Selective Stimulation Of Indigenous Biosurfactant-producing Microbes In The Oil Reservoir

The biosurfactant production is one of the dominant mechanisms of microbialenhanced oil recovery （MEOR）. At present, there are mainly two kinds of applicationof biosurfactants in MEOR, which are the injection of fermentation production（ground method） and exogenous microorganisms （exogenous microbial flooding）,respectively. Compared with ground method and ex-situ microbial flooding, in-situmicrobial flooding is more inexpensive, process simple, and environmental friendly.However, there are few studies concerning the detection of biosurfactant-producingmicrobes accurately. Especially，it is hard to regulate and control the indigenousbio-producing microbes rationaly.In this paper, a new method was constructed, which can analyse function genes ofindigenous biosurfactant-producing microbes of oil reservoir qualitatively andquantitatively. Based on the analysis to the bio-producing microbes of Shengli OilField, the Zhan3block was choosen as the object of study to selectively stimulateindigenous biosurfactant-producing microbes. As the result, a formula of activatorswas determined. This paper showed the influence of the stimulationbiosurfactant-producing microbes to the relative permeability profile, rock wettability,and oil recovery. Finally, the physicochemical properties and structure of thebiosurfactants produced by indigenous microorganisms were also analysed. The mainresults are as followings.1、There are two types of degenerate primers which are the surfactin/lichenysin（srfA3/licA3） gene involved in lipopeptide biosurfactant production in members ofBacillus subtilis/licheniformis group and the rhlR gene involved in regulation ofrhamnolipid production in pseudomonads. There were ten well produced fluids fromfive blocks of Shengli Oil Field, which were detected and analysed concerninglipopeptid/glycolipid biosurfactant-producing microbes. The methods of culture andcloning and sequencing were used subsidiarily to judge the species of the microorganisms. The results were as followings, The srfA3/licA3gene involved inlipopeptide biosurfactant production has been detected in most of the reservoir brinesand lipopeptide biosurfactant-producing microbes should be the target to biostimulatethe production of lipopeptide biosurfactants by indigenous microorganisms.2、Target at the detection of lipopeptid-producing microbes, the technology ofReal-Time PCR was applied into quantitative analysis of lipopeptid-biosynthesis genewhich was standardized with srfA3/licA3gene. The real-time fluorescence quota PCRmethod which was for the test of lipopeptid-biosynthesis gene were fleet, sensitive,and specific, which can be used into laboratory and field tests.3、 The feasibility study about biosurfactants biosynthesized by indigenousmicroorganisms was applied at Zhan3block, the comprehensive results of real-timefluorescence quota PCR and cloning and sequencing showed thatlipopeptid-biosynthesis gene has something to do with Bacillus licheniformis. Withthe addition of external carbon source, the stimulating agent formulas of inorganic andorganic nitrogen sources were selected through Orthogonal experimental design,respectively. The formula of organic nitrogen source was determined as corn syrupdry powder0.4%, glucose0.5%, soluble starch0.2%, peptone0.4%, which wasnamed as JHJ-1. The experiment result of the application of JHJ-1to Zhan3blockwas that lipopeptid-biosynthesis gene was maximized as2.6×106copies/uLDNA,surface tension was reduced to38.0mN/m minimally, the biggest diameter ofexpanding oil circle was7.0cm, emulsification index was less than5%. The formulaof inorganic nitrogen source named as JHJ-2was that sucrose2%, NH₄Cl0.6%, yeastextract powder0.03%, corn syrup dry powder0.05%，KH₂PO₄0.06%, Na₂HPO₄0.2%.With the application of this formula, lipopeptid-biosynthesis gene was maximized as1.6×10⁵copies/uLDNA, emulsification index reached up to96%.After thebiostimulation of JHJ-1, the sample had simples microbial community, and thepredominant bacterium was Bacillus licheniformis. Nevertheless, after thebiostimulation of JHJ-2, there would be more complex result, the predominantbacteria were Bacillus licheniformis and Bacillus thermoamylovorans. 4、The influences to the relative permeability curve and wettability were studied,after the biostimulation of biosurfactant-producing microbes. The model test was alsoexperimentalized to evaluate the biostimulation to oil recovery.The oil-water relativepermeability curves of Zhan3reservoir before and after biostimulation ofbiosurfactant-producing microbes by JHJ-1and JHJ-2were measured under thereservoir condition.The result show that the equal-permeability point on the curve ismoving to the right which indicate the core hydrophilicity is increased after microbeflooding. The oil-phase relative permeability is much higher than water flooding.Biostimulation of biosurfactant-producing microbes can drive residual oil effectively.The rock relative wettability indexs are increased which indicate cores hydrophilicityare increased after microbe flooding.The ability of improving oil discovery bybiostimulation of biosurfactant-producing microbes is estimated using core floodingexperiments. Biostimulation of biosurfactant-producing microbes with JHJ-1andJHJ-2can improve oil discovery10%and13.6%respectively on the basis ofwater-flooding under the conditions of Zhan3reservoir.5、The physicochemical properties and the structure of the biosurfactant in thesample which was biostimulation by JHJ-2was analyzed.The yield of biosurfactant inthe sample is0.47g/L.Critical micelle concentration（CMC） of the biosurfactant is33.3mg/L,at the concentration,the surface tension of the biosurfactant solution is38.5mN/m. The emulsifying activity index is1.25U with the sample was diluted100timeswhich indicate the sample emulsifying activity is strong.By means of TLC、IR、HPLCand MS，the structure of the biosurfactant in the sample which was biostimulation byJHJ-2was elucidated to be a type of lipopeptide.The results of this study will privide the technologic support for directionalcontrol of reservoir indigenous microorganism for improving oil discovery.Inaddition,it will provide the theoretical and technologic support for indigenousmicroorganism oil displacement technology bring into practice in Zhan3block.