Biosurfactant-producing Bacteria In Oil Reservoir: Strain Isolation, Optimization Of Culturing Condition, And Characterization Of The Products

Biosurfactant, mainly produced by microorganisms or plants, are varieties of hydrophilicand hydrophobic compounds. Biosurfactant has attracted more and more attention due to thebiodegradebility, low toxicity, and high surface activity compared to chemical counterparts.There are diversity of microorganism such as biosurfactant-producing bacteria in the reservoirs.The microbial metabolic products, like biosurfactants, biopolymers, etc, can lower the freezingpoint of crude oil, decrease the oil flow resistance, improve the fluidity of crude oil, and enhancethe recovery of the crude oil.In this thesis,11biosurfactant-producing strains were isolated from produced water inDaqing oil reservoir. A high-yield biosurfactant-producing strain DY-9was identified and chosenaccording to the decrease in surface tension and oil spreading diameter measurement. Analysis of16S rDNA sequence and the phenotypic and physiological characteristics showed that DY-9was99%similar to Bacillus licheniformis.The biosurfactant produced by DY-9was separated, and purified through extraction. Themain structure of DY-9was similar to surfactin by analysis of the profiles o TLC, HPLC-MS andNMR. The critical micelle concentration was10mg/l, which was far lower than that of thepreviously reported. The emulsion value of the paraffin emulsion effect remained around55%after96hours. The biosurfactant produced by DY-9was stable in25to100℃, and at pH2to12.The surface tension of biosurfactant produced by DY-9was below30mN/m when it wassubjected to high salinity (10%of NaCl).In order to further improve the biosurfactant yield, single factor combined with responsesurface method was used to optimize the fermentation conditions of DY-9. Through the singlefactor method, the optimal carbon source, nitrogen source, temperature, pH, and oxygen requiredwas determined, respectively. Then, significant factors were selected by Plackett-Burmansoftware, and the path of the steepest ascent was utilized to approach the optimal region. Underoptimal culturing conditions, the yield of biosurfactant by DY-9was increased by19.6%.