砷污染环境微生物群落分析及耐砷菌Bacillus sp.JH-5-4特征研究

发布时间：2019-06-27 19:16

【摘要】：含砷金矿尾矿堆积通常会导致砷(Arsenic,As)污染物向周围环境释放,造成周围环境砷污染,这种污染现象已经受到了全球性的关注。现有研究表明微生物在砷元素循环中发挥重要作用,而砷对微生物群落结构及多样性的影响研究较少。为了探究砷污染物对微生物群落结构多样性及耐砷潜力的影响,本项研究采集了中国辽宁丹东地区某金矿尾矿库农田土壤样品及河流沉积物样品,使用高通量测序手段对该环境下微生物群落结构和分布进行研究,通过PCR克隆文库方法对耐砷基因arsB和ACR3的丰度和系统发育进行分析,对稳定携带arsB、ACR3功能基因的耐砷菌进行分离培养,探究该菌种在实验室环境下的生长情况和基本特征。主要研究成果如下:(1)砷污染造成样品内微生物群落多样性降低,门水平变形菌占比上升。属水平上,陆地土壤样品中污染环境优势菌种为Sulfuricurvum,未污染环境中优势性菌种为Candidatus Solibacter(Acidobacteria酸杆菌门)。河流环境沉积物样品中严重污染环境优势菌种为Clostridium,污染程度轻地区优势性菌种为Comamonadaceae。(2)arsB、ACR3功能基因亲缘关系较近菌种主要为β变形菌及γ变形菌。arsB功能基因亲缘关系较近菌种为假单胞菌,在河流沉积物样品中分布多,分布受地理位置影响大;ACR3基因在农田土壤中分布多,总体分布范围广泛,多样性高,分布受样品种类及地理位置影响小。ACR3与产硫酸杆菌亲缘关系近,砷和硫存在偶合现象。(3)分离耐砷菌种一株:Bacillus sp.JH-5-4。该菌种稳定携带arsB、ACR3耐砷基因,两种基因分别来自于不同菌种,在培养过程中使培养基pH上升,单位细菌数量中携带arsB基因拷贝数高于ACR3基因1000倍以上。培养体系中耐砷基因拷贝数随砷浓度升高而升高。培养系中添加林可霉素(10μL),在砷浓度为5mM时抑制arsB,其余条件均提高两种耐砷基因拷贝数。
[Abstract]:The accumulation of arsenic-bearing gold tailings usually leads to the release of arsenic (Arsenic,As) pollutants to the surrounding environment, resulting in arsenic pollution in the surrounding environment, which has attracted global attention. Existing studies have shown that microorganisms play an important role in arsenic cycling, while arsenic has little effect on microbial community structure and diversity. In order to investigate the effect of arsenic pollutants on the diversity of microbial community structure and arsenic tolerance potential, the farmland soil samples and river sediment samples of a gold tailings reservoir in Dandong area, Liaoning Province, China were collected. The microbial community structure and distribution in this environment were studied by high throughput sequencing. The abundance and phylogeny of arsenic resistant genes arsB and ACR3 were analyzed by PCR clone library method, and the stable carrying arsB, was analyzed. The arsenic-tolerant bacteria of ACR3 functional gene were isolated and cultured to explore the growth and basic characteristics of the strain in laboratory environment. The main results are as follows: (1) arsenic pollution reduces the diversity of microbial communities and increases the proportion of deformed bacteria at gate level. At the genus level, the dominant strain in land soil samples was Sulfuricurvum, in the unpolluted environment, and the dominant strain in the unpolluted environment was Candidatus Solibacter (Acidobacteria acid bacteria). The dominant strains in river environmental sediment samples were Comamonadaceae. (2) arsB,ACR3 functional genes. The genetic relationship of arsB functional genes was mainly 尾-deformable bacteria and gamma-deformed bacteria. The genetic relationship of arsB functional genes was Pseudomonas, which was more distributed in river sediment samples, and the distribution was greatly affected by geographical location. ACR3 gene is widely distributed in farmland soil, with wide distribution range and high diversity, which is less affected by sample species and geographical location. ACR3 is closely related to sulfuric acid producing bacteria, and arsenic and sulfur have coupling phenomenon. (3) one arsenic tolerant strain, Bacillus sp.JH-5-4., is isolated from farmland soil. (3) one arsenic tolerant strain, ACR3, is isolated from farmland soil. (3) one arsenic tolerant strain, ACR3, is isolated from arsenic tolerant bacteria. The strain stably carries arsB,ACR3 arsenic-tolerant gene, and the two genes come from different strains respectively. During the culture process, the pH of the culture medium increased, and the number of copies of arsB gene carried per unit bacterial number was more than 1000 times higher than that of ACR3 gene. The copy number of arsenic-tolerant gene in the culture system increased with the increase of arsenic concentration. When lincomycin (10 渭 L) was added to the culture line, the other conditions of inhibiting arsB, at arsenic concentration of 10 渭 L increased the copy number of arsenic resistant genes.
【学位授予单位】：中国地质大学(北京)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172

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