| Oil and gas are important energy and strategic resources. Microbial oil and gas survey technique playing a role as novel and advanced oil exploration methodology, which has attract growing concern for its fast and cost-effective advantages. While methane can originate from non-petroleum sources, the only natural sources of butane are oil and gas fields. The increased presence of butane-oxidizing microorganisms in a given soil sample and high butane degradation abilities can be used by oil prospectors as an accurate indicator of a proximal petroleum reservoirs. So butane-oxidizing microorganisms resources and butane degradation characteristcs can serve microbal prospecting powerful theoretical and practical significance.In this thesis, butanol was used as only carbon source to mearsure butane-oxidation microorganism abundance in Jianghan oil field, Puguang gas field and Bohai Sea oil field.16S rRNA gene clone library was constructed to unveil microbial community structure characteristics in Puguang gas field; Buane and butanol were used as only carbon source to isolate butane-oxidizing bacteria. After identification, butane degradation characteristics of most part of isolated butane-oxidizing bacteria were analized intensively. bmoX gene sequences were amplified from parts of them. Our study is beginning to shed light on the mechanisms by bmoX gene took part in butane-oxidation bacteria partner syntrophic cooperation interactions.(1) The number of culturable butane-oxidation microorganisms in oil and gas field is around1～2×105CFU/g soil, significantly higher than that in non-petroleum soil. And almost all butane-oxidation microorganisms are bacteria, which showed that the abundance of butane-oxidizing bacteria can indicate the oil and gas environment. Results of clone library indicated that proportion (12%) of Actinobacteria in Puguang gas field was significantly higher than that in general soil.98%OTU showed high similarity with uncultured bacteria, indicating that the bacterial community composition characteristics in oil and gas fields has obvious environmental habitat, and a large number of unknown microbial resources are encompassed in it.(2)43butane-oxidizing bacteria were isolated from Puguang gas field, Jianghan oil field, Daqing oil field, Songyuan oil field and Bohai sea oil field. The main parts of it are Rhodococcus (7strains), Bacillus (12strains), Pseudomonas (12strains), Arthrobacter (2strains) and Acinetobacter (2strains). Among which, bacteria of the genus Arthrobacter and Bacillus are first found as advantage butane-oxidizing bacteria. Quantitative results from GC showed that high butane oxidation ability bacteria are all in genus Rhodococcus. Most bactieria in genus Bacillus are low ability ones. Study on utilization of various growth substrates by Arthrobacter3-2showed that strain3-2exhibited relatively high performance for growth supported by butane, butyrate and1-butanol as carbon sources. However, slow growth velocity was supported by other alkanes with two to ten carbons (except pentane). The pathway of butane metabolism by Arthrobacter3-2was determined to be butaneâ†'1-butanolâ†' butyraldehydeâ†'butyrate.(3)4bmoX gene sequences were amplified from isolated butane-oxidation bacteria. Amino acid sequences comparison found that BmoX of Rhodococcus3-6and Rhodococcus3-13have high similarity with PrmA from propane-oxiation bacteria (98%and96%respectively). BmoX of Bacillus3-7have has as high as95%similarity with MmoX from methane-oxidizing bacteria. BmoX of Arthrobacter3-2has80%similarity with BmoX from butane-oxidizing bacteria.(4) Butane degradation manners and characteristics of isolated butane-oxidation bacteria were intensively studied. Butanol accumulation phenomenon was found in butane degradation pathway. Butane degradation abilitis of isolated butane-oxidation bacteria were affected by their butanol degradation abilities and tolerance. Syntrophic cooperation among butane-oxidation bacteria in butane degradation procedure was first discovered. That is butanol-oxidizing bacteria released butane monooxygenase competitive inhibition by using butanol as carbon source to grow, thereby the butane degradation abilities of butane-oxidizing bacteria were significantly improved, in the end, the degradation ability differences among isolated butane-oxidizing bacteria were eliminated.(5) bmoX gene expression in syntrophic cooperation procedure was measured through RT-qPCR technique. Results found that bmoX gene expression was upregulated under syntrophic cooperation’s help, and also it was a fantastic indicator for butane degradation ability. Further combining the results of the insertional inactivation of bmoX that resulted in a mutant Arthrobacter3-2strain incapable of growth with butane, which improved that bmoX is a key and essential gene. Preliminary indicated that bmoX gene has potential function to used as a molecular probe for microbial oil and gas exploration in the future. |
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