Antarctic Marine Petroleum Hydrocarbons In Low-temperature Degradation Of The Bacteria Shewanella Sp.nj49 Dioxygenase Gene

In recent years, the Antarctic environmental pollution is aggravating with the increase of human activities, especially for the increase of Antarctic tourism and scientific research activities. Petroleum hydrocarbon pollution is one of the most remarkable problems for the Antarctic environment pollution. A lot of petroleum products entered into the Antarctic waters in a variety of ways and caused huge threat for the Antarctic environment. Bioremediation is an important way to eliminate offshore oil hydrocarbon pollution, however, the normal temperature microbe is hard to exert its biological repair function because of the unique environment conditions of Antarctic ocean systems such as high salt, low temperature and the strong ultraviolet radiation. In order to lay the foundation for the application of low-temperature bioremediation technology of marine oil hydrocarbon pollution, the low temperature bacteria that can degrade petroleum hydrocarbon and be separated from Antarctic sea ice samples as the research object.The psychrophilic bacterium NJ49 that can degrade alkane and aromatic hydrocarbons efficiently and be separated from Antarctic sea ice samples as materials strains with hexadecane used as sole carbon source. The alkane degradation characteristics of Antarctic psychrophilic degradation bacterium, Raman spectroscopy real-time monitoring of petroleum hydrocarbon microbial degradation, localization of degradation enzyme, purification and physical and chemical properties of hydroxylase and molecular characterization of oxygenase were researched. The results were as follows:Antarctic psychrophilic degradation bacterium Shewanella sp. NJ49 could degrade hexadecane efficiently at low-temperature environment. The variation of individual cells of degradation bacteria in the process of alkane degradation and the variation of products in the process of cells growth were monitored synchronously by Laser optical tweezers Raman spectroscopy. The representative materials of the Raman spectrum peak for Antarctic psychrophilic bacterium NJ49 were preliminary inferred. The phospholipids and oleic acid were mainly located in the peak of 783 cm- 1 and 812 cm^-1 and the lipid was located in the peak of 1004 cm^-1. The peak of 1156 cm^-1 was mainly represented the glucan. The genetic material of nucleotide was located in the peak of 1370 cm^-1 and the protein was located in the peak of 1435 cm^-1 and 1516 cm^-1. The new cognition of alkane degradation of Antarctic psychrophilic bacteria was obtained from the level of individual and colonial cells.The research of localization of degradation enzyme of Antarctic psychrophilic bacteria indicated that micro-biological degradation of alkane needed the enzymatic reaction of oxidase system. The extracellular enzyme played key role in the process of hexadecane degradation of Antarctic psychrophilic bacterium NJ49. It accomplished the preliminary degradation of hexadecane by the terminal oxidation and produced the products of n-hexadecanal and cetyl alcohol. The intracellular enzyme played little role in the process of hexadecane degradation. The analysis result of GC-MS was in accordance with this conclusion.The extracellular enzyme was obtained by the cell resting technology. Four enzymes of different molecular weight (greater than 100000, 30000^-100000, 10000-30000 and 5000^-10000, respectively) were collected by ultrafiltration and then determined and compared the enzymatic activity. Gas chromatography analysis indicated that the enzyme of molecular weight of 30000^-100000 was the key enzyme of hexadecane degradation. Polyacrylamide gel electrophoresis revealed purified hydroxylase was obtained finally. It was composed ofα,βandγsubunits but the contents were less.Monooxygenase can degrade oil hydrocarbon and exist in Antarctic psychrophilic bacterium NJ49. The gene full-length sequence of 1176 bp of monooxygenase was cloned by genome walking method. The expression results of real-time fluorescence quantitative PCR technology showed concentration of hexadecane, temperature, high salt and ultraviolet radiation influenced the ability of hexadecane degradation with varying degrees for Antarctic psychrophilic bacterium NJ49. The maximum relative express quantities of monooxygenase were 20 mg/L of hexadecane, 15 ?C of temperature gradient, 90‰NaCl 2 h of time gradient of high salt, 6 h of time gradient of ultraviolet radiation, respectively.In addition, The gene full-length sequences of 924 bp of dioxygenase was also cloned by genome walking method from Antarctic psychrophilic bacterium NJ49. The expression results of real-time fluorescence quantitative PCR technology revealed that concentration of naphthaline, temperature and ultraviolet radiation affected the ability of naphthaline degradation with varying degrees for Antarctic psychrophilic bacterium NJ49. The maximum relative express quantities of dioxygenase were 50 mg/L of naphthaline, 15 ?C of temperature gradient, 4 h of time gradient of ultraviolet radiation, respectively.