The Research Of Surfactin Bio-surfactant High-yield Strains And Its Mechanism In Enhanced Oil Recovery

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, NH₄NO₃ 4.0g, Na₂HPO₄·12H2O 3.0g, KH₂PO₄ 3.0g, FeSO₄ 6.8μmol,ZnSO₄ 0.038mmol,CaCl₂ 0.5mmol,MgSO₄ 0.2mmol,MnSO₄ 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), Ca²⁺/Mg²⁺(0.0-1.0×105mg/L), and temperature (50¹20℃). 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.