Studies On The Screening Of MEOR Microbial Strain, Rhamnolipid Performance, And Rhamnosyltransferase Gene Cloning And Expression

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.