Investigation Of Isolation Of Cold-adapted Oil Degrading Marine Bacteria And Response Of Bacterial Community To Oil Contamination

In the Bohai and North Yellow Seas,the average sea surface temperature in winter is approximately 2?,during which time the seawater may freezes.Several oil spills have occurred in these bodies of water,and these oil spills pose a great hazard to marine ecosystems and human health.An equivalent level of oil contamination may have a greater impact in cold environments than warm ones due to the increased persistence of petroleum pollutants and greater difficulties associated with oil spill responses.The degradation of petroleum pollutants by bacteria is a promising technology for cleaning up oil spills at low temperatures.Some isolated marine bacteria have been shown to efficiently degrade oil at 0?.However,most of these bacteria have been isolated from Polar Regions,and few are from China.Because indigenous bacteria are more adapted to local environments than non-native species,it is best to use autochthonous bacteria for oil spill bioremediation.Thus,it is necessary to isolate indigenous bacteria from China.In addition,to optimize oil bioremediation it is important to assess the in situ effects of oil-degrading bacteria with respect to oil degradation and study the response of the bacterial community to oil contamination.This study focus on the isolation of cold-adapted oil degrading marine bacteria and response of bacterial community to oil contamination in China.The results showed:1.Six cold-adapted oil degrading marine bacteria were isolated,including Pseudoalteromonas sp.QY-1,Pseudoalteromonas sp.QN-1,Rhodococcus sp.QY-2,Planococcus sp DC-1,Cobetia sp.QF-1 and Pseudoalteromonas sp.DC-2.The six bacteria could degrade petroleum hydrocarbon at 0? in the laboratory.Strain Rhodococcus sp.QY-2 was the most effective bacterium in degrading crude oil,after 60 days of growth in the medium containing 5 gL-1 of crude oil and 10%(v/v)bacteria broth at 0?,the degradation efficency of crude oil was 52.6±2%.Strain Pseudoalteromonas sp.QN-1 was the most effective bacterium in degrading naphthalene,after 30 days of growth in the medium containing 2 gL-1 of naphthalene and 10%(v/v)bacteria broth at 0?,the degradation efficency of naphthalene was 80.6±2.5%.2.The genome of Cobetia sp.OF-1 was 4,084,184 bp with 57.44%GC content.The genome contained 3,513 predicted protein-coding sequences,and the total length of genes was 3,470,121 bp.Genes involved in the degradation of petroleum hydrocarbons were annotated,and genes encoding enzymes required for the metabolism of several xenobiotics,such as chloroalkane,chloroalkene,chlorocyclohexane and chlorobenzene were present.The presence of genes required for rhamnolipid biosurfactant synthesis and regulation were also observed.In addition,some cold adaption genes were annotated,such as the gene encoding cold shock protein CspA.3.From Jannury 30 th to March 29th,2015,a simulation experiment of oil spill bioremedation was conduced at the dock of Dalian Marinetime Univeristy.During the study time,the average sea surface temperature was 4.5?.The response of bacterial community to oil contamination was investigated based on high throughput sequencing.The abundance of bacteria from the familigy Flavobacteriaceae and Colwelliaceae and genera Oleispira,Pseudoalteromonas,Polaribacter and Arcobacter were markedly increased in bioremediation samples,indicating that these bacteria were key players in oil degradation.In addition,the relative abundances of functional genes involved in petroleum hydrocarbon degradation pathways were markedly increased in bioremediation samples.4.From March 13th to April 27th,2015,an in situ bioremediation study was conducted at an intertidal rocky shore site near the Donggang central business district of Dalian,which was contaminated by crude oil at the end of February 2015.During the study period,the sea surface temperatures ranged from 4.0 to 7.5?.An oil bioremediation agent was prepared by mixing six cold-adapted,oil-degrading isolates fermentation broth.The agent was sprayed onto rock surfaces,and after one and a half months of in situ oil bioremediation,an oil degradation rate of up to 75.1±8.9%was observed compared to that observed from natural weathering.The response of the bacterial community to oil contamination was investigated using high throughput sequencing,with the results showing that Cyanobacteria was the dominant genus in this area.The relative abundances of the functional genes involved in petroleum hydrocarbon degradation pathways were markedly increased.For example,the genes encoding the enzyme P450,which is involved in alkane degradation,were increased 8-fold,while the genes encoding polyaromatic hydrocarbon degrading enzymes were increased 3-fold.