| Mairne crude oil pollution is a worldwide problem, alkane is an important content in the crude oil. As the activities of human in polar area and other cold environments have increased, it’s important to study the petroleum degrading in the cold areas. Previous study indicated that Thalassolituus is usually dominant in oil-degrading consortia from surface seawater of the Arctic Ocean and the reason haven’t been found yet. This thesis mainly focused on the alkane-degrading and cold adaption mechanism of genus Thalassolituus at low temperature. Two Thalassolituus strains named R6-15and4BN06-13and three reference strains belonging to genus Alcanivorax (named A. borkumensis SK2, A. dieselolei B-5and A. jadensis T9) were used in this study. The alkane degradation mechanism of these strains were contrastively studied with the tetradecane as the representative alkane substrate at15℃and25℃. This thesis analysized the mechanism of alkane-degrading through genomic sequencing. Meanwhile, the biodiversity of cultivable Thalassolituus strains was performed in the surface seawater from the western Pacific Ocean and the coastal sea of Ximen Bay.At physiological and biochemical levels, strain R6-15and4BN06-13were able to utilize very restricted spectrum of carbon substrates for growth, including sodium acetate, Tween-40, Tween-80and C8-C36aliphatic hydrocarbons. When used tetradecane as sole carbon source at15℃, the growth of strain R6-15and4BN06-13was better than25℃, and also better than strains Alcanivorax SK2and B-5. Compared to25℃, two Thalassolituus strains grew faster when used sodium acetate as the sole carbon source at25℃, indicated that Thalassolituus is psychrotolerant Either at15℃and25℃, the ability of hydrophobicity of cell surface of Thalassolituus strains was better than SK2, but worse than B-5, when they served with tetradecane or not. When strain R6-15was induced by alkanes, it could decrease the surface tension of the cultures from59.43to49.09mN/m at25℃, but it failed to deceased these values at15℃. In contrast, strain R6-15couldn’t decrease the surface tension when the alkane substrates were absent. The major cell fatty acids of strain R6-15were C14:0, C16:0and16:1w6c/16:1w7c. Meanwhile, whether exist alkanes or not, the proportion of unsaturated fatty acids (16:1w6c/16:1w7c) of strain R6-15will significantly increase both at15℃, compared with25℃. In order to see if Thalassolituus would be dominant at low temperature, we carried on competitive culture experiments, using tetradecane as sole carbon source at15℃and25℃. The results of competitive culture experiments indicated that strain R6-15became to be the predominant bacteria in the co-cultures, which was inoculumed with strain R6-15and the in situ community of surface seawater from the Xiamen coastal sea. For strain4BN06-13, it dominated in the25℃co-cultures. When cultured Thalassolituus strains with Alcanivorax jadensis T9at15℃and25℃, Thalassolituus was the dominant member in these co-cultures obtained from two temperatures.In order to better understand the alkane-degrading mechanism, the genome of strain R6-15was sequenced. The whole genome was3,764,053bp with a G+C content of46.6%. The similarity between strain R6-15and the type strain Thalassolituus oleivorans MIL-1(T) was96.92%at the whole genome sequence level. Two alkane hydroxylase genes(alkB) and two flavin-binding monooxygenase genes(almA) were found in its genome, respectively. These two kinds of gene showed more than99%similarity at amino acid residues with their counterparts in type strain T oleivorans MIL-1. Phylogenetic analysis results showed that the alkB and almA genes originated from genus Thalassolituus separately clustered into the independent branches, which indicated that they may evolve independently. In addition to the alkane-degrading genes, many cold adaption genes were also found in its genome, such as cold shock protein gene(Csp), a dedicated protein-folding system using DnaK-DnaJ-GrpE、 GroESL、ClpB and SecB, and cold shock dead-box protein A gene(csdA). The existence of these genes indicated that strain R6-15may own the protein fold system which will support it grow at low temperature.In addition to the above results, the biodiversity of the cultivable strains of genus Thalassolituus was also surveyed. Three isolates were obtained from the oil-enriched surface seawater of the Western Pacific Ocean during the DY-27cruise, and from the surface seawater of Xiamen coastal sea. Phylogenetic analysis results of16S rRNA genes indicated that two of these three strains were candidate new species of genus Thalassolituus.To sum up, this thesis is the first time to study the characteristics of Thalassolituus strains which utilized alkanes to grow and their cold adaption mechanism at low temperature. The results will help us understand the reason why Thalassolituus strains always dominated in the oil-degrading consortia at cold marine environments, and will provide reference to their application for crude oil polluted cold marine environments. |
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