博碩士論文 104326006 詳細資訊




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姓名 陳詠菁(Yong-Jing Chen)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 Shewanella oneidensis MR-1 於水相均質系統中還原二價汞之機制探討
(Endogenous flavins dominate extracellular reduction of Hg(II) to Hg(0) by Shewanella oneidensis MR-1 in aqueous phase)
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摘要(中) 部分異化性金屬還原菌(dissimilatory metal-reducing bacteria, DMRB)如Shewanella及Geobacter等可在低汞濃度下進行與mer無關的生物性汞直接還原作用,但其背後運轉的機制截至目前為止仍未確認。由於DMRB是沈積物系統(即底泥與地下含水層環境)中掌控污染物質移動與轉化最重要的微生物族群之一,因此深入了解此還原作用的機制層對於未來的相關系統的環境管理與整治將有所助益。有鑑於此,本研究以 Shewanella的Mtr (即metal-reducing)胞外呼吸作用的電子傳遞途徑為模型,利用基因圖譜已全面解密的S. oneidensis MR-1野生菌株,以及分別剔除其細胞外膜表面的多血紅素細胞色素蛋白(即MtrA、MtrB、MtrC及OmcA)的生成基因與黃素類化合物(即flavins)的分泌基因而得的突變菌為試驗模式生物mtrA、mtrB、mtrC、omcA、mtrC/omcA與bfe,進行低汞濃度的暴露試驗,以測試「Shewanella菌屬可利用胞外電子傳遞時所需的外膜細胞色素蛋白將Hg(II)還原成Hg(0),且此還原效率可因所分泌的內源性黃素類化合物的協助而增強」之假說與機制。實驗結果證實S. oneidensis MR-1的細胞外膜色素蛋白雖可將胞外水溶液中的Hg(II)還原為Hg(0),但其反應效率與程度遠不及內源性的黃素類化合物的作用;且當MR-1與外源性的電子梭物質AQDS一起培養時,Hg(II)的還原作用只在厭氧狀態且AQDS達一定濃度下才較為顯著。這些結果說明Shewanella在進行胞外的重金屬轉化時,主要依賴的還是自身所分泌的黃素類電子穿梭物;此外,當環境中存在著具有微生物可利用性的氧化還原活躍介質時,可能會更進一步影響汞在環境中的形態與分佈。
摘要(英) Shewanella oneidensis MR-1 is an important model strain of dissimilatory metal-reducing bacteria (DMRB) that has been shown to reduce low-levels ( 0.3 M) of aqueous inorganic Hg(II) under both aerobic and anaerobic conditions via pathways independent of mer activities. Yet, the electron transport mechanism that underpins this Hg redox transformation process is unclear. Given that DMRB like Shewanella are prevalent in soil and sedimentary environments, as well as that Hg(II) reduction by DMRB may be a critical process in these settings that controls the Hg content in the system profile, a better understanding of the processes modulated by DMRB that govern the fate and transport of Hg in the environment is crucial for future environmental management and remediation efforts. In this study, by using MR-1 and its mutant strains lacking the gene(s) encoding the protein(s) associated with the Mtr respiratory pathway, including ∆omcA, ∆mtrA, ∆mtrB, ∆mtrC and ∆omcA/∆mtrC, and the mutant that lacks the primary bacterial FAD exporter (i.e., ∆bfe), laboratory incubation experiments were carried out to test the hypothesis that while the outer-membrane multi-heme cytochromes of S. oneidensis MR-1 may directly involve in the reduction of Hg(II) to Hg(0), this Hg redox transformation process is facilitated in the presence of endogenous (i.e., flavins) and exogenous (i.e., AQDS) electron shuttles. Results show that (i) Hg redox chemistry in the MR-1 culture was a function of Hg-to-cell ratios, suggesting that this (ratio) factor may need to consider when assessing the spread and impact of Hg in the aquifer; (ii) although outer-membrane cytochromes directly participated in the reduction of Hg(II) of Hg(0), endogenous flavins might play a more important role in this process; (iii) the importance of electron shuttling in the conversion of Hg(II) to Hg(0) by MR-1 was further supported by the results of short-term incubations showing that synthetic flavins and AQDS significantly facilitated Hg(II) reduction with an effect of riboflavin > FMN > AQDS; (iv) in the absence of terminal electron acceptors, MR-1 not only remained viable but also maintained the capacity of Hg(II) reduction after incubation with externally provided electron shuttles for 12 hr.
關鍵字(中) ★ 異化性金屬還原菌
★ 胞外電子傳遞途徑
★ 多血紅素色素蛋白
★ 黃素類化合物
★ 二價汞還原
關鍵字(英) ★ dissimilatory metal-reducing bacteria
★ extracellular electron transport
★ multi-heme cytochromes protein
★ flavin compounds
★ Hg(II) reduction
論文目次 摘要 I
Abstract III
致謝 V
目錄 VII
圖目錄 IX
表目錄 XIII
第一章 前言 1
1-1 研究緣起 1
1-2 研究目的 3
第二章 文獻回顧 5
2-1 全球汞的宿命及傳輸 5
2-2 汞的毒性及危害 6
2-3 地下水相關案例及潛在因素 7
2-4 生物地球化學反應 9
2-4-1 非生物性汞還原作用 10
2-4-2 生物性汞還原作用 11
2-5 異化性金屬還原菌 14
2-5-1 胞外電子傳遞途徑 15
2-5-2 Shewanella oneidensis MR-1之Mtr電子傳遞途徑 17
第三章 研究方法 21
3-1 實驗架構 21
3-2 實驗藥品及配製 22
3-3 模式生物及菌株活化 24
3-4 Shewanella oneidensis MR-1之培養液製備 25
3-5 化學物種組成模擬軟體 27
3-6 平板技術及光學密度曲線法 28
3-7 總汞回收率測試 28
3-8 汞暴露實驗 30
3-8-1 生物性汞還原試驗 30
3-8-2 過濾試驗 31
3-9 總汞分析 31
第四章 結果與討論 35
4-1 吹氣捕捉系統回收率試驗 35
4-2 生物性汞暴露試驗 37
4-3 水化學物種組成之汞暴露試驗 42
4-3-1 不同培養液之汞還原反應 43
4-3-2 過濾試驗 49
4-4 突變菌之汞暴露試驗 52
4-4-1 野生型MR-1及△bfe 52
4-4-2 Mtr 電子傳遞途徑 (△mtrC及△omcA) 55
4-4-3 Mtr電子傳遞途徑 (△mtrA及△mtrB) 58
4-5 內源/外源性電子穿梭物之汞暴露試驗 61
4-5-1 MR-1之內源/外源性電子穿梭物汞暴露試驗 62
4-5-2 △bfe 之化學性電子穿梭物汞暴露試驗 67
4-5-3 無添加電子接受者之汞暴露試驗 69
4-6 環境意義 72
第五章 結論與建議 75
5-1 結論 75
5-2 建議 76
參考文獻 79
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指導教授 林居慶(Chu-Ching Lin) 審核日期 2018-11-28
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