Studies On Isolation Of Oil-Degrading Bacteria And Microbial Remediation Of Oil Contaminated Soil

Soil oil-pollution becomes a globlly environmental problem, and is more and more concerned by scientists in recent years. The bioremediation technique relying on the microbial metabolic degradation of contaminants, with its advantage of low cost and no second pollution developed rapidly and has been a major enviromental treatment method with a promising application prospects. But at present, many problems of microbial remediation technology, such as lack of efficient petroleum degrading strains, unreasonable method of building strains group, faultiness of bioremediation technique, limit its practical application. In this research, high efficient petroleum degrading strains were screened from Daqing oil-contaminated soil based on the microbial diversity analysis through T-RFLP technology. And the oil-degradating stains group was structured by analyzing the crude oil degradation components. Remediation effect on oil-contaminated soil was studied in order to assess repair ability of the group. The research results were listed below:(1) Microbial community structure had obvious regularity fluctuations with the increase of oil pollution degree. T-RFLP scanning data showed the numbers of competitive T-RFs fragment declined, and the overall competitive T-RFs were198bp,185bp,133bp,121bp etc.MDS ranking and clustering analysis of CLUSTER indicated the soil with oil content around4975mg·kg-1(B group) had formed a relatively stable microbial communities structure, where the oil-degrading strains were most likely to be isolated.(2)10strains with higher oil degradation abilities were selected from Daqing oil field where the oil content was4975mg·kg-1, and then these strains were numbered from W-1to W-10, of which Strain W-7had the highest oil-degradation rate of25.28%. Strains groups with the top five higher degradation rates were selected to make GC-MS analysis on the products after their degradation of crude oil. The results showed that those strains had different degradation ranges of carbon chains. W-3had the degradation ranges of carbon chains from C21to C32. W-2could degradate C16, C15and C20. And C12～C17were more easily degradated by W-5while W-7had advantage in short carbon chains degradation.Therefore, W-2, W-3, W-5and W-7were selected in order to build degrading bacteria group for the obvious complementary in their degradation carbon chains.(3) The Strain W-8which could use naphthalene as the sole carbon source was isolated from10oil-degrading bacterials. The Strain W-8was preliminarily identified as Serratia sp. by the morphology, physiological-biochemical characteristics and sequencing analysis of its16S rDNA. The optimal temperature and pH for its growth were35℃and7.5. It could tolerate the high concentration of NaCl and naphthalene. Under the treatments of30g*L NaCl, the degradation rate of naphthalene was80.9%after culture for3days when the naphthalene concentration was100mg·L-1, even the degradation rate reached15.8%when 800mg·L-1. According to the analysis of GC-MS and the absorbance measured of different substrates, the W1strain could utilize other aromatic hydrocarbons, such as phenol, methylbenzene, benzoic acid,1-Naphthalenol, acetone and octane for growth. It also could degrade aliphatic hydrocarbon of C20～C23, C33～C36in crude oil. The pathway of naphthalene degradation was measured by scanning the metabolic intermediates of degradation with UV-Vis. The possible degradation pathway of W1strain was catechol pathway. Naphthalene was first turned into salicylic acid, and then pyrocatechol, small molecule substance by opening benzene ring, finally entered the tricarboxylic acid cycle.(4) Cultivation conditions of the strains group was optimized by an orthogonal experiment, in which the best inoculation proportion was1:1:1:1, the optimal nitrogen source was NH4NO3and the best source of phosphorus was KH2PO4, The optimum temperature for the growth of strains group is30℃, and their growth environment is neutral to acid. GC-MS analysis result displayed that all strains in the group exserted a harmonious effect on oil degradation. Oil-pollution remediation simulation experiment shows N, P and the other nutrients in soil were the main factors limiting effect of microbial degradation.