| Petroleum pollution has been a serious problem in environments, especially in the marine environments . The annual world production of crude oil is 70 million barrels per day, with approximately 50% of this being transported by sea. Occasionally oil tanker accidents result in large-scale marine and coastal pollution. Oil pollution is likely to remain a significant threat to marine and coastal wildlife and ecosystems. It has long been recognized that many microorganisms can use medium or long-chain n-alkanes as sources of carbon and energy, which has stimulated many studies on the usefulness of these organisms in the bioremediation of oil spills and contaminated sites. It's very important and necessary to isolate and culture and detect petroleum-degrading microorganisms from the polluted environment. In many microorganisms the first step for alkane degradation is the terminal oxidation of the molecule by an alkane hydroxylase, which has been proven to be useful and versatile biocatalyst, opening the possibility of its use in the industrial production of fine chemicals.By enrichment with crude oil or diesel as the sole carbon source, the diversity of petroleum-degrading bacteria in the surface water of the Strait of Malacca were surveyed. Ten samples resulted in 10 petroleum -degrading consortia, whose structures were analyzed by the combined methods of DGGE and CFU counting. DGGE revealed quite diversity in bacterial population. We isolated 48 strains in total and they belong to 17 different genus. They belong to Proteobacteria, Acinobacteria, Flavobacteria, respectively. Members of theγ-Proteobacteria(59%)were found to be most abundant.α-Proteobacteria(33%) were the second dominant bacteria. The results showed that the dominant strains were detected phylogenetically close to Alcanivorax, Pseudomonas , Rhodococus, Marinobacter, Novosphingobium, Halomonas, Thalassospira, Salinisphaera, Parvibaculum respectively. Most of these dominant populations were reported as petroleum-degraders. Alcanivorax, Pseudomonas, Novosphingobium had a wide distrbution, especially Alcanivorax, Marinobacter, Novosphingobium were important petroleum-degraders in marine environment. Among them, isolate A-11-3 showed the predominant biodegradability to diesel fuel and was able to utilize various n-alkanes as the sole carbon source, ranging in chain length at least from C8 to C36. On the basis of 16S rRNA gene sequence similarity, B-5 was shown to belong to theγ-Proteobacteria. Highest similarity values were found with Alcanivorax borkumensis(96.5%), A. venustensis ISO4T(94.22%), A. jadensis T9(94%), A.dieselolei B-5T(94.82%),respectively. According to results of physiological and biochemical tests, cellular fatty acid composition, comparisons of 16S-23S internal transcribed spacer sequences,the sequence of gryase gene gryB and comparisons of the partial deduced amino acid sequence of alkane hydroxylase, both strains were affiliated to the genus Alcanivorax but were differentiated from recognized Alcanivorax species. Therefore, a novel species, Alcanivorax Malaccasis sp. nov., is proposed.The alkane hydroxylase in B-5 encodes a functional hydroxylase.A B-5 alkB gene knockout mutant was constructed. But it was surprised to find that the alkane-degrading capability and the region of alkanes the mutant use was the same with the widetype strain. This result proved that there were other type alkane hydroxylase systems in B-5. By the method of degenerated primers and Thermal asymmetric interlaced PCR , a CYP153 alkane hydroxylase gene,which encoding a 367aa polypeptides fragment was amplified, and a full-length of alcohol dehydrogenaes(ADH) ,which is the second key enzyme in the course of alkane-degrading, was amplified too. To detected whether there were more other alkane hydroxylase systems in B-5 , whether the putatived CYP153 P450 encodes a functional alkane hydroxylase and responds for which length of alkanes , P450 gene was knockout in B-5 and B-5△alkB. Further analysis is on the progress.
|
PDF Full Text Request