【作者】 吴学玲；

【导师】 邱冠周； 刘新星；

【作者基本信息】 中南大学 ， 微生物学， 2007， 博士

【摘要】 本研究选育对重金属等毒性离子具有抗性的高效浸矿菌株，并对抗性菌株对重金属等毒性离子的耐受能力、耐受机理、抗性相关基因及基因差异表达、阴离子对细菌的影响等进行研究。用9K培养基分别以Fe²⁺及元素S作为能源，对来自江西德兴铜矿、湖北大冶铜矿、广西大厂等多个矿区的矿坑水样本进行了硫化矿浸矿细菌的富集、筛选、驯化、分离纯化，得到能氧化Fe²⁺及元素S的纯化菌株。提取纯化细菌基因组DNA，通过PCR聚合酶链反应扩增出其16S rDNA片段，测定其16S rDNA片段的核苷酸序列，用BLAST对其16S rDNA序列进行比较分析以鉴定选育出的浸矿细菌并构建进化树。在9K培养基中分别加入Cu²⁺，Ag⁺，Hg²⁺，Pb²⁺，Mg²⁺等重金属离子，通过重铬酸钾滴定法测定培养基中的Fe²⁺浓度来确定所分离的菌株在重金属离子抑制情况下对Fe²⁺的氧化能力变化并筛选出重金属抗性菌株。对抗性菌株的最高耐受能力进行测定并在最高耐受浓度时对Fe²⁺的氧化能力进行测定和分析。对筛选出的重金属抗性菌株进行紫外诱变以获得具有更高的Fe²⁺氧化能力及更高重金属离子耐受能力的突变菌株。根据GenBank中的抗性基因序列自行设计抗性基因引物，对实验菌株的重金属抗性基因（抗铜基因、抗砷基因及抗银基因）进行PCR扩增、分子克隆及序列测定，用BLAST搜索工具对上述抗性基因进行分析；对上述抗性基因编码的蛋白质进行分析并构建进化树。在不同铜离子浓度时对抗铜基因差异表达进行分析并进行铜蓝浸矿实验。在以Fe²⁺为能源的9K培养基中加入不同锌盐，研究在同种阳离子存在时阴离子对浸矿菌株的Fe²⁺氧化能力的影响。16S rDNA的序列分析结果表明，本研究的实验菌株均为嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans，简称为At．ferrooxidans）。实验菌株对重金属离子的耐受能力测定结果表明，驯化后筛选出对重金属离子有较高抗性的抗性菌株，对Cu²⁺，Ag⁺，Hg²⁺，Pb²⁺，Mg²⁺的最高耐受能力分别为32000 mg／L，240 mg／L，0.9 mg／L，3500 mg／L，22500 mg／L。而野生菌株对上述离子的最高耐受浓度分别为19000mg／L，60 mg／L，0.1 mg／L，400 mg／L，13500 mg／L，说明驯化后抗性菌株对重金属离子的耐受能力明显增强。对具有较高Cu²⁺抗性和Ag⁺抗性的抗性菌株进行紫外诱变，获得的突变菌株生长性能稳定，比诱变前菌株及野生菌株具有更高的Fe²⁺氧化能力和更强的耐受重金属离子性能。其对Cu²⁺和Ag⁺的耐受能力分别是野生菌的2—3倍，是驯化菌的1.1—1.3倍。对其抗铜机理及抗银机理研究的结果表明：At．ferrooxidans有抗铜基因AFE0454 copper resistance protein；但没有发现抗银基因silC。在不同铜离子浓度时抗铜基因表达有明显差异。铜蓝浸矿实验发现M26^#的浸矿能力明显高于DC^#和26^#，在CuS浓度为9％时M26^#浸出率可达66.87％。对抗铜基因编码的蛋白质进行分析发现了结构域CopD和PcoD。结构域CopD编码抗铜蛋白，而PcoD的预测产物是铜离子导出蛋白。PcoD与CopD均与Cu²⁺抗性有关。通过自行设计的抗砷基因引物对另一个抗性基因抗砷基因arsH进行PCR扩增、克隆及测序。结果显示，实验菌株的抗砷基因arsH的序列与模式菌株At．ferrooxidansATCC23270的序列有14个碱基不同。对arsH基因编码的arsH蛋白进行分析，找到了一段以硫作为信号的NADPH（还原性辅酶Ⅱ）依赖的FMN（黄素单核苷酸）还原酶的功能域。推测At．ferrooxidans菌中arsH的功能可能与arsC基因编码的还原酶的氧化还原作用有关。除了重金属离子对At．ferrooxidans菌的影响，阴离子对细菌生长活性及Fe²⁺氧化能力也有重要影响，其影响的强弱顺序为：SO₄^2-≦Cl^-＜NO^3-，不同菌株对阴离子的耐受能力有明显差异。更多还原

【Abstract】 To screen bioleaching bacteria which are resistant to heavy metals and other toxic ions, and to study the microorganisms’ resistance capacities, resistance mechanisms, responsible resistance genes, and the effects of anions. The sulphides bioleaching bacteria were obtained from acid mine drainage （AMD） samples collected from Dexing Copper Mine in Jiangxi Province, Daye Copper Mine in Hubei Province, Dachang Copper Mine in Guangxi Province, China by enrichment, screening, adaptation, and isolation using 9K medium with Fe²⁺ and sulfur as energy sources. Genomic DNA of the isolates was extracted and the 16S rDNA was amplified using PCR technique. The PCR products were compared and analyzed with the known sequences published in GenBank using the BLAST search tool of the National Centre for Biotechnology Information database; the bioleaching bacteria were identificated; the evolutionary relationships were described and the homolog trees were constructed. 9K medium containing several of heavy metal ions (Cu²⁺, Ag⁺, Hg²⁺ , Pb²⁺ , Mg²⁺) was used to culturemetal-resistant bacteria; the ferrous ions concentrations were determined by potassium dichromate titration methods to examine the bacterial oxidation capacities of ferrous ions. The highest toleranceconcentrations to heavy metal ions for metal resistant bacteria were determined and at which the oxidation capacities of ferrous ions of these bacteria were analyzed. The metal resistant bacteria were UV induced mutated to obtain mutants which have higher oxidation capacities of ferrous ions and higher resistance to heavy metal ions. Resistance gene primers were designed according to the data in gene bank. The resistance genes were amplified by PCR （Polymerase Chain Reaction） and the resulting PCR products were cloned and the cloned productswere sequenced. The cloned results were compared and analyzed with the known sequences published in GenBank using the BLAST search tool of the National Centre for Biotechnology Information database. The amino acid sequences of protein were compared with the sequences in the NCBI database, the evolutionary relationships were described and the homolog trees were constructed. The effects of anions were inverstigated when 9K medium containing ZnCl₂, ZnSO₄, Zn（NO₃）₂,respectively, was used to enrich resistant bacteria.The results of 16S rDNA sequences analysis showed that the isolates in this study are all Acidithiobacillus ferrooxidans（At. ferrooxidans）. The bacterial resistance capacities to heavy metal ions were determined and the results showed that the tolerate concentration levels of the adaptation bacteria were 32000 mg/L (Cu²⁺ ), 240 mg/L （Ag⁺）, 0.9 mg/L (Hg²⁺ ), 3500 mg/L (pb²⁺), 22500 mg/L (Mg²⁺ ), respectively, while the tolerate concentration levels of the wild bacteria were 19000 mg/L (Cu²⁺), 60 mg/L （Ag⁺）, 0.1 mg/L (Hg²⁺), 400 mg/L (Pb²⁺), 13500 mg/L (Mg²⁺ ), respectively, which means the adaptation bacteria have higher resistance capacities than that of the wild types. The adapted resistant bacteria were UV induced mutated and the mutants grew well and had higher resistance capacities to heavy metal ions and higher oxidation capacities of ferrous ions than that of the adapted bacteria. Their tolerate capacities to Cu²⁺ and Ag⁺ were 2-3 times higher than that of the wild bacteria and 1.1--1.3 times higher than that of the adapted types, respectively. The resistance mechanisms to Cu²⁺ and Ag⁺ were studied and the results showed that there is copper resistant gene CopD gene but fails to find silver resistant gene silC gene in At. ferrooxidans. The amino acid sequence coded by copper resistant gene was analyzed and two domains were found named CopD and PcoD. The domain CopD coded copper ion resistance protein D and the domainPcoD has a conserved sequence and presumed as a copper ion exporting protein. CopD and PcoD are both responsible to copper resistance. Another gene, arsenic resistance arsH gene of At. ferrooxidans, was amplified by PCR（ Polymerase Chain Reaction ） and the resulting PCR products were cloned and sequenced. The cloned results were analyzed and compared by NCBI search tool. The amino acid sequence of arsH protein of At. ferrooxidans was compared with sequences in the NCBI database. The results indicated that there are 14 bp of the arsH gene of the tested strain which are different from those of model At. ferrooxidans ATCC 23270. arsH protein coded by arsH gene was analyzes and a functional domain named FMN _red was found which was proposed to be FMN reductase regarding sulfur as a signal. It is presumed that arsenic-resistance arsH gene is functionally related to redox of arsC reductase.Besides the effects of heavy metal ions, anions also obviously impact the oxidation capacities of ferrous ions of these bacteria and the effect orderly NO₃^-＞Cl^-＞SO₄^2-. And the bacterial tolerance levels to anions had visible differences from different strains.更多还原