一株嗜热厌氧杆菌的分离、鉴定及其代谢特征

Isolation, identification and metabolic characterization of an anaerobic thermophilic bacterium

摘要:

【目的】分离、保护油藏嗜热微生物资源, 解析其主要的代谢特征。【方法】利用Hungte厌氧分离技术从大港油田埕海一区油层采出液中分离出厌氧菌株BF1。通过生理生化特征分析、16S rRNA基因序列比对与电化学分析, 确定BF1的分类地位及其S元素代谢对腐蚀电流的影响。【结果】菌株BF1为严格嗜热厌氧革兰氏阴性杆菌, 顶端产芽孢、不运动, 菌体大小为0.42 μm×(1.6?5.4) μm, 单生、成对或成串生长。其温度生长范围为45 °C?75 °C (最适温度60 °C); pH生长范围在4.5?8.5 (最适pH 6.5) 之间, 比生长速率(μm) 0.99 h?1, 倍增时间为42 min。能利用葡萄糖、松三糖、棉子糖、甘露糖、乳糖、纤维二糖、果糖、核糖等碳水化合物, 利用葡萄糖发酵的产物是乙醇、乙酸、CO2及少量的H2。菌株BF1能还原亚硫酸盐与硫代硫酸盐产生H2S, 其耐受上限分别为50 mmol/L和75 mmol/L; 还原硫代硫酸钠(50 mmol/L) 后其极化电阻由2 099 Ω/cm2降低至776 Ω/cm2, 腐蚀电流由9.936e-006 A提高至3.25e-005 A。细胞膜脂肪酸主要由高级饱和脂肪酸组成, 含量最丰富的为十五烷酸占70.6%。菌株BF1的DNA (G+C)mol%含量为34.0%, 其16S rRNA与Thermoanaerobacter pseudethanolicus DSM 2355T相似性最高, 为98.3%, 与T. brockii subsp. brockii DSM 1457T次之, 为98.0%。菌株BF1的许多生理、生化特征与T. pseudethanolicus DSM 2355T 和T. brockii subsp. brockii DSM 1457T有着明显的差别, 如倍增时间、最适生长温度及底物利用等; 而菌株BF1的细胞膜脂肪酸组成与T. pseudetha-nolicus DSM 2355T也不相同。【结论】菌株BF1可能是Thermoanaerobacter属中的一个新种, 其确切分类地位还需要进一步进行DNA分子杂交; 其代谢元素硫提高腐蚀电流密度, 可能会对油田管道与设备造成腐蚀。

Abstract:

[Objective] To isolate, protect thermophilic microbial resources from petroleum reservoirs and analyze these main metabolic characterization. [Methods] The strain BF1 was isolated by Hungte anaerobic technique from Chenghai 1 Unit of Dagang oil field in China. Its taxonomic status determined by physiological, biochemical and 16S rRNA gene sequence analysis. Its effect of sulfur metabolism on the corrosion current was measured by electrochemical analysis. [Results] The strain BF1 was Gram-negative, strictly thermophilic anaerobic, top-sporulating, non-motile, rods, 0.42 μm×(1.6?5.4) μm, grew solitary, in pairs or in chains. Growth occurred at 45 °C?75 °C (optimum 60 °C), at pH 4.5?8.5 (optimum 6.5). Specific growth rate (μm) was 0.99 h?1 and doubling time was 42 min. Substrates included glucose, melizitose, raffinose, mannose, lactose, fructose and ribose. The main products of glucose fermentation were CO2, H2, acetate and ethanol. The strain could reduce thiosulfate and sulfite to sulfide, and its tolerance limits were 75 mmol/L and 50 mmol/L, respectively. The electrochemical impedance reduced from 2 099 Ω/cm2 to 776 Ω/cm2 and the corrosion current increased from 9.936e-006 A to 3.25e-005 A after thiosulfate (50 mmol/L) was reduced. The fatty acids were mainly composed of saturated long chain fatty acids, with C15:0 the most, accounting for 70.6%. The G+C content of DNA was 34.0 mol%. The 16S rRNA gene sequence analysis indicated that the closest phylogenetic relatives were Thermoanaerobacter pseudethanolicus DSM2355T and T. brockii subsp. brockii DSM1457T, 98.3% and 98.0%, respectively. However, the strain BF1 was different with T. pseudethanolicus DSM2355T and T. brockii subsp. brockii DSM1457T in doubling time, optimum temperature and substrates utilized, and different with T. pseudethanolicus DSM 2355T in fatty acid profile. [Conclusion] The strain BF1 may be a new species of Thermoanaerobacter genus, the exact taxonomic status of it requires DNA hybridization further. The corrosion current density improved by its metabolic element sulfur may cause corrosion of oil pipelines and equipments.