本省南部某污染場址同時有五氯酚、汞、戴奧辛等污染問題，對生態環境有很大的傷害，也危及鄰近居民的身體健康。因此本研究針對該場址汞污染，開發生物復育法，以現地微生物來進行處理。因為污染場址現地的汞污染濃度高達50 ppm，因此在篩選菌種方面，設計篩選所使用的培養基含有60ppm的汞離子 (Hg2+)，在分離到耐汞菌株之後，再利用對merA gene專一的primers以PCR檢測是否帶有merA gene。其次，再進行16 S rDNA比對鑑定出所篩出菌株的菌種。本研究篩選出三株菌，編號分別為B37、A45與A46。菌種鑑定B37為Enterobacter cloacae、A45與A46均為Pseudomonas sp.，且此三菌株均具有merA gene。因污染場址鄰近海岸鹽度較高，需評估菌株耐鹽性，經過測試此三菌株對於海水鹽度 (3.5%) 具有耐受性，且最高都能耐受100 ppm汞濃度。以RT-PCR與real time PCR檢測，發現三菌株的merA gene都能夠被10 ppm汞離子所誘導表現。汞離子去除批次實驗，發現在60 ppm汞濃度下，在NB、PMM、LB培養基中分別以A45、三菌混合、B37組別處理效果最好，12天後分別可以揮發掉54.4%、89.0%、88.6%的汞離子，顯示此三株菌可以作為未來生物復育之候選菌。

A combination of PCP, mercury and dioxin polluted site in Southern Taiwan causes great damage to the natural environment and endangers the health of nearby residents. The goal of this study is to isolate mercury resistant microorganisms from the polluted site and develop an in situ bioremediation technique. Because the concentration of mercury pollution in the polluted site is about 50 ppm, the medium used to isolate bacteria was designed to contain 60 ppm mercury ion. After mercury-resistant bacteria were isolated, PCR specific primers were applied to detect whether the bacteria contain merA gene or not. Identification of bacteria were based on their 16S rDNA sequences. In this study, three bacteria were isolated, designated as B37, A45 and A46. Species identification showed that B37 was closely related to Enterobacter cloacae while A45 and A46 were associated with Pseudomonas sp.. All three bacterial strains were also confirmed to grow well in 100 ppm Hg2+ media as well as under seawater salinity (3.5%). RT-PCR and real time PCR analysis revealed that the expression of merA gene could be induced by 10 ppm mercury ion in all 3 bacterial strains. Mercury ion removal batch studies exhibited that A45 strain in NB medium, combination of 3 strains in PMM medium, and B37 strain in LB mediums possessed the best treatment effect. After 12 days of incubation, 54.4%, 89.0% and 88.6% of mercury ions could be removed respectively. This study shows that these three bacterial strains are useful candidates in future bioremediation.

審定書 i
誌謝 ii
摘要 iii
Abstract iv
目錄 vi
圖目錄 x
表目錄 xii
第一章 前言 1
1.1 研究緣起 1
1.2 研究目的 2
第二章 文獻回顧 3
2.1汞簡介 3
2.2 汞化合物對人體的危害 6
2.2.1 元素汞對健康的影響 6
2.2.2 有機汞對健康的影響 8
2.2.3 無機汞對健康的影響 10
2.3汞污染的處理 11
2.3.1固定法 11
2.3.2熱處理法 11
2.3.3 微生物處理 12
2.4 mer gene組的調控 13
2.5 菌株的篩選鑑定與特性測試 15
2.5.1分子生物技術鑑定菌株 15
2.5.2細菌16S核糖體核糖核酸 (Ribosomal RNA, rRNA) 於菌種鑑定及分類上的應用 16
2.5.3 DGGE (Denaturing gradient gel electrophoresis) 變性梯度膠體電泳 17
2.5.4 merA gene的檢測 19
2.5.5 反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 19
2.5.6 即時定量聚合酶連鎖反應 (Real time PCR, Q-PCR) 20
2.6 微生態系統研究 (microcosm study) 21
第三章 實驗材料與方法 22
3.1菌株的篩選方法 22
3.2 菌株DNA的萃取 22
3.3檢測merA gene 23
3.4 DGGE (Denaturing gradient gel electrophoresis) 25
3.4.1 供DGGE使用所需準備的16S rDNA片段 25
3.4.2 DGGE操作方法 26
3.5 Clone library製作 28
3.6菌種與merA基因鑑定 30
3.6.1菌種鑑定所需的16S rDNA片段 31
3.6.2 16S rDNA clone 檢測 32
3.6.3 merA gene clone檢測 33
3.7 耐汞菌特性測試 33
3.7.1耐鹽度的測試 33
3.7.2耐汞能力測試 34
3.7.3 生化測試 34
3.7.4 merA gene表現 38
3.7.4.1 RNA的抽取 38
3.7.4.2 核糖核酸反轉錄 (RNA reverse transcription) 39
3.7.4.3反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 40
3.7.4.4即時定量聚合酶連鎖反應 (Real time PCR, Q-PCR) 40
3.8 菌株還原揮發汞離子能力測試 42
3.9 汞消化萃取與檢測方法 43
第四章 結果與討論 45
4.1可還原汞離子菌株篩選 45
4.1.2 耐汞菌株篩選 45
4.1.2 檢測merA gene 45
4.1.3以DGGE刪除重覆的菌株 46
4.1.4 菌株merA gene sequences定序比對 47
4.1.5 菌株16S rDNA sequences定序比對菌種鑑定 48
4.2 耐汞菌特性測試 49
4.2.1 耐鹽度的測試 49
4.2.2 耐汞能力測試 52
4.2.3 菌株生化測試 54
4.2.4 菌種merA gene之表現 55
4.2.4.1以反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 分析merA gene之表現 55
4.2.4.2 以即時定量聚合酶連鎖反應 (Real time PCR, Q-PCR) 分析merA gene之表現 57
4.2.5 菌株還原揮發汞離子能力測試 58
第五章 結論與建議 64
參考文獻 67
圖表 77

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