Study On Isolation And Identification Of Hydrocarbon Degrading Bacteriaand TheInterdependence Mechanismbetween Strains

In this study, petroleum hydrocarbon degradation flora were cultured in the bio-trickling filter, in which seeded with waste activated sludge from a municipal wastewater treatment plant, and diesel oil as the sole carbon source. A high-efficiency hydrocarbon degrading bacterium X2b was isolated from the bio-trickling filter. Morphological, physiological and biochemical identification experiments indicated that X2b was bacillus cereus sp. The study on environmental influencing factors showed that X2b preferentially degraded C9-C14of petroleum hydrocarbon, compared to C15-C23of petroleum hydrocarbon. The optimal NaCl concentration for X2b degrading petroleum hydrocarbon was2%. Moreover, X2b still maintained higher activity, and the degradation rate achieved75%after30d under5%NaCl concentration. The optimum initial pH for X2b degrading petroleum hydrocarbon was7.0. The degradation ability of strain X2b significantly inhibited under pH<6.0and pH>8.0, and the influence of acidic environment was greater than alkaline environment. X2b achieved higher degradation rate (91.8%and89.9%) after3d under the conditions of initial diesel oil concentration of1680mg/L and4200mg/L However, the degradation of various diesel oil components was obviously inhibited under initial diesel oil concentration of16800mg/L, which indicated that excessive initial concentration was unfavorable for the degradation of X2b. This study also extracted genomic DNA and plasmid DNA of Bacillus cereus X2b, and gel electrophoresis was used to determine initial DNA fragments. PCR products of target gene alkB were cloned and identified followed by DNA sequencing. Combined sequencing results with analysis of BLAST, the size of alkB conserved sequence was547bp. The conserved sequence of alkB’s sequence homology with four reported alkB alkane hydroxylase gene was86%,99%,99%and95%, respectively. X2b had the potential ability for the bioremediation of oil pollution in marine environment for high salinity tolerance and facultative anaerobic characteristics.A high-efficiency bio-surfactant producing bacterium X1A-2was isolated from the bio-trickling filter, which reduced the surface tension of fermentation broth from 67.6mN/m to about33.0mN/m by surfactant. Morphological, physiological and biochemical identification experiment indicated that X1A-2was Gordon’s genus. The results of environmental impact factors for bio-surfactant production showed that X1A-2adapted to pH from6.0to8.0, but the surfactant producing capacity of X1A-2was obviously inhibited under extreme pH. X1A-2grew well under the conditions of below4%NaCl concentration, the temperature range of25℃～40℃, but X1A-2grew slowly under lower temperature conditions. The presence of petroleum hydrocarbon had little effect, while glucose and other carbohydrate carbon sources had certain inhibition effect on surfactant production. In the simulation experiments for oil pollution, the optimal condition of X1A-2producing bio-surfactant was pH7.5, temperature30℃and2%NaCl concentration.A lipopeptide was isolated and purified from the fermentation broth of bacterium X1A-2. The results showed that the molecular weight of the bio-surfactant was1022Da, its fatty acid was C13β-hydroxy fatty acid, and the link order of amino acids was Glu-Leu-Leu-Val-Asp-Leu-Ile, which was a lipopeptide biosurfactant and C13surfactin of surfactin series. The results also showed that surfactin series of lipopeptide were generated by Gordon’s genus (Gordonia).The metabolites of strain X2b were analyzed under the conditions of n-hexadecane as the sole carbon source. The results showed that hexadecylamine alcohol and3-hydroxy-decane acid was existed in the metabolites. The analysis of metabolites combined the synthetic pathway of surfactin in vivo and the identification of the surface surfactant inferred that the coexistence of two strains mechanism:the primary metabolite of X2b, that is,3-hydroxy fatty acids opened the production mechanism of surfactin. The surfactin generated by X1A-2further promoted the dispersion of petroleum hydrocarbon in the water, which led to better degradation effect for petroleum hydrocarbon.The results explained why the bio-surfactant producing bacteria were easily found in petroleum hydrocarbon degradation flora from the microbial mass transfer point of view, and the phenomenon that X1A-2could grow and reproduce in the bio-trickling filter, which didn’t have the hydrocarbon degradation capacity. The conclusion provided very valuable reference for the bioremediation of marine oil pollution.