嗜酸性氧化亞鐵硫桿菌突變株生理、生化特性與亞鐵氧化基因之研究

Abstract

Acidithiobacillus ferrooxidans 屬於好氧化學自營菌 (Chemolithotrophic bacteria)，可藉由氧化亞鐵離子 (Fe2+) 成為鐵離子 (Fe3+) 得到生長所須能量。由於此菌具有絕對嗜酸性(acidophilic)，可生存於高濃度之金屬離子，因此被廣泛應用於微生物溶出法(金屬經由微生物代謝作用，或間接與微生物代謝產物反應後從固體介質中溶出)之相關工業，如採礦業，環境工程等。 本實驗室先前以 Acidithiobacillus sp. CP1為野生株，用隨機突變的方式得到一株具有約10倍之亞鐵氧化率及高生長速率突變株 Acidithiobacillus sp. A47-1. 本計畫將分為生理、生化活性及亞鐵氧化酵素、基因等層次來探討 A. ferrooxidans A47-1突變株的亞鐵氧化機制。 在酵素及基因分子層面，本計畫將藉由比對突變前後菌株之重要亞鐵氧化相關基因如rus、 cyc2等基因序列，來分析亞鐵氧化分子作用機制是否改變，並利用大腸桿菌系統表現相關酵素，或直接由突變株中純化相關蛋白進行酵素動力學研究。 在微生物生理及生化活性測試方面，將先比較具高氧化力突變株在不同生長因子影響下的亞鐵氧化效率，如亞鐵濃度、pH、溫度、氧及二氧化碳濃度等。再者將以化學反應之高氧化性結合生物反應之再生循環，利用硫酸鐵 (Fe3+) 溶液和硫化氫氣體進行吸收及氧化作用，將生成之產物亞鐵 (Fe2+) 溶液再以A. ferrooxidans A47-1 突變株進行生物再生作用，最後將再生產物 (Fe3+) 迴流到化學反應器內和硫化氫進行反應，完成吸收-氧化-再生之化學-生物密閉循環系統，藉由觀測反應器內鐵離子變化，及硫化氫去除效果來研究該突變株對亞鐵氧化之生理及生化條件，最後應用此系統進行沼氣中硫化氫去除，純化後的沼氣將可用於沼氣發電。

Acidithiobacillus ferrooxidans belongs to aerobic chemolithotrophic bacterium, which acquire energy from Fe2+ oxidation. This microorganism has being widely applied to mining and environmental engineering technology. In our previous study, NTG (N-methyl-N』-nitro-N-nitrosoguanidine) was applied to wild type strain Acidithiobacillus sp. CP1 for random mutation. High ferrous oxidation ability mutant had been isolated. In this study, we will compare the Fe2+ oxidation activity caused by the following factors: Fe2+ concentration, pH, temperature, and O2/ CO2 concentration between the wild type strain CP1 and the mutant strain A47-1. In addition, we have developed a system, which combined the advantages of both the fast H2S oxidation rate in chemical tank and the regenerable Fe2+/ Fe3+ conversion in biological tank to remove H2S. Therefore, this system will be also applied to study physiological and biological properties in both strains via measuring Fe2+/ Fe3+ conversion and H2S removal rate. In order to study the Fe2+ oxidation mechanisms in mutant strain Acidithiobacillus sp. A47-1, we will compare gene sequences and amino acids variations caused by randon mutagenesis as well as measuring enzyme activity. Genetic study on functional enzymes for ferrous oxidation will be conducted. The ferrous oxidation relative genes from mutant strain A47-1 will be cloned and constructed, and the sequences will be compared with the wild type strain CP1. After that we will overexpress these genes that contained mutant sites, or the mutant enzymes can be isolated directedly from the mutant strain to compare with the wild type enzymes.