Isolation,optimization And Application Of Petroleum Degrading Bacteria

Petroleum is a kind of complex compound with teratogenic,carcinogenic and mutagenic effects.After entering the soil,petroleum will lead to negative impacts such as soil hardening,bituminization and nutrient imbalance.The remediation of petroleum pollution has been studying as an important issue in the field of environmental science,and the microbial remediation is considered as one of relatively economic and eco-friendly remediation methods.Therefore,this study aims to isolate efficient petroleum degrading bacteria from petroleum contaminated soil and optimize their degradation conditions for petroleum hydrocarbons.Meanwhile,the degradation mechanism of petroleum hydrocarbons by bacteria was discussed.Additionally,the dominant strains were applied to remediate the petroleum contaminated soil in the field,which could provide strain resources and theoretical basis for the bioremediation of petroleum pollution.The main conclusions are as follows.(1)Seven strains that could degrade petroleum,with the capacity of petroleum degradation rate more than 30%,were obtained from isolation and purification.There were three strains named Acinetobacter sp.Y183,Pseudomonas sp.Y187 and Bacillus sp.N47 selecting to optimize their petroleum hydrocarbon degradation conditions.The optimum growth conditions of Acinetobacter sp.Y183 were temperature 30℃,Na Cl content 0 g/L,p H = 8,petroleum concentration 10 g/L.The optimum nitrogen source was 1 g/L ammonium chloride.Under the optimum conditions,the amount of petroleum degradation increased from 1.047 g in 10 days to 3.231 g in 5 days.For the Pseudomonas sp.Y187,the optimum growth temperature was 30℃,Na Cl content was 0 g/L,p H = 8,petroleum concentration was50 g/L,and the optimum nitrogen source was 0.5 g/L ammonium nitrate or ammonium sulfate.The amount of petroleum degradation increased from 0.912 g in 10 days to 8.987 g in 5 days at the optimum conditions.For the Bacillus sp.N47,the optimum growth condition temperature was 30℃,Na Cl content was 20 g/L,p H = 8,petroleum concentration was 30g/L,and the optimum nitrogen source was 4 g/L ammonium nitrate.The amount of petroleum degradation increased from 1.470 g in 10 days to 2.149 g in 5 days under these conditions.(2)The degradation mechanism of petroleum hydrocarbon via three dominant strains was explored through the oil displacement test.It was found that Pseudomonas sp.Y187 could produce biosurfactant,which belonged to the type of lipopeptide verified by the thin layer analysis.Furthermore,based on the PCR technology,the strains Pseudomonas sp.Y187 and Bacillus sp.N47 existed in aromatic-ring-hydroxylating dioxygenase gene(RHDα)and benzoic acid 1,2-dioxygenase gene(ben A)and alkane oxygenase gene(alk B)and alkane hydroxylase gene(alk Bw),respectively.However,no degradation genes were found in Acinetobacter sp.Y183.(3)Three strains above mentioned was selected set eight treatments to investigate their ability to degrade petroleum hydrocarbons in petroleum contaminated soil.The results showed that the petroleum contaminated soil added with single strain N47 or Y187 could shorten the degradation time of petroleum hydrocarbons,which decreased from 75 days to14 days compared with the control(no strain addition).The combination treatment with Y187 and Y183 strains could decrease the degradation time of petroleum hydrocarbons from75 days to 30 days.Additionally,the degradation rate of petroleum hydrocarbons increased2.37% when tested soil applied with N47 and Y183 strains.In summary,three strains of petroleum hydrocarbon degrading bacteria obtained in this study have significant application in petroleum pollution remediation,especially Pseudomonas sp.Y187 and Bacillus sp.N47,which have strong capacity of oil displacement and petroleum hydrocarbon pollution remediation.Therefore,this study provides important bacterial sources and theoretical support for the remediation of petroleum pollution.