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研究生(外文):Wen-Hung Huang
論文名稱:結合土壤縮模試驗與 Real-time PCR 探究水田主要汞甲基化菌群
論文名稱(外文):Investigation of primary mercury methylating guilds in rice paddies using soil incubation microcosms and qPCR techniques
外文關鍵詞:rice paddiesmethylmercuryqPCRsulfate-reducing bacteria
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近幾年的研究顯示除了水產食品之外,稻米的攝食亦是人類暴露於甲基汞的另一重要途徑,然而目前對於水稻田系統中操控甲基汞的生成及稻作植株對甲基汞的攝取與累積於米粒的生物地質化學機制所知仍有限,為深入探討此一課題,本實驗室自 2014 年開始對台中火力發電廠周邊水稻田進行調查,以根圈土壤與合成培養液進行縮模試驗,並從中觀察到硫酸鹽還原菌極可能為主要的汞甲基化菌群。然而此試驗並非直接採用現地孔隙水,且未分析微生物族群的細部結構,其結果可能無法完整的反應水稻田系統內部汞循環與轉化的實際狀況。因此,本研究接續先前的研究,並採用現地根圈土壤與孔隙水重複與追蹤調查前期的縮模試驗,並依可能的厭氧菌群其特定營養條件添加各自相應的促進或抑制劑,於相同時空環境下同時進行汞的甲基化與去甲基化試驗,另外亦藉由qPCR相對定量技術,從基因層面探討主要左右甲基汞生成的厭氧菌群。研究結果顯示,不同控制條件下的甲基汞去除率相近,表示水田環境中的甲基汞累積程度主要取決於汞的甲基化潛勢;化學分析與 qPCR 相對定量的綜合結果再次顯示本研究所選的水稻田環境是以 Deltaproteobacteria 中的硫酸鹽還原菌為主要汞甲基化菌群。而此研究結果或許可供後續水稻田環境系統中的汞污染防治與管理策略的擬定參考。
Recent studies have shown that in addition to marine foods, rice consumption is another significant route of human exposure to methylmercury. However, knowledge of the mechanism that underpins mercury methylation in rice paddies as well as the uptake and accumulation of methylated mercury in rice plants is still limited. To explore this subject, we began with field studies that measured relevant geochemical and microbial parameters pertaining to mercury cycling in a suite of rice paddies proximate to the Taichung coal-fired power station in 2014; soil incubations with synthetic growth media were also carried out. It was observed that sulfate-reducing bacteria (SRB) were likely the primary mercury methylators under the investigational conditions. However, because these tests did not directly use the pore water from the rice fields, nor did them analyze the detailed structure of the microbial population, the obtained results might not be able to thoroughly reflect the actual picture of mercury biogeochemical transformations in the paddy systems. Therefore, in this study we modified our previous set-ups by using not only the rhizosphere soil but also the porewater of our study sites to probe the potential of in situ mercury methylation that is closer to the reality. Additionally, quantitative real-time PCR (i.e., qPCR) was incorporated into the investigation to obtain the information of microbial activity at the molecular level in terms of gene copy numbers. Results show that the methylmercury degradation rates under different experimental conditions were similar, indicating that the degree of methylmercury accumulation in paddy fields might depend mainly on the methylation intensity. The combined results of chemical analysis and qPCR showed that the principal mercury methylating guild of the selected paddy fields might still be Deltaproteobacteria, in particular SRB. The outcome of this study may serve as a reference for the prevention and management strategies of mercury methylation in arable land systems.
摘要 i
Abstract ii
目 錄 iv
圖 目 錄 vi
表 目 錄 vii
第一章 前言 1
1-1 研究緣起 1
1-2 研究目的 6
第二章 文獻回顧 7
2-1 環境中汞的來源、型態與危害 7
2-2 濕地為汞甲基化的熱點場址 11
2-3 參與汞甲基化反應的微生物 13
2-4 環境因子對汞甲基化的影響 17
2-5 水稻田的甲基汞生成與風險 22
第三章 研究方法與設備 26
3-1 研究架構流程 26
3-2 場址概述 27
3-3 實驗藥品與試劑 29
3-4 現地採樣規劃與樣品前處理 30
3-5 化學分析 32
3-5-1 鐵分析項目 32
3-5-2 硫酸鹽分析 33
3-5-3 孔隙水甲基汞分析 33
3-6 微生物縮模試驗 35
3-7 分子生物實驗 41
3-7-1 DNA 萃取 41
3-7-2 即時聚合酶鏈鎖反應 42
第四章 結果與討論 46
4-1 水稻田環境地化參數分析 47
4-2 汞甲基化縮模試驗結果 50
4-3 去甲基化縮模試驗結果 55
4-4 分子生物實驗結果 58
4-4-1 DNA isolation test 58
4-4-2 Real-time PCR 實驗結果 61
4-5 分生實驗與縮模試驗之探討 69
4-6 研究與環境層面意義 71
第五章 結論與建議 73
5-1 結論 73
5-2 建議 74
參考文獻 75
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