Degradation Of Alkanes Based On Bacillus Genome Editing

Petroleum hydrocarbon is one of the most important energy materials,and it is the raw material for various industries.With the developments of the economy,people’s demand for refined petroleum products is increasing,resulting a lot of serious pollution.Due to the stable chemical structure,petroleum is usually difficult to be degraded.The oil spill not only pollutes the environment,but also threaten life on the earth.Traditional physical and chemical repair techniques include machinery,burying,and washing,but these techniques are expensive and inefficient.In contrast,bioremediation technology is attracting more and more attention,in that it is cost-effective and environmental-friendly.Among the factors that affect the efficiency of petroleum biodegradation,the petroleum metabolism activity by microorganisms is the limiting factor.Therefore,obtaining a highly efficient alkane-degrading bacterium is the prerequisite for petroleum bioremediation.In this study,a strain with high alkane degradation efficiency was successfully screened.The strain was designated as Bacillus velezensis Fast30,based on 16 S r RNA BLAST and physiological tests.The degradation rate of 0.1% crude oil by Fast30 was 40.25% within 9days.In order to improve the performance of this bacteria,the genes rhl AB that are responsible for the synthesis of rhamnolipids as well as the green fluorescent protein gene sf GFP were introduced into strain Fast30,and the positive mutant was named as Bacillus velezensis Fast30-YQ.After medium optimization,the yield of rhamnolipids reached 683±30mg/L,and the emulsifying ability is 2.07 times that of the wildtype strain.The green fluorescence of strain Fast30-YQ was stable even after 9 days of degradation,which facilitated tracing,enumeration and observation of the target bacteria during remediation.Furthermore,the degradation condition was explored,and the optimal degradation condition turned out to be: p H 7.0,8% inoculum,200 rpm,0.4% crude oil,37°C,degradation for 9 days.The optimized degradation rate of 0.4% crude oil by engineered bacteria was 55.21%.In summary,this study successfully obtained an efficient alkane-degrading bacterium,and by genome editing,the degradation efficiency of crude oil was increased by 32.05%,which opens a new avenue to the bioremediation of petroleum pollution.Besides,this study further explored the effect of degradation conditions on the degradation rate by our bacterium,and provided a theoretical basis for practical application.By and enlarge,the strain and the protocol in this work can also be used to improve the efficiency of oil extraction.