| In recent years, the soil pollution caused by the petroleum becomes more and more serious. In the process of crude oil extraction, its processing and transportation, large quantities of oil and its products were sent into the soil, which caused soil pollution. It does great harm to human and even the entire biosphere, and is becoming a worldwide environmental problem. Bioremediation, as a potential clean technology, with the advantages of high efficiency and low cost, increasingly draw universal attention from international society in the research work of controlling the oil contamination. Research on bioremediation is very active in the world in recent ten years, and even in some countries, this technique had been applied to actual remediation of soil pollution, which has already got remarkable achievements.Three highly efficient petroleum degrading strains, named DH-5, DH-8and DH-9, were screened and isolated from the soil around the long term extracting well in Shengli oilfield. The crude oil degradation rates from DH-5, DH-8and DH-9were42.39%,40.42%and43.30%after incubating for7days, respectively. After morphological observation, physiological and biochemical identification, and molecular biology identifications of16S rDNA of isolated high efficient oil-degrading bacteria were performed, DH-5, DH-8and DH-9were found to belong to Pseudomonas, Citrobacter and Klebsiella, respectively. Then the three strains’capacity of utilizing different substrates was studied by using common petroleum products and typical straight-chain alkanes and aromatic hydrocarbons as the sole carbon source. We found that strain DH-5grew well on long-chain alkanes such as liquid paraffin (C16-C20), strain DH-9grew well on short-chain alkanes such as hexane (C6), and both of the strains could utilize cycloparaffins (such as Cyclohexane and Cyclohexanone) well. However, strain DH-8grew poorly or even showed no growth on alkanes, but it showed strong growth on naphthalene and Phenanthrene, while strains DH-5 and DH-9grew poorly on the two pAHs. All the3highly oil-degrading bacteria strains could use various components of crude oil, and the capacity of their utilizing hydrocarbons was in the order of DH-5> DH-9> DH-8.Environmental condition for petroleum degradation of the two petroleum hydrocarbon degraders DH-5and DH-9, which used alkane compounds well, were further optimized by orthogonal experiment. It was found that strain DH-5’s optimal conditions for degradation were0.5%crude oil,500mg·L-1N sources,50mg·L-1P sources,15mg·L-1arginine (Arg),7.0in pH at30℃, and its highest petroleum degradation was63.40%; while strain DH-9’s optimal conditions for degradation were0.3%crude oil,400mg·L-1N sources,150mg-L"1P sources,5mg·L-1aspartic acid (Asp),7.5in pH at30℃, and its highest petroleum degradation was63.77%. It indicated that the abilities of oil degradation by the two strains had been improved significantly after futher optimization.In order to study their degrading effect, oil’s four components (hydrocarbons, aromatic hydrocarbons, colloid and asphaltene) before or after their orthogonal optimization treatments were analyzed by column chromatography. When natural degradation rate had been deducted from total, degradation rates of colloid and asphaltene by strain DH-9was significantly higher than those of strain DH-5before optimizing condition, and after orthogonal experiment, the degradation rates of hydrocarbons, aromatic hydrocarbons, colloid and asphaltene by strain DH-5increased significantly,62.01%,58.86%,46.62%and9.33%respectively, while the degradation rates of oil’s four component by strain DH-9were also increased to63.97%,62.03%,37.16%and14.67%respectively. Above all, the capacity of their utilizing oil’s four component was in the order of saturated hydrocarbons> aromatic hydrocarbons> colloid> asphaltene.In order to understand the microbial metabolism on oil, the composition and content of the fatty acids in metabolites of crude oil were determined by gas chromatography (GC). It was found that acetic acid, propionic acid, butyric acid and valeric acid were all detected in the medium, and acetic acid was the major short carbon chain organic acid. Besides, the content of acetic acid in medium of strain DH-5was obviously higher than in the medium of the other two stains. Also,13types of long-chain fatty acids were detected in the medium. The variety and content of these fatty acids produced by the3strains were different, and strain DH-9produced more types of fatty acids than the other two strains. The carbon numbers of the fatty acids were concentrated in C8-C24produced by strain DH-9, and the carbon numbers of the fatty acids were concentrated in C8-C18produced by strains DH-5and DH-8. However, the fatty acids produced by the three strains were almost alkanoic acids and olefine acids. Thus, it concluded that the monoterminal oxidation pathway and the direct dehydrogenation pathway were probably the oil-degrading pathways of the above-mentioned3high oil-degrading bacteria strains.Cyclohexanone degradation rates of strains DH-5, DH-9and A-1(preserved strain of our lab) were94.84%,93.60%and94.39%after incubating for3days, respectively. Then TLC, FT-IR and GC/MS analysis methods were used to determine the metabolites of cyclohexanone by the three strains. Cyclohexanol, caprolactone, phthalic acid and adipic acid were all detected, it confirmed that cyclohexanone was likely to be oxidated to8-caprolactone by cyclohexanone monooxygenase, and then s-caprolactone was transformed to adipic acid for β-oxidation by esterase and dehydrogenase.2,5-dihydrofuran was found in the metabolites of strain DH-5, which Indicated a novel metabolic pathway on cyclohexanone of strain DH-5. Besides, cyclohexanone monooxygenase gene (chnB) fragment of strain DH-5was also cloned successfully, it provided theoretical basis for studying the function of cyclohexanone monooxygenase and the metabolic process of the... |
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