| Four efficiency oil-degrading strains which had screened in the laboratory including Raoultella planticola, Serratia marcescens, Bacillus cereus, Klebsiella variicola were applied to constructing mixed bacteria in order to improve the remediation of oil contamination.Response Surface Methodology(RSM) based on Box-Benhnken Design(BBD) was applied to design the experiments and optimize the ratio of mixed bacterial consortium. Biodegradation products of oil compounds by pure and mixed bacterial cultures were analyzed, which provided theoretical foundations for the study on degradation metabolism of hydrocarbons and screen for functional microorganism. The natural organic materials(wheat straw, biochar and activated carbon) were selected as carriers to obtain immobilizied strains by absorption-entrapping method.Immobilized microorganisms were applied in the remediation of simulating oil-contaminated soil in the indoor pot. The repairing effect of each immobilized strain, the soil microbial community structure and distribution profiling were discussed.Optimization of hydrocarbon-degrading bacterial group ratio by RSM based on BBD showed that the maximum removal efficiency of petroleum hydrocarbon achieved 68.83% when the inoculation quantity of four strains(Rp, Sm, Bc, Kv) is 4%, 2%, 2% and 2%, respectively. The degrading activity of the mixed strain kept strongly in a large range of salinity and p H. Best salinity is 0.5% and p H is 7.5. When the salinity was 5%, 40.37% of the objective pollutants were still degraded. So it can be used to repair the oil contaminated seawater.Pure bacteria degraded 41.83~54.87% of diesel and the optimal mixed strains removal ratio was 64.27%, which were cultured for 5 days at 30℃ using 0# diesel oil as the sole carbon source. GC-MS analysis showed that Raoultella planticola and Klebsiella variicola can degrade C12~C16 straight chain alkanes and some branched ones, while Bacillus cereus and Klebsiella variicola can degrade C12~C22 straight chain alkanes. A suspension of strains oxidized n-hexadecane to a mixture of internal monohexadecenes. The latter exhibited a cis configuration and carbonyl, and the mixture consisted of the following: cis-9-hexadecenal; cis-11-hexadecenal. The dehydrogenation of hydrocarbons appeared to be related to the formation of unsaturated fatty acids in Bacillus cereus and Klebsiella variicola.It is postulated that double bond and carbonyl insertion may represent an early step in a new pathway of n-Hexadecane degradation.Absorption-entrapping immobilized strain had higher oil removal efficiency than the free strain in a relatively short time. About 99.76% oil has been removed by the biochar immobilized mixed strains in 5 days. The removal ratio of wheat straw, biochar and activated carbon immobilized ball without mixed strains followed as 49.08%, 61.48% and 63.12%, respectively. The immobilized microorganism bead was similar as a microreactor consisting of adsorption and biodegradation. Scanning electron micrographs images showed that the outside and inside of biochar and activated carbon immobilized ball had a large number of pores and full of skeleton structure. SEM images also indicated that cells were successfully entrapped in the beads and grown well.The experiment of immobilized microorganism simulated repairing oil contaminated soil showed that the immobilized strains had better removal of oil than free strain and the bioremediation effect were varied by using different carriers. The effect of wheat straw immobilized mixed strain was the best after repairing sterilized oil-contaminated soil 42 days, the oil degradation extent of it was 54.92%, nearly 20% higher than the one by non-immobilized mixed strains; at the same time, biochar immobilized strains have better removal of oil(54.19%) than wheat straw and activated carbon in unsterilized oil-contaminated soil, nearly 30% higher than free mixed strains. That wheat straw can enhance soil nutrient content was significantly impact factor for improving oil mixed bacteria degradation rate in the absence of indigenous bacteria competition. However, in the unsterilized soil, biochar was the best carrier to provide optimum growth environment to resist the growth of indigenous bacteria competition.Urease activities of wheat straw immobilized strains have the highest values in the sterilized treatment. It reaches 0.75 mg?g-1?24h-1, which was 3.26 times greater than that in control group. We also found that urease activities of biochar immobilized strains was highest 1.15 mg?g-1?24h-1 in the unsterilized treatment, which was 2.05 times greater than that in control. Soil urease activity showed a positive correlation with oil degradation rate.The Shannon-wiener index of soil and changes of bacterial community structure monitored by terminal restriction fragment length polymorphism(T-RFLP) technique showed that the diversity in sterilized treatment soil decreased. The change in unsterilized treatment soil was first increased then decreased.The efficiency of oil removal was sensitive to bacterial diversity. Firmicutes and Proteobacteria from immobilized mixed strain played an important role in the process of petroleum biodegradation.The total of Firmicutes and Proteobacteria about biochar immobilized mixed strain in sterilized treatment and unsterilized treatment were 5.37%(Firmicutes 2.25%; Proteobacteria 3.12%) and 3.73%(Firmicutes 0.48%; Proteobacteria 3.25%), respectively. Compared with straw and activated carbon, biochar contributed more to the growth of Firmicutes and Proteobacteria. We also found that Bacilli and Gammaproteobacteria were higher in wheat straw immobilized mixed strain in sterilized treatment and biochar immobilized mixed strain in unsterilized treatment, respectively.Clustering analysis based on the T-RFLP profiles showed that bacterial community structures similarity of wheat straw immobilized mixed strain in sterilized treatment and biochar immobilized mixed strain in unsterilized treatment were relatively high. The same similarity to biochar and active carbon immobilized mixed strain. However, compared with other five treatments, community similarity of wheat straw immobilized mixed strain in unsterilized treatment was low. |
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