Molecular Breeding of High Ability Iron-oxidizing Bacteria

由于对环境的负荷比传统的火法冶金工艺小，利用铁氧化菌的生物浸出工艺有望成为下一代金属冶炼工艺。然而，很难找到具有高生物浸出能力的铁氧化菌。如果存在这种细菌，则生物浸出也适用于浮选精矿等高品位矿石的处理。本研究的目的是大幅度提高铁氧化细菌的能力。利用偶联抗汞基因的载体质粒作为转化子的选择性标记，考察了电穿孔条件。通过对预处理条件的设计和缓冲液的改进，使菌株Y4-3转化为铁氧化菌成为可能。用含铁氧化酶基因的载体质粒转化该菌株。然而，铁的氧化速率没有明显的提高。检测了载体质粒的稳定性。即使传代10次以上，也证实载体DNA稳定存在。即使在汞离子不存在的情况下，这种稳定性也是一样的。将野生型菌株与转化型菌株置于同一容器中培养，分析二者的竞争关系。在不施加汞离子选择压力的情况下，菌株在培养液中的转化比例迅速下降，只留下野生型菌株。除抗汞基因外，银抗性基因作为一种可选择的基因标记受到了广泛的关注。在10株铁氧化菌中，菌株E-24对银的抗性最强。对菌株E-24的抗性机制进行了研究，发现了银离子向体外放电的可能性。通过对耐药机理的研究，推测银离子向胞外释放。人们曾尝试克隆抗大肠杆菌的银基因，但以失败告终。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