导　　师： 郭勇

学科专业： 082203

授予学位： 博士

作　　者： ;

机构地区： 华南理工大学

摘　　要： 硫氧化细菌在硫的地球化学循环中扮演着重要的角色,是自然界中硫的生物氧化的主导者,在制革、印染造纸、石油化工、化学品制造等工业废水的生物治理中起着极其重要的作用。对含硫废水处理系统中硫氧化细菌群落结构的分析、高效污染物降解菌的筛选和硫氧化特性的研究有利于解析环境中硫氧化细菌的组成和丰度,优化污水处理系统硫化物降解工艺。本论文针对基于PCR的微生物分子生态学技术应用上的局限性,构建了基于微生物活细胞检测的环境微生物分子生态学研究方法;研究了印染废水处理系统中细菌、古菌和硫氧化细菌的群落结构;通过好氧和厌氧富集,富集和筛选到了能氧化硫代硫酸盐的硫氧化细菌;并对所筛得的高效硫氧化菌的生理生化和生物学特性进行了研究。 叠氮溴化丙锭/(Propidium Monoazide, PMA/)是一种对DNA具有高度亲和力的光敏DNA染料,不能透过完整的细胞膜,只能选择性地修饰细胞死亡后暴露出来的DNA分子,能有效抑制热处理、异丙醇处理等致使细菌细胞膜破裂而导致的死细胞DNA的PCR扩增;PMA能有效抑制大片段DNA的PCR扩增,而对于小片段的DNA/(例如﹤200 bp/),PMA不能有效或完全抑制其PCR扩增;针对该问题构建的PMA-巢式PCR-DGGE方法可以有效抑制环境样品中死细胞DNA的PCR扩增,提高DGGE检测的灵敏性,可实时有效地研究环境微生物群落结构和动态变化,在复杂样品的研究中也有应用价值。 细菌和古菌16S rRNA基因的克隆文库表明印染废水处理系统中细菌主要由Gammaproteobacteria /(73 /%/), Anaerolineae /(6 /%/), Bacilli /(5 /%/), Deltaproteobacteria /(7 /%/), Clostridia /(4 /%/), Bacteroidetes /(1 /%/)和Chlorobia /(1 /%/)组成,古菌主要由Methanomicrobia /(99 /%/)和Thermococci /(1 /%/)组成,细菌比古菌的多样性高;基于硫氧化菌功能酶基因dsr、sox和sqr的克隆文库表明Alphaproteobacteria Sulfur-oxidizing bacteria are the main participant in sulfur biological oxidation, which pay an important role in the sulfur geochemical cycle and biological treatment of industrial wastewater from tanneries, textile dying and printing factories, paper mills, petrochemical refineries, liquefied petroleum gas plants and so on. The microbial ecology analysis of sulfur-oxidizing bacteria in sulfide-rich wastewater treatment, and isolation of bacteria with high biodegradation ability, and characterization of the sulfur-oxidation for sulfur-oxidizing bacteria are helpful for revealing the microbial community and abundance of sulfur-oxidizing bacteria in the environment and optimizing the wastewater treatment process. In this Ph.D research project, we first constructed a viable cells based microbial ecology method to overcome the limitation that traditional PCR-based molecular techniques cannot discriminate viable cells from dead cells in microbial community analysis, and used this method to study the bacteria, archaea, and sulfur-oxidizing bacteria communities in printing and dyeing wastewater treatment. Then, we enriched and isolated sulfur-oxidizing bacteria by aerobic and anaerobic enrichments. Finally, the physiology, biochemistry, and biological characteristics of a sulfur-oxidizing bacterium with high sulfur-oxidizing ability were studied. Propidium Monoazide /(PMA/) is a membrane impermeant dye that selectively penetrates into dead cells with compromise membranes, and covalently cross-linked to DNA strain after intercalating into it, which strongly inhibits the PCR amplification of DNA from heat-killed and isopropanol-killed cells. However, PMA treatment can not completely suppress dead cells from PCR amplification when the targeted gene is too short /(<200 bp/). Then, a method that PMA treatment in combination with two-step nested PCR was designed to overcome this problem. The results suggested that the PMA-nested PCR-DGGE method could detect only viable cells in microbial ecology analysis, and enhance the

关 键 词： 硫氧化细菌 活细胞 克隆文库 微生物群落 富集筛选

领　　域： [环境科学与工程] [环境科学与工程]