【作者】 申丽；

【导师】 邱冠周； 刘学端；

【作者基本信息】 中南大学 ， 生物工程， 2011， 博士

【摘要】 我国矿产资源是以低品位多金属复杂原生硫化矿为主,传统选冶技术成本高,环境污染严重已经不能满足经济的需求,而微生物冶金通过利用以矿物为能源的微生物的作用,氧化分解矿物使金属离子进入溶液,进一步分离、提取金属具有成本低、流程短、设备简单、低污染等优势逐渐成为解决资源开发与环境保护矛盾的新途径。但生物冶金过程存在浸出速度慢、浸出周期长,对某些重金属缺乏抗性的问题,特别是对于复杂低品位的原生硫化矿而言,需要有高氧化活性的微生物才能破坏其晶格,而如何快速鉴别与筛选高效浸矿微生物成为解决生物冶金浸出速度慢、浸出率低这一难题的关键；另一方面,生物冶金过程的多样性也决定了浸矿体系微生物种群与功能及其影响因素的复杂性,有效的浸矿微生物在浸矿体系中没有高效表达。上述两个难点是制约微生物冶金技术发展的瓶颈,因此,如何强化细菌浸出成为生物冶金发展的重要课题,需要用更新、更有力的生物技术来研究浸矿微生物本身特性。嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)是目前研究得最多的浸矿细菌,在矿堆、浸矿系统中普遍存在,好氧嗜酸,可以将Fe2+氧化成Fe3+,将还原态的硫氧化成SO42-,是生物冶金中最常用的菌种,也是完成全基因组序列测定的唯一浸矿细菌,这样我们就可以在全基因组水平上对A. ferrooxidans进行全面系统的研究。本文首先基于TIGR公布的A. ferrooxidans ATCC 23270全部序列信息共选得3217个寡聚核苷酸探针,挑选人类基因和植物基因作为阴性对照或定量对照,并以A.f菌的基因组DNA作为阳性对照,设计了58mer寡核苷酸全基因组芯片,整个芯片包含3270个探针。我们还对此芯片进行了评估,此芯片的杂交最佳温度为45℃；检测灵敏度为5ng, 100ng时已经达到饱和,且信号值与浓度之间具有很强的线性关系(r2=0.992),可以作为定量判定的依据；没有发现与非靶标基因间的非特异性交互杂交现象,表明此寡核苷酸芯片具有良好的特异性。利用构建的全基因组芯片对不同浸矿活性的菌株进行了基因层面的分析,在3217个基因中发现有967个基因为不同的菌株所特有,其余的2250个(70%)基因为所有A.f菌共有,可以作为是否是A.f菌的判据；另发现320个高氧化活性菌特征基因,在其中还找到135个与亚铁和硫氧化以及抗性等功能有关的基因,这些基因作为A.f菌冶金性能好坏的判据。据此建立了“嗜酸氧化亚铁硫杆菌及其活性的基因芯片检测方法”国家标准(GB/T20929-2007)。为了进一步了解有效的浸矿微生物在浸矿体系中高效表达的途径,本文基于我们已经克隆和测序获得的嗜酸性微生物序列和NCBI已知的相关序列设计了1194个50mer寡聚核苷酸探针,其中包括16S rRNA探针140个、功能基因探针1054个,这些功能基因探针的靶标序列分别涉及到碳固定,氮代谢,硫代谢,铁氧化,金属抗性、电子传递)和外膜蛋白相关的功能基因,人类基因在芯片中用来作为阴性对照和定量对照。此外,基因芯片上所涉及的这些靶标序列几乎包含目前在自然酸性环境和浸出系统中已知的27个属和55种嗜酸性细菌的全部基因序列,能够有效的分辨酸性生态系统中微生物群落结构和功能的差异性。本文还使用基于16S rRNA基因的RFLP技术研究了AMD微生物群落的地理分布,以及使用第二代功能基芯片研究了其微生物群落的系统发育和功能多样性、组成和结构；结果表明检测的不同的AMD样地其微生物群落无论在系统发育多样性、功能多样性还是组成和结构上都不同,周围的环境参数可能是造成这种现象主要的原因,尤其是Fe、S、Ca、Mg、Zn和Cu的浓度以及pH值是其最主要的影响因素；这项研究提供了有力的手段去了解酸性矿坑水的地理分布、多样性、组成、结构和功能同时对进一步发展生物冶金技术从废矿石和低品位矿石中回收有价金属离子提供有力的科学支撑。更多还原

【Abstract】 The mineral resources in China are mainly Low-grade complex and original metal sulfide-based. The traditional mineral separation and smelting technology with high cost and environmental pollution can no longer meet the demands of the economy, however, micro-organisms through the use of mineral-metallurgical energy microorganisms, oxidative decomposition of the metal ions into the mineral solution, and further separation, extraction of metals with low cost, raw materials low demand, the process short, simple equipment, low pollution and other advantages to has become a new way to solve the problem between resource development and environmental protection.However, the bioleaching metallurgical process is slow, leaching cycle is long, lacks resistance to some heavy issues. Especially for the native complex of low-grade sulfide ore, the biological oxidation requires more energy, which requires a higher oxidation activity of the microorganisms to destroy its crystal lattice, but how to quickly identify and screen high leaching microorganisms become key factors to the problem of the slow bioleaching and the low leaching rate; on the other hand, the biological diversity of metallurgical process also determines the leaching system and function of microbial populations and the complexity of factors, effective microorganisms in the leaching system are not highly expressed. The two difficulties restrict the development of microbial metallurgy, therefore, how to strengthen the development of bacterial leaching becomes important issue in biometallurgy, It is necessary to use newer, more powerful techniques to study the bio-leaching characteristics of the microorganism itself.A. ferrooxidans is the most studied bioleaching bacteria. It could oxidize Fe2+ to Fe3+, reduced sulfur in the oxidized SO42-, are prevalent at low and middle temperatures and in the FeS2 environment (mine heap leaching system), and is a very important for industrial production and environmental protection of the chemoautotrophic bacteria, is the most commonly used bio-metallurgical strain. It is the completion of genome sequencing the only bioleaching bacteria, so that we can comprehensive study on the whole genome of A. ferrooxidans. Based on the published TIGR A. ferrooxidans ATCC 23270 sequence information,3217 oligonucleotide probes were selected. And specific human genes and plant genes were selected as negative controls and quantitative controls. With A.f bacterial genome to DNA as a positive control,58mer oligonucleotide whole genome chip were designed, which contains 3270 probe. To verify the practicality and find the best hybridization conditions, we evaluated this chip. The results show:The best temperature for the hybridization is 45℃; the signal at a concentration of 100ng value has already reached saturation and test sensitivity is 5 ng, moreover, the signal value Concentration has a strong linear relationship (r2=0.992), probe found no non-target genes with non-specific interaction between the hybrid phenomenon indicating that the oligonucleotide microarray has a good specificity.We also use the whole genome micro array constructed leaching activity of different strains of gene-level analysis. Through this experimental analysis,3217 genes were found to have 967 genes specific for different strains. The remaining 2250 (70%) were shared by total A.f bacterial gene, which can be used as the criterion of whether it is A.f; Among the other 320 genes with high oxidation activity characteristics of bacteria,135 related with resistance to ferrous iron and sulfur oxidation and other functions related genes. The bacterial genes could be used as criteria to judge the metallurgical properties of A.f bacteria. Thus a " The methods of testing Acidithiobacillus ferrooxodans and its activity by microarray technology " National Standards (GB/T20929-2007) was established.For to understand how effective microorganisms in the leaching system are highly expressed, we have cloned and sequenced the obtained sequences and NCBI acidophilic micro-organisms related known sequences of the 1194 oligonucleotide probes, including 140 16S rRNA probes, functional gene probe 1054. The functional gene probe target sequences were related to carbon sequestration, nitrogen metabolism, sulfur metabolism, iron oxide, metal resistance, electron transfer and outer membrane protein-related genes. In this chip human gene were used as negative controls and quantitative controls. In addition, gene chip containing target sequences involved nearly all the present leaching systems in the natural acidic environment, the 27 known genera and 55 species of acidophilic bacteria of all gene sequences, which can effectively distinguish acidic microbial community structure of ecosystems and functional differences.This work used 16S rRNA gene-based RFLP technology to study the geographical distribution of microbial communities in AMD, and used functional genome arrays of the second generation to study its microbial community diversity in the system development and function, composition and structure; The results show that in detection of the different AMD plots, the microbial community in terms of phylogenetic diversity, functional diversity or composition and structure are different, the surrounding environmental parameters may be the main reason for this phenomenon, especially Fe, S, Ca, Mg, Zn and Cu concentrations and the pH value is the most important factor; This study provides a powerful means to understand the geographical distribution of acid mine water, diversity, composition, structure and function, meanwhile, provide a strong scientific support to the further development of bio-metallurgy technology from the waste rock and ore in the recovery of the low taste of metal ions.更多还原