【作者】 曹琳辉；

【导师】 刘学端；

【作者基本信息】 中南大学 ， 微生物学， 2007， 硕士

【摘要】 矿物开采导致地表水和地下水的污染,其原因主要是由于矿物的暴露,使得硫化物加速溶解导致金属离子含量丰富的酸性水的产生,这些污染水通常被称为酸性矿坑水(AMD)。酸性矿坑水通常含有高浓度的金属离子如铁,铜,锌,镁及非金属离子如硫,砷等。虽然AMD是一种高酸性,热的,含有高浓度的硫酸盐和有毒金属离子的极端环境,但是许多微生物生存在这种环境中,这些微生物的活动促进AMD的形成并导致环境污染。同时,AMD环境中生存的微生物对金属的生物浸出也具有重要的意义。金属浸出现在被认为是主要是由铁离子和质子执行浸出反应的一个化学过程。微生物的作用是产生浸出化学物质和为浸出反应发生提供空间,当微生物附着在矿物表面时能够产生胞外多聚物(EPS),生物氧化反应在EPS层比溶液中反应更为迅速有效,EPS层是生物氧化反应场所。因此,研究酸性矿坑水中的微生物群落结构及其功能活动具有重要的意义。本文利用16SrRNA和gyrB基因作为分子标记的RFLP技术对江西银山铅锌矿的微生物群落结构进行了研究,并比较了两种分子标记分析结果的差异,同时还利用本实验室构建的寡核苷酸基因芯片对江西德兴铜矿和银山铅锌矿的微生物群落结构及微生物功能活动以及环境变量对其产生的影响进行了研究。首先利用16S rRNA作为分子标记分析了银山铅锌矿中AMD样品的微生物群落结构。结果显示,所有的克隆都分布在7个不同的系统发育分支上(Cyanidium caldarium,Alpha-Proteobacteria,Gamma-Proteobacteria,Delta-Proteobacteria,Acidobacteria,Actinobacteria,Nitrospira),3个样点的微生物均存在较高的多样性,其中C.caldarium是一种能耐受高浓度金属离子的红藻,只存在于YSK1中,而Delta-Proteobacteria则只在YSK2中存在,而且在这个矿区的样品中只检测到少量的Acidithiobacillus ferrooxidans,通常这种细菌在AMD微生物群落中为优势种群,并且在YSK1中完全检测不到,这可能是由于受该样点的低pH值影响。化学分析结果显示YSK2和YSK3具有相似的环境因子但他们与YSK1具有较大的区别,系统发育分析结果表明YSK2和YSK3相对于YSK1具有更高的群落相似性,这一结果表明微生物群落结构受地理化学参数,特别是铁,硫浓度和pH值有较大影响。同时我们还发现3个样点中微生物群落结构的差异与其生存环境之间存在着一定的相关关系。进一步利用gyrB基因作为分子标记分析了银山铅锌矿AMD样品的微生物群落组成,并与16S rRNA的分析结果进行了比较。结果显示,两种分子标记分析的微生物群落结构在整体上的组成虽然很相似,但是以gyrB基因为分子标记从微生物群落中所发现的OTU数目(68个)明显高于16S rRNA为分子标记的OTU数量(52个),而且gyrB基因的多样性指数也明显高于16S rRNA的多样性指数。建立在AMD样品的系统发育树分析显示,gyrB基因代表了更高水平的遗传多样性。结果表明gyrB基因能够更好的区分一些16S rRNA无法辨别的近亲菌株,具有比16S rRNA更好的辨别力,其丰度更高,可能更适合于微生物群落的多样性分析。基因芯片技术因其独特的优势已被广泛应用于微生物群落结构与功能的研究中,本研究利用实验室自行构建并评估了的寡核苷酸基因芯片对德兴铜矿6个样点和银山铅锌矿3个样点的酸性浸矿水样品进行了分析。并使用除趋势对应分析(DCA),典范对应分析(CCA),偏典范对应分析(Partial CCA),Mantel测试等统计学方法来研究样品地球化学性质对微生物群落结构和功能活动的动力学影响,总共检测到373个16S rRNA和功能基因信号,对这些杂交结果的分析表明酸性矿坑水样品中存在着高度的多样性和异质性,其微生物群落主要由Acidithiobacillus ferrooxidans、Leptospirillum sp.、Acidithiobacillusthiooxidans、Acidothermus cellulolyticum、Thermoplasmataceaearchaeon、Sulfolobus sp.、Sulfobacillus sp.、Thiomonas sp.、Hydrogenobacter acidophilus、Alicyclobacillus herbarius、Acidianussp.、Actinobyces naeslundii、Microthrix parvicella、Acidobacteria、Desulfobibrio longus、Acidiphilum spp.、Holophaga sp.、Thermomicrobium roseum和Metallosphaera sp.等19个不同的种和属组成。统计学方法和基因聚类分析表明微生物群落结构和功能活动受环境变量(主要是pH,Fe,Ca,Cu和S)的高度影响,具有相似环境变量的样品也具有相似的微生物群落结构以及微生物功能活动。更多还原

【Abstract】 Deep mining and surface mining of metal ores results in the contamination of ground and/or surface waters. The most well documented type of water pollution associated with mining is that which results from the accelerated oxidative dissolution of exposed minerals, principally sulfides, giving rise to acidic, metal-enriched waters generally referred to as "acid mine drainage" (AMD). Usually the acid mine drainage have a high concentrations of metal ions such as iron, copper, zinc and some nonmetal such as sulfur and arsenic. Despite the extreme acidity, heat, and high concentrations of sulfate and toxic metals, a diverse range of microorganisms populate AMD environments. These microorganisms can accellerate the generations of AMD, and result in the pollutions of environment. Also the microorganisms thrived in AMD that have a important significance in bioleaching, they can used to process the mineral and as a new source of bio-molecular in industry. So, study the microbial community and their functional activity of acid mine drainage have a great importance. In present study, we use RFLP technology based on the 16S rRNA and gyrB gene as the molecular marker to study the microbial compositions of Yinshan lead-zinc mine, Jiangxi province. And compared the difference of analysis result of two molecular marker. We also use the microarray that have been constructed and evaluated by our laboratory to detect the microbial community compositions and functional activities of Dexing copper mine and Yinshan lead-zinc mine and study the environmental factors how to influence the microbial community.Firstly, 16S rRNA genes were used as molecular marker to evaluate the diversity of microbial communities from three different AMD site in Yinshan lead-zinc mine. The result showed that the predominant microorganism in Yinshan mine were Cyanidium caldarium, Alpha-Proteobacteria, Gamma-Proteobacteria, Delta-Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira. All three site have the high community diversity, C. caldarium is a red alga that can tolerate higher metal ions, it only presented in site YSK1, and Delta-Proteobacteria only presented in YSK2. Also we can only detected few Acidithiobacillus ferrooxidans which usually is the dominant populations in AMD, maybe this effected by the lower pH value. YSK2 and YSK3 have the similar environment factors and have a large different with YSK1 from chemical analysis result. From the phylogenetic analysis results both based on 16S rRNA gene, it suggested there are higher community similarity between YSK2 and YSK3 than with YSK1, this show that the distribution of the microbial communities was influenced by geochemical parameters, especially controlled by the iron, sulfur ions and pH values. And then we use the gyrB gene as molecular marker to carry out the diversity research and compare the analysis result by 16S rRNA. Dispite the microbial compositions have a similarity, we found the OUT number resulted from gyrB gene higher than 16S rRNA. The diversity index of gyrB gene also larger than 16S rRNA, but the microbial community abundance acquired from gyrB analysis was extremely higher than obtained through 16S rRNA analysis in terms of Shannon diversity index. Through gyrB gene, we will be able to differentiate closely related microorganisms in phylogenetic analysis, the result revealed that gyrB shows better performance in research concerning the correlation between community structure and geographic chemical parameters.Microarray technology has been widely used in researches concerning the function and structure of microbial community due to its unique advantages. We use the microarray that have been constructed and evaluated by our laboratory to detect the microbial community compositions and functional activities of the nine acid mine drainage samples of Dexing copper mine and Yinshan lead-zinc mine. At the same time we use the statistical methods such as detrended correspondence analysis (DCA), canonical correspondence analysis (CCA) and Partial CCAs to detect the dynamic influence of environmental factors to microbial community compositions and functional activity. A total of 373 genes (including 16S and functional genes) showed significant hybridization with at least one of the nine samples. These result indicated there have higher diversity and heterogeneity in AMD. These community composed by 19 different genus or species, including Acidithiobacillus ferrooxidans, Leptospirillum sp., Acidithiobacillus thiooxidans, Acidothermus cellulolyticum, Thermoplasmataceae archaeon, Sulfolobus, Sulfobacillus sp., Thiomonas sp., Hydrogenobacter acidophilus, Alicyclobacillus herbarius, Acidianus sp., Actinobyces naeslundii, Microthrix parvicella, Acidobacteria, Desulfobibrio longus, Acidiphilum spp., Holophaga sp., Thermomicrobium roseum and Metallosphaera sp.. The result by statistical and cluster analysis revealed the microbial community and funtion high influenced by the environmental factors(mainly are pH, Fe, Ca, Cu and S) .更多还原