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采用黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物作为主要能源物质在50°C条件下分别培养中度嗜热细菌混合物,研究其细菌多样性。提取细菌基因组总DNA,采用PCR结合限制性酶切片段多态性分析(RFLP)方法进行细菌16SrRNA基因的系统发育分析,比较不同能源条件下富集培养的混合细菌群落构成的差异。从3个培养物中共获得阳性克隆303个并进行RFLP分析,对29种不同酶切谱型的克隆插入序列进行测定和系统发育分析。大部分序列与已报道的浸矿微生物16SrRNA序列相似性较高(89.1%~99.7%),归属于硫化叶菌属的耐温氧化硫化杆菌(Sulfobacillus thermotolerans)和热氧化硫化杆菌(Sulfobacillus thermosulfidooxidans),嗜酸硫杆菌属的喜温硫杆菌(Acidithiobacillus caldus),钩端螺旋菌属的嗜铁钩端螺旋菌(Leptospirillumferriphilum)以及unculturedforest soil bacterium、uncultured proteobacterium。其中Acidithiobacillus caldus,Sulfobacillus thermotolerans,Leptospirillumferriphilum3种细菌为三类能源物质培养物中的优势细菌类群。L.ferriphilum在黄铁矿培养体系(53.8%)和硫酸亚铁和硫粉为能源的培养体系中(45.9%)中丰度最高;在以黄铜矿为能源物质的培养体系中,S.thermotolerans的比例大幅上升(70.1%)。关键词:中度嗜热细菌;生物多样性;生物浸矿;喜温硫杆菌(Acidithiobacillus caldus);耐温氧化硫化杆菌(Sulfobacillus thermotolerans);嗜铁钩端螺旋菌(Leptospirillumferriphilum)
The mixture of pyrite, chalcopyrite, ferrous sulfate and sulfur powder was used as the main energy substance to cultivate moderate thermophilic bacteria mixture under the condition of 50 ° C to study the bacterial diversity. The bacterial total DNA was extracted. The phylogenetic analysis of bacterial 16S rRNA gene was carried out by PCR and restriction fragment analysis (RFLP). The differences of composition of mixed bacterial community under different energy conditions were compared. A total of 303 positive clones were obtained from 3 cultures and subjected to RFLP analysis. The cloned insert sequences of 29 different digest patterns were determined and phylogenetic analysis was performed. Most of the sequences showed high similarity to reported 16SrRNA sequences (89.1% -99.7%) of the leaching microorganisms, Sulfobacillus thermotolerans and Sulfobacillus thermosulfidooxidans belonging to the genus Sulfolobus, Acidithiobacillus caldus, Leptospirillum ferriphilum, and uncultured forest soil bacterium, uncultured proteobacterium. Among them, three kinds of bacteria, Acidithiobacillus caldus, Sulfobacillus thermotolerans and Leptospirillum ferriphilum, were the dominant bacterial groups in three types of energy material culture. L. ferriphilum had the highest abundance in the culture system of pyrite (53.8%) and ferrous sulfate and sulfur powder (45.9%). In the culture system with chalcopyrite as energy source, The proportion of thermotolerans increased significantly (70.1%). Key words: moderate thermophilic bacteria; biological diversity; bioleaching; Acidithiobacillus caldus; Sulfobacillus thermotolerans; Leptospirillum ferriphilum;