| Biosurfactants were widely used in enhanced oil recovery and environmental bioremediation due to its properties of high surface activity,safe and nontoxic,biodegradable.The vast majority of studied biosurfactants-producing bacteria are aerobic microorganisms.But in some application field of anoxic environment,such as in situ production of biosurfactant for enhancing oil recovery in oil reservoirs and in situ bioremediation of river sediment,biosurfactants-producing bacteria which can produce biosurfactant under anaerobic conditions are ungently needed.Biosurfactants-producing bacterial strains which can produce biosurfactant under anaerobic conditions are scarce.The treatment of sewage produced after chemical flooding is high-cost and difficult.The development of environmentally friendly oil recovery technology also needs biosurfactants-producing bacteria which can produce biosurfactant under anaerobic conditions.In this dissertation,Pseudomonas aeruginosa strain which can produce rhamnolipids under anaerobic conditions was isolated.Culture medium optimization and gene modification were used to enhance the anaerobic production of rhamnolipids.The enhanced oil recovery mechanism of P.aeruginosa strains producing rhamnolipids under anaerobic conditions was studied.The application potential of enhancing oil recovery by P.aeruginosa strain was also evaluated.The research results are of strategic significance to develop inoculant of microorganisms producing biosurfactant under anaerobic conditions and to develop environmentally friendly oil production technology.A facultative anaerobic bacterial strain SG was isolated from Xinjiang oil reservoir production water.It can produce 8237.5 mg/L of rhamnolipids under aerobic conditions and produce 222.6 mg/L of rhamnolipids under anaerobic conditions.Combined with the morphological description and 16 S rDNA sequence(GenBank accession number KJ995745)analysis,strain SG was identified as Pseudomonas aeruginosa SG.Strain SG can well anaerobically produce rhamnolipids at temperatures(30℃~40℃),pH values(6.0~7.5),and salinities(lower than 30 g/L of NaCl),respectively.Under anaerobic conditions,strain SG reached the stable phase of cell growth in 34 h,and the highest yield of rhamnolipids occurred at 192 h.Oil spreading method was established as a rapid and reliable method to quantitatify rhamnolipids in culture.There was a significant linear regression relationship(P<0.001)between the diameter of formed oil spreading circle and the concentration of rhamnolipids(100-800 mg/L).Bacterial cells and other metabolites in culture have no effect on discharge of oil spreading method.The best carbon source and the best nitrogen source for anaerobic production of rhamnolipids by strain SG were glycerol and nitrate,respectively.Limited nitrogen source and phosphorus source are not conducive to anaerobic production of rhamnolipids.The medium optimized by response surface method contained 70.32 g/L of glycerol,4.87 g/L of NaNO3,5.49 g/L of KH2PO4,6.97 g/L of K2HPO4·3H2O,0.40 g/L of MgSO4·7H2O,0.13 g/L of CaCl2,1.0 g/L of KCl and 1.0 g/L of NaCl.Using the optimized medium,683.4 mg/L of rhamnolipids was produced by strain SG under anaerobic conditions.Using molecular biology technology,the fusion genes Popr-rhlAB were constructed by replacing the original promoter of rhlAB genes by the strong promoter PoprL of the coding gene of peptidoglycan-associated lipoprotein.Then increasing the copies of the fusion genes Popr-rhlAB in strain SG,an engineered strain Pseudomonas aeruginosa PoprAB was constructed.In this study,anaerobic production of rhamnolipids by strain SG was improved;gene modification of bacterial strains producing rhamnolipids under anaerobic conditions was achieved.The engineered strain PoprAB can produce 1094.6 mg/L of rhamnolipids under anaerobic conditions,which is 60.2% more than the yield of strain SG.The rhamnolipids product produced by strain SG under aerobic conditions has a critical micelle concentration(CMC)of 60 mg/L;the rhamnolipids product produced by strain SG under anaerobic conditions has a CMC of 80 mg/L.Anaerobic rhamnolipids product of strain SG has better emulsifying activity(EI24 = 80.33%)to crude oil than that of aerobic rhamnolipids product(EI24 = 62.30%),which demonstrates that using the anaerobic rhamnolipids product of strain SG for MEOR is more advantageous.Results of HPLC-MS analysis showed that the aerobic rhamnolipids product contained three kinds of mono-rhamnolipids(54.76% of relative abundance)and six kinds of di-rhamnolipids(45.24% of relative abundance).Anaerobic rhamnolipids product contained four kinds of mono-rhamnolipids(95.69% of relative abundance)and three kinds of di-rhamnolipids(4.31% of relative abundance).The different structure and composition between the aerobic and anaerobic rhamnolipids product of strain SG led to the different activity of these two kinds of rhamnolipids product.Through the reservoir simulation experiments,the oil displacement mechanisms of bacterial strains producing rhamnolipids under anaerobic conditions were illuminated.The potential of ehnacing oil recovery by bacterial strains producing rhamnolipids under anaerobic conditions was also verified.The wild type strain SG and engineered strain PoprAB can steadily grow and efficiently produce rhamnolipids under simulated reservoir conditions.Addition of the engineered bacterial strain PoprAB firstly changed the microbial community structure in simulated reservoir system,and then made the microbial community structure get close to the level of the initial stage.Genus of Pseudomonas was always one of the domain genera during culture process.Results of physical simulation core displacement experiment showed that wild type strain SG and engineered strain PoprAB can enhance extra oil recovery values of 7.26% and 9.15% on the basis of first water flooding,respectively.All the results confirm that using the bacterial strains which can produce rhamnolipids under anaerobic conditions for in situ MEOR is promising and feasible.In this paper,strains Pseudomonas aeruginosa SG and P.aeruginosa PoprAB provide microbial resources for development of new-style MEOR technology based on in situ production of rhamnolipids in oil reservoirs. |
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