油藏最好的技术,对充分利用现有油矿藏资源,意义重大。另外,在石油的勘探与开采,运输与储存,炼制与使用过程中,常会造成石油外泄或不当排放,对环境造成严重的污染。应用烃降解微生物对污染地区进行生物修复因其具有适用范围广、操作简单、效果显著、费用低、无二次污染等优点而被认为是解决复杂烃类污染的最彻底最有效的手段。铜绿假单胞菌所产生的鼠李糖脂生物表面活性剂具有良好的表/界面活性和乳化活性,能形成胶束溶液,乳化分散烃类物质,降低原油粘度;对烃类的增溶作用能促进微生物对烃类的摄取,从而提高微生物对烃类的降解效率;此外,作为生物表面活性剂又具有无毒、可生物降解的优点。这都使其成为在MEOR和石油污染的生物修复领域中最佳的生物表面活性剂。不同来源的铜绿假单胞菌其鼠李糖脂的组成,性能,产量差距均较大,寻找合适石油工业的产脂菌株及其复配菌株具有重要价值。另外,铜绿假单胞菌虽然可高效产脂,但它是人体条件致病菌,不适合大规模工业应用。而利用基因工程的方法,将鼠李糖脂合成途径中的关键酶——鼠李糖基转移酶转入其它无害采油微生物受体中,构建产鼠李糖脂的工程采油微生物直接应用于生产可以有效避免菌株毒性。关于鼠李糖基转移酶基因在采油菌中的表达研究,少见国内外报道。在此方面的研究可为基因工程菌在微生物采油和石油烃污染的生物修复中的应用提供理论基础。本文主要从上述两点展开进行研究工作,主要研究内容和结果如下:1.产鼠李糖脂的原油降解菌的筛选与鉴定以原油为唯一碳源和能源,从油田样品中分离筛选多株兼性厌氧原油烃类降解菌,通过表面张力测量从中筛选出产糖脂生物表面活性剂的原油降解菌SH6。通过形态学观察、生理生化实验和16S rDNA序列分析鉴定其为铜绿假单胞菌（Pseudomonas aeruginosa）。该菌生长于原油培养基中,能很好的乳化分散原油,并将原油发酵液的表面张力降至37.4 mN/m。经GC-MS分析,P.aeruginosa SH6可以利用C₁₃～C₂₈较宽范围的原油烃组分,其原油降解率为18.8%,原油粘度降低率为47.7%。利用TLC、IR及蓝平板法分析,证明其产生的生物表面活性代谢产物为鼠李糖脂。2.鼠李糖脂理化性质和乳化活性的研究制备P.aeruginosa SH6所产的鼠李糖脂粗提样品。利用HPLC-MS分离鉴定了其所产鼠李糖脂同系物中的组分。结果表明,以甘油为唯一碳源时,P.aeruginosa SH6所产鼠李糖脂中主要包含8种同系物,都由1～2分子鼠李糖和1～2个C₈～C₁₂碳链长度的β-羟基饱合或不饱合脂肪酸组成。该鼠李糖脂粗品CMC为166.7 mg/L,在CMC时的表面张力为29.5 mM/m。该糖脂表现极高温度（-20～120℃）和盐度耐受性（小于50%NaCl,20%MgCl₂和CaCl₂）及优良的环境稳定性,并可在一定的pH范围内（1～7）保持活性。对液体石蜡、甲苯、植物油和原油等疏水性有机物具有良好的乳化性能。这些性质使得其在石油工业和石油污染修复领域具有良好的应用前景。3.碳氮源对鼠李糖脂生产的影响就不同种类的碳源和氮源对P.aeruginosa SH6产脂的影响进行了研究。结果表明,P.aeruginosa SH6可以利用多种类型的碳氮源产脂。其中以葡萄糖与菜籽油（1:1）为复合碳源,酵母粉与硝酸钠为复合氮源,且碳氮比为18:1时产脂量最高,达5.40 g/L。保持此最优碳氮比加大碳氮源浓度时,在碳源浓度为7%时产脂量最高,达6.37 g/L。这些研究为进一步的鼠李糖脂发酵生产条件的优化和应用不同碳氮源时所产鼠李糖脂组分与理化性质的研究提供了依据和基础。4.鼠李糖基转移酶基因的克隆及在大肠杆菌中的表达研究对P.aeruginosa SH6的鼠李糖基转移酶结构基因rhlAB和rhlABRI操纵子进行克隆与鉴定。构建含鼠李糖基转移酶rhlAB和rhlABRI的重组质粒pSAOR2和pJDOR4,利用鼠李糖基转移酶自身启动子使其在大肠杆菌E.coli SM10、E.coliDH5α中进行表达。结果表明,只转入结构基因rhlAB的两种大肠杆菌产脂量均很低;而转入rhlABRI操纵子的两宿主产脂量均有所提高,其中E.coli DH5α产脂量最高达105.2 mg/L。仅管是应用自身启动子而不是强启动子进行表达,这一产量在重组大肠杆菌表达鼠李糖脂的研究中仍是较高的,并且可以避免使用IPTG带来的成本问题和操作上的麻烦。对鼠李糖基转移酶基因进行克隆及在大肠杆菌中表达,为该基因在采油菌中的表达研究提供基础。5.鼠李糖基转移酶基因在采油微生物中的表达研究构建了采油微生物接合转移系统,利用该系统成功地将含鼠李糖基转移酶rhlAB和rhlABRI的重组质粒pSAOR2和pJDOR4转移进入采油微生物受体菌中并进行了验证。鼠李糖基转移酶基因在采油微生物中的表达情况与其在大肠杆菌中的表达情况基本一致。只转入结构基因rhlAB的两种采油菌产脂量极低;而转入rhlABRI操纵子的两宿主产脂量均有不同程度提高,其中Pseudomonas sp.JH4（pJDOR4）产脂量最高为101.8mg/L。这一表达量虽然没达到期望的高效表达的水平,但还有进一步研究的空间,并且也为采油基因工程菌在微生物采油和石油烃污染的生物修复中应用的可行性研究提供了理论依据和实验基础信息。6.其它优良MEOR菌株的研究在采油微生物的分离筛选过程中得到一株新的降蜡菌LH3,经鉴定为友谊戈登氏菌。该菌株在好氧和厌氧条件下均能利用C₁₈～C₃₆的烃组分生长从而降解石蜡,好氧时降蜡率约达18.0%,厌氧时降蜡率仅为好氧条件下的1/8。该菌最适生长温度为37～40℃;在5%盐浓度下能很好地生长,对10%高盐浓度有一定的耐受能力;0.2%鼠李糖脂表面活性剂可以明显促进其生长和提高降蜡率。此外,该菌能够降解石油,降油率达到10.4%,降粘率达到44.7%。这些结果显示,G.amicalis LH3作为微生物清防蜡菌剂有较好的应用前景,在原油污染的生物修复和MEOR助采方面也具有很好的应用潜力。此外,后续的研究中可将其作为鼠李糖脂复配菌株或采油工程菌的受体菌株进行深入的研究。更多还原

【Abstract】 Nowadays,the decreasing of the world’s petroleum reserves brings serious energy crisis.In fact,it is generally accepted that only about 30%of the oil in the underground can be recovered using conventional technology.The great mass of crude oil,especially those high viscosity oil is remained in oil reservoir.However, microbial methods（microbial enhanced oil recovery,MEOR）are proved to be effective in recovering high viscous and residual oil.Therefore,it is significent to investigate MEOR to enhance crude oil recovery and make the best use of existing oil reservoir resources.In addition,spills from the production,processing and transport of crude oil pollute seriously the ecological environment.Bioremediation of hydrocarbon contaminated soil is proved effective.Rhamnolipid produced by Pseudomonas aeruginosa has good properties including the rapid reducing of surface tension and interfacial tension between water and oil; emulsifying and dispersing oil hydrocarbon;reducing oil viscosity and increasing the bioavailability of hydrophobic compounds to accelerate microbial biodegradation and transformation of long-chain hydrocarbons.Moreover,the notable advantages of rhamnolipid of biodegradability and low toxicity are optimal for applications in MEOR as well as in bioremediation.Rhamnolipid is found to be produced mainly by Pseudomonas aeruginosa up to now,whereas the composition,performance and yield of rhamnolipids from different P.aeruginosa strains were different.It is significant to screen the rhamnolipid producing strains for application in petroleum industry.Moreover,P.aeruginosa is not a safe industrial strain beacause of its opportunistic pathogenicity.It may be a effective meathod to avoid virulence of P.aeruginosa by genetic engineering means using other non-pathogeneic microorganism to construct rembinant rhamnolipid producing strain.The aim of this work is to screen the strain which has good features for MEOR and can produce rhamnolipid,clone the key enzyme for rhamnolipid synthesis and express it in heterologous host of E.coli or in MEOR strains.Very little information was available concerning on expression of the key enzyme for rhamnolipid synthesis of rhamnosyltransferase gene in MEOR strains.This work will provide us a theoretic and experimental preparation for subsequent researches on heterologous production of rhamnolipid biosurfactant in the MEOR microorganisms.The main research works and results were summarized as follows:1.Screening and isolation of facultative anaerobic crude oil-degrading microorganisms producing rhamnolipidA facultative anaerobic crude oil-degrading strain producing rhamnolipid named SH6 was isolated from oil field samples.It was identified by morphology methods, physiology and biochemical tests and 16S rDNA analysis as Pseudomonas aeruginosa.P.aeruginosa SH6 could grew well using crude oil(C₁₃～C₂₈)as the sole carbon source and stimulated the emulsification and dispersion of oil.It could degrade 18.8%of the crude oil,decreased oil viscosity by 47.7%,and decreased the surface tension of the fermentation broth to 37.4 mN/m.It is demonstrated that P. aeruginosa SH6 could produce rhamnolipid using TLC,IR and blue plate analyzing methods.2.The researches on the physical and chemical characteristics of rhamnolipidThe components of crude rhamnolipid biosurfactant produced by P.aeruginosa SH6 was analyzed using HPLC-MS.Eight rhanmolipid homologs of the biosurfactant mixture produced by P.aeruginosa SH6 using glycerol as the sole carbon source were isolated and identified.The rhamnolipid homologs is composed of one or two rhamnose molecules linked by one or twoβ-hydroxy fatty acids of saturated or unsaturated alkyl chain between C₈～C₁₂.The critical CMC concentration of the rhamnolipid is 166.7 mg/L,the surface tension is 29.5 mM/m.The rhamnolipid shows excellent surface activity at high or low temperature（-20～120℃）,high salinity（<50% NaCl,20%MgCl₂ or CaCl₂）and pH（1～7）and it also displays good stability and emulsifying activity to many kind of hydrophobic organic substance,such as liquid paraffin,toluene,peanut oil and crude oil.These features suggest that the potential of the kind of rhamnolipid in MEOR and in bioremediation for oil hydrocarbon contaminated soils. 3.Effects of carbon and nitrogen sources on rhamnolipid productionDifferent kinds of carbon and nitrogen sources on cell growth and rhamnolipid production were studied.P.aeruginosa SH6 could produced rhamnolipid using many kinds of carbon and nitrogen sources.Single-factor experiments show that the optimal carbon sources were the 1:1（w/w）mixture of glucose and rapeseed oil,the optimal nitrogen sources were sodium nitrate and yeast extract,and the optimal C/N ratio was 18 for rhamnolipid production,with the highest rhamnolipid yield of 5.40g/L.The optimal concentration of carbon and nitrogen sources were studied with the constant C/N ratio of 18,the maximal rhamnolipid production of 6.37 g/L was achived using concentrated medium.The results provide essential informations for the optimizition of medium compositions and fermentation conditions.4.Cloning and expression research of rhamnosyltransferase geneRhamnosyltransferase gene clusters of rhlAB and rhlABRI of P.aeruginosa SH6 were amplified,identified and cloned into E.coli JM109.Two recombinant plasmids of pSAOR2 and pJDOR4 were constructed and expressed in E.coli SM10,E.coli DH5αunder the control of its own promoter.The results showed that rhamnolipid production was increased slightly in both E.coil DH5αand SM10 after transfer of rhlAB gene cluster,but the production was obvious enhanced after the transfer of the whole rhlABRI gene cluster.The maximal rhamnolipid production of 105.2 mg/L was obtained by E.coli DH5α（pJDOR4）,which is the highest yield produced in recombinant E.coli without IPTG induction.The results of this study provided useful information for future researches on heterologous production of rhamnolipid biosurfactant in other non-pathogeneic MEOR microorganisms.5.Expression of rhamnosyltransferase gene in MEOR microorganismsA conjugative gene mobilization system was constructed for gene transformation of rhamnosyltransferase gene to MEOR microorganisms and two recombinant plasmids of pSAOR2 and pJDOR4 were transformed succeesfully in MEOR microorganisms. Rhamnolipid production was improved in the strains containing pJDOR4.The maximal rhamnolipid production of 101.8 mg/L was obtained by Pseudomonas sp. JH4（pJDOR4）.Although the production level needs to be increased for real applications,the result still can provide some useful information for the future construction of recombinant strains in applications of MEOR or bioremediation for oil hydrocarbon contamination.6.Performance studies of other excellent hydrocarbon degradation bacteriumA novel acultative anaerobic paraffin and oil degrading strain,Gordonia amicalis LH3,was isolated and identified.It could grew well using C₁₈～C₃₆alkalines as the carbon source in aerobic or anaerobic conditions.In aerobic condition,it utilized about 18.0%of paraffin.In anaerobic condition,paraffin utilization was about 1/8 of aerobic condition.The strain sustained the salinity up to 5%of NaCl and the temperature upper than 40℃.Addition of 0.2%of rhamnolipid increased the growth and paraffin degradation of G.amicalis LH3.The strain could also grow on oil, decreased oil viscosity by 44.7%and degraded oil by 10.4%under aerobic condition. The results showed that G.amicalis LH3 is potential in paraffin control,MEOR and in bioremidition of hydrocarbon-polluted environments.更多还原