高鐵氧化效率之嗜酸性硫鐵桿菌

Abstract

Acidithiobacillus sp. CP1為嗜酸性化學自營菌，生長速度緩慢，常用於生物浸礦、礦坑廢水、氧化還原實驗上。本實驗室先前冺用Acidithiobacillus sp. CP1氧化二價鐵發現其可能為Acidithiobacillus ferrooxidans，因此結合化學反應之高氧化性及生物反應之再生循環，用來處理硫化氫，但由於生長速度緩慢，使系統效率受限於生物反應，因此藉由突變得到可快速生長及快速氧化二價鐵之菌株，提升生物反應轉換效率。 本實驗冺用NTG(N-methyl-N’-nitro-N-nitrosoguanidine) 對Acidithiobacillus sp.CP1進行隨機突變，並冺用點菌塗盤、液態培養及連續式培養方式，篩選出具生長快速及具高氧化二價鐵能力之突變株Acidithiobacillus sp. W3，篩選結果之突變株在培養108小時內鐵氧化效率是野生株的10倍，細胞生長速度在培養108小時內突變株也為野生株之8倍，而突變株的停滯期(lag phase)也從原本108小時提前至培養24小時內達到。在突變株Acidithiobacillus sp. W3生理生化活性測詴中，我們測詴數個條件如基本鹽類濃度、亞鐵離子濃度、CO2濃度等，找出最適生長條件，在實驗結果中得到當培養在含亞鐵離子10gL-1及一倍鹽類濃度基質溶液中，每分鐘通入50ml空氣並混合10%二氧化碳為Acidithiobacillus sp. W3最佳生長條件。在基因的表現方面，比較不同生長時期之鐵氧化基因rus operon 及 pet operon表現，經由qPCR的結果中發現兩個operon在突變株中表現量都高於野生株，尤其在停滯期(lag phase)的表現差異最大，相關基因的表現差異大約在10至250倍之間。

The high concentration H2S contained in biogas is corrosive and hinders biogas application. The combined chemical- biological H2S removal system contains rapid H2S oxidation in chemical reaction and cyclic Fe3+ regeneration in biological reaction for biogas purification. The microbial strain, Acidithiobacillus sp. CP1, applied in this system is acidophilic, chemoautrophic and oxidize Fe2+ to Fe3+, yet the ferric ion regeneration efficiency was limited due to the slow bacteria growth rate. In this study, NTG(N-methyl-N’-nitro-N-nitrosoguanidine) was applied to wild type strain Acidithiobacillus sp. CP1 for random mutation selection in order to get the rapid grow mutants. We picked up the rapid-formed colonies to proceed the continuous culture selections, and obtained the best performance mutant strain named Acidithiobacillus sp. W3. The iron oxidation of the△W3 strain is 10-fold higher to the WT strain after 108 hours culture.The cell growth velocity of the W3 strain is 8-fold higher to the WT strain by shorten the lag phase III period from 108 hours to 24 hours. We also investigated the optimal growth conditions including salt concentrations, CO2 concentrations, Fe2+ starting concentrations, and aeration conditions. The results showed that the best growth condition is 10% CO2 contained in 50 ml/min air aeration and 10gL-1 ferrous ion on 1-fold basal salt medium. In the gene expression aspect , we also analysis the iron oxidation related rus and pet operons to identfy the differences in mutant and wild type strain in varies conditions. By qPCR analysis, the results showed the two operons are dominantly expressed in the mutant strain, especially in the lag phase, the related genes expression are 10 to 250 fold comparing to the wild type strain.