Molecular Breeding of High Ability Iron-oxidizing Bacteria

由于对环境的污染比传统的火法冶金工艺要小，因此利用氧化铁细菌的生物浸出工艺有望成为下一代的金属冶炼工艺。然而，很难找到具有较高生物浸出能力的氧化铁菌。如果有这样的细菌存在，细菌浸出也适用于浮选精矿等高品位矿石的加工。本研究的目的是为了大幅度提高氧化铁细菌的能力。以含汞抗性基因的载体质粒为转化子的选择标记，考察了电转化条件。通过设计前处理条件和对缓冲液的改进，使氧化铁菌Y4-3的转化成为可能。该菌株还被含有铁氧化酶基因的载体载体转化。但在tr-…中没有观察到铁氧化速率的提高。更具变形能力。对载体载体的稳定性进行了检测。证实了载体DNA的稳定存在，即使进行了10次以上的传代。即使在不存在汞离子的条件下，这种稳定性也是相同的。将野生型菌株与转化子在同一容器中培养，并对两者的竞争关系进行分析。在不施加汞离子选择压力的情况下，转化子在培养液中的比例迅速下降，只有野生型菌株留在培养液中。除汞抗性基因外，银抗性基因也是一个可选择的标记。在所检测的10株氧化铁菌中，菌株E-24的耐银性最强。对菌株E-24的抗性机制进行了研究，发现银离子释放到体外的可能性。作为研究耐药机制的结果，预计银离子被释放到细胞外。对大肠杆菌的银抗性基因进行了克隆，但以失败告终。较少

Since the load to the environment is smaller than conventional pyrometallurgy process, bioleaching process using iron-oxidizing bacteria is expected as a next-generational metal smelting process. However, it is difficult to find the iron-oxidizing bacteria with high bioleaching ability. If such bacterium exists, the bioleaching becomes also applicable the processing of high-grade ore such as the flotation concentrate. The purpose of this study is to drastically improve the ability of iron-oxidizing bacteria. Using vector plasmids of which the mercury resistance gene was coupled as a selectable marker of transformants, the condition of the electroporation was examined. By contrivance of the pretreatment condition and improvement on the buffer solution, it was possible to transform the iron-oxidizing bacterium, strain Y4-3. This strain also transformed by vector plasmids containing iron oxidizing enzyme gene. However, the improvement of iron oxidizing rate could not be observed in the tr … More ansformant. The stability of vector plasmid was examined. It was confirmed that vector DNA existed stably, even if the subculture over 10 times was carried out. This stability was same even in the condition that the mercury ion did not exist. Wild type strain and the transformant were cultured in the same vessel, and both competition relation was analyzed. The ratio the transformant in the culture solution rapidly decreased, when it did not put on the selection pressure of mercury ion, and only the wild type strain remained in the culture. As a selectable marker except for the mercury resistance gene, the silver resistance gene was noticed. The strain E-24 showed the highest silver resistance in the 10 strains of iron-oxidizing bacteria tested. On strain E-24, the resistance mechanism was examined, and the possibility of discharging the silver ion to extracorporeal was found. As a result of examining the resistance mechanism, it was anticipated that the silver ion was discharged to extacellular. The cloning of the silver resistance gene to Escherichia coli was tried, and it ended in the failure. Less