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题名: 迪茨氏菌高效降解石油烃机理及在环境管理和石油工业中的应用研究
作者: 王兴彪
学位类别: 博士
答辩日期: 2012
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 吴钢
关键词: 原油 ; crude oil ; 长链烷烃 ; long-chain alkane ; 降解 ; biodegradation ; 迪茨氏菌 ; metabolites ; 代谢产物 ; Dietzia sp.
其他题名: The efficient degradation mechanism of petroleum hydrocarbons by Dietzia sp. and applied research in environmental management and petroleum industry
中文摘要:     本论文研究了迪茨氏菌属菌株Dietzia sp.DQ12-45-1b降解原油和长链烷烃的机理。取得的主要工作进展如下: (1)从大庆油田采油三厂井排水中分离得到一株对原油和烷烃具有强降解活性的细菌,命名为DQ12-45-1b,形态学,生理学和系统发育学分析证明,该菌株属于放线菌门(Actinobacteria),与迪茨氏菌属菌株Dietzia cercidiphylli 的序列相似度达99.358%,确定该菌为迪茨氏菌属细菌(Dietzia); (2)活性筛选发现,该菌对原油,正构烷烃(C6-C40),支链烷烃(HMN)和芳烃(菲,屈,萘和芴)均具有较好的降解活性,经过21天培养,每100ml发酵单位,菌株DQ12-45-1b对正构烷烃C14,C16,C20,C24,C28,C32,C36和C40的降解量分别达到了121.41,81.17,40.85,31.57,15.06,31.54,10.75和13.87mg,表现出了很强的活性; (3)研究了菌株DQ12-45-1b对原油的降解活性。发现该菌株降解利用大庆油田原油时,对不同长度的烷烃具有选择性,优先利用碳链数≤25(培养5天)和≥28(培养22天)的烃类物质。 (4)测定了菌株降解不同长链烷烃发酵液和细胞活性,包括发酵液排油圈活性,表面张力,浊度,乳化活性,有机酸变化和菌体细胞疏水活性。扫描电镜观察菌体细胞同不同烷烃作用的过程,发现菌株降解三十六烷时,细胞表面产生明显疏水活性,与烷烃颗粒有大量吸附。 (5)提取了烷烃降解发酵液中的表面活性物质并做了活性测定和结构分析。GC-MS结构鉴定实验发现,菌株降解十六烷,二十四烷和三十六烷过程中,分别主要产生糖脂,磷脂和脂肽三类表面活性剂,对不同来源的糖脂进行结构分析发现,十六烷,二十四烷和三十六烷降解发酵液中的糖脂各不相同,分别是α-D-吡喃葡萄糖苷-β-D-呋喃果糖,6-脱氧甘露糖和2-甲肟-葡萄糖酸,同时发现其疏水链中的脂肪酸组成也有很大的差异; (6)对十六烷降解发酵液中的四种主要代谢产物纯化和活性测试,发现四种代谢产物在菌种降解十六烷过程中对表面张力的下降都有贡献,贡献最大的是两种糖脂表面活性剂,临界胶束浓度分别达到0.25ug/L和2000ug/L; (7)检测了菌株降解三种不同烷烃糖脂合成基因表达情况,发现细胞降解烷烃活性越强,基因表面活性剂基因表达量越高,整体上,菌株降解利用十六烷过程中糖脂基因的表达量最大; (8)分析了降解长链烷烃微生物中糖脂和脂肽合成基因的分布,发现降解长链烷烃的微生物细胞中都有表面活性剂合成基因存在,说明包括菌株DQ12-45-1b在内的长链烷烃降解菌中存在的表面活性剂合成基因是一个普遍现象。 (9)对以菌株Dietzia sp. DQ12-45-1b为代表的迪茨氏菌属菌株在石油污染治理和石油工业的应用前景作了展望,分析了石油工业和环境修复方面的工业化应用的需求同迪茨氏菌属之间的相关性,对菌株的环境适应能力和强活性在工业应用中的潜在价值进行了讨论。
英文摘要:     The article was focused on the degradation mechanism of a bacterium strain, DQ12-45-1b grew in different long-chain alkanes,All data provide the necessary support for development of microbial enhanced oil recovery and oil remediation. (1) Morphological, physiological and phylogenetic analyses were all carried out for the strain DQ12-45-1b. Phylogenetic analysis of 16S rRNA gene sequence suggested the strain DQ12-45-1b belonged to Actinobacteria, forming a stable clade with the type strains of all species in the genus Dietzia and showing the highest 16S rRNA sequence similarity of 99.358% with Dietzia cercidiphylli. (2) When crude oil, n-alkanes, branched alkane and aromatic hydrocarbons were used as the sole carbon sources for the strain DQ12-45-1b, growth was observed in all cases. The degradation of C14, C16, C20, C24, C28, C32, C36 and C40 was 121.41, 81.17, 40.85, 31.57, 15.06, 31.54, 10.75 and 13.87 mg, respectively. (3) The oil degradation activity of the strain DQ12-45-1b was tested. At day 5, the hydrocarbons with a chain-length of ≤ 25 were degraded. But at day 22, the ratios of hydrocarbons with carbon numbers ≤ C22 and ≥ C28 decreased. (4) The activities of the culture broth and cells of the strain degrading different long-chain alkanes, n-Hexadecane, n-Tetracosane and n-Hexatriacontane, respectively, The interaction between the cells and different alkane particles was also studied using scanning electron microscopy, and the data showed the cells surrounded thickly on the surface of C36 particles when the strain grew on C36 degrading broth. (5) Different biosurfactants were extracted from C16, C24 and C36 fermentation broth for activity determination and structural analysis. GC-MS data showed the strain DQ12-45-1b produced glycolipid, phospholipid and lipopeptides as major metabolite, respectively. The hydrophobic fatty acid of the glycolipids also different each other. (6) Isolation and activity test of the four major metabolites extracted from the C16 culture broth were carried out. Four metabolites contributed different to the decrease of culture surface tension, two major glycolipid biosurfactants, A and C, contributed most with critical micelle concentration was 0.25 ug/L and 2000 ug/L, respectively. (7) The expression of glycolipid synthesis related gene was detected. The stronger of the cell degradation activity, the higher expression of the glycolipid synthesis related gene. Generally speaking, the highest gene expression occurred in the cell when C16 used as sole carbon source. (8) The distribution of the glycolipid and lipopeptide synthesis related gene in long-chain alkane degrading bacteria was carried out, all strains contained glycolipid or/and lipopeptide synthesis related gene, there was a common phenomenon that biosurfactant synthesis gene existed in long chain alkane degrading bacteria. (9)The application prospects in oil pollution control and oil industry was made for the genus Dietzia, the needs of the oil industrial application and environmental restoration were analyzed, the environmental adaptability and strong activity of the strains in genus Dietzia and potential value in industrial applications were discussed.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35101
Appears in Collections:城市与区域生态国家重点实验室_学位论文

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Recommended Citation:
王兴彪. 迪茨氏菌高效降解石油烃机理及在环境管理和石油工业中的应用研究[D]. 北京. 中国科学院研究生院. 2012.
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