Bioremediation Of Crude Oil Contaminated Sediments Using Acinetobacter Sp.strain HC8-3S-9

Petroleum is one of the most important natural resources in modern society.However,the oil spill accidents have threatened the human life and ecosystem balance during the process of oil mining,smelting,transport and use.Bioremediation has drawn a growing rate of interests over the years.Compared with conventional physical or chemical methods,the use of microbial metabolic is eco-friendly and cost effective.When using microorganism to degrade petroleum hydrocarbons,it is necessary to evaluate the ecological risks of exogenous microorganisms in order to prevent the adverse effects.Therefore,it is essential to study the response of petroleum hydrocarbon degrading bacteria to environmental factors.The research object is the oil-degrading strain of Acinetobacter sp.HC8-3S,which was selected from the bottom sediments near Penglai 19-3 oil platform.A recombinant plasmid pTn-oKm-egfp containing kanamycin resistance was constructed to insert egfp gene into the Acinetobacter sp.HC8-3S genome.Then,a degrading strain with green fluorescent protein was obtained.A seawater-sediment microcosm was conducted to simulate the process of oil spilling,and the environmental factors in the degradation process were monitored in real time.The results were as follows.?1?The degradation rate constant of the HSO experimental group?Acinetobacter sp.HC8-3S-9+sterilized seawater and sediments+oil?were the lowest with the value of 0.032.The value in ON experimental group?oil+native sediment and seawater?was0.045.The degradation rate constant of the ONH group?oil+native sediment and seawater+Acinetobacter sp.HC8-3S-9?was the highest,with the value of 0.047.The results showed that exogenous engineering bacteria would accelerate the degradation of petroleum hydrocarbons.After 60 days,the content of petroleum hydrocarbon decreased to 10.52%of the initial value.?2?The PO₄^3-content in water samples decreased during the oil degradation.In the experimental group of oil,the content of PO₄^3-was higher than that of the oil-less experimental group.Similarly,the NH₄⁺content in the experimental group of oil maintained at a high level??8 mg/L?15 days ago.However,the NH₄⁺content of each group was extremely low?<0.3 mg/L?15 days later.The variation trend of NO₃^-and NO₂^-in water samples were approximately the same.The contents of NO₃^-and NO₂^-were lower?<0.3 mg/L?15 days ago,while increased and had a significant peak?>1.5mg/L?after 15 days.The correlation between degradation rate and environmental factors in the process of the degradation showed that the degradation rate was positively correlated with the dissolved oxygen content,with the correlation coefficient of 0.45.However,the degradation rate were negatively correlated with temperature,total organic carbon?TOC%?,phosphorus and NH₄⁺,with the correlation coefficients of0.63,0.85,0.66,0.61,respectively.?3?Visualizing the Acinetobacter sp.HC8-3S-9 in the microcosm using fluorescent microscopy.The results showed that all of them have significant luminescence characteristics within the experimental temporal scale of 60 days.The egfp copy numbers were detected by real-time PCR.During the process of petroleum degradation,the copy number of egfp gene decreased,and the final copy number reached about 10%of the initial value.It showed that the green fluorescent protein could be used to characterize the survival,reproduction,development and spatial distribution of the degrading bacteria.DNA samples from the microcosm were extracted,and the microbial community changes of microbial communities were analyzed by high throughput sequencing and bioinformatics.The results showed that the proportion of Proteobacteria was the largest at the phylum level,followed by Firmicutes,Chloroflexi,Nitrospirae Acidobacteria and Planctomycetes.There existed significant differences among the Chloroflexi,Acidobacteria and Gemmatimonadetes.Partial Least Squares Discriminant Analysis?PLS-DA?were conducted on OTU level.The results showed that,the ON experimental group,the HSO experimental group and the control group were able to distinguish well.However,the difference between the ONH experimental group and the HN experimental group was not obvious.These results indicating that under the condition of Acinetobacter sp.HC8-3S-9existence,the addition of oil does not result in obvious group differences.This study favorably completed the expected research objective of clarifing the ecological relationship between exogenous petroleum hydrocarbon degrading bacteria and native bacteria in the microcosmic environment.Moreover,this research expanded the category of the germplasm resources of petroleum hydrocarbon degrading bacteria.The study results built up the corresponding theoretical basis for the microbial remediation of petroleum hydrocarbon.