結合土壤/孔隙水縮模培養、總體基因體與即時聚合酶鏈鎖反應探究水田根圈系統內主要汞甲基化菌群組成;Using root soil & pore water microcosm incubations coupled with metagenomic and qPCR techniques to probe primary Hg-methylating guilds in the paddy rhizosphere

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Title:	結合土壤/孔隙水縮模培養、總體基因體與即時聚合酶鏈鎖反應探究水田根圈系統內主要汞甲基化菌群組成;Using root soil & pore water microcosm incubations coupled with metagenomic and qPCR techniques to probe primary Hg-methylating guilds in the paddy rhizosphere
Authors:	廖怡婷;Liao, Yi-Ting
Contributors:	環境工程研究所
Keywords:	水稻田;甲基汞;即時聚合酶鏈鎖反應;總體基因體;硫酸鹽還原菌;rice paddies;methylmercury;qPCR;metagenomics;sulfate-reducing bacteria
Date:	2017-08-22
Issue Date:	2017-10-27 16:24:04 (UTC+8)
Publisher:	國立中央大學
Abstract:	近期文獻雖顯示攝食稻米是人類除了魚類食品外暴露於甲基汞的另一重要途徑，但目前對於水田生態系統內操控甲基汞(在根圈土壤)的生成、攝取與最終累積(於米粒)的生物地球化學機制所知仍極為有限。為深入了解此一課題，實驗室於2014年開始在台中火力發電廠周圍的水稻田進行採樣調查，並從根圈土壤的縮模培養試驗觀察到硫酸鹽還原菌極可能是現地主要的汞甲基化菌群；然而，此部分的試驗由於並非直接使用孔隙水，而是採用適合絕大多數厭氧菌生長的合成培養液進行實驗，加上微生物族群的細部結構並未分析，因此所得的結果尚未能實際且完整的窺得水田此敏感生態系統內部汞循環轉化的全貌。有鑑於此，本研究此次即利用現地所採的根圈土壤與其孔隙水重複前期的縮模培養，在針對可能的厭氧菌群依其獨特的生理條件添加各自生長所需的促進或抑制物質，於厭氧狀態下同時進行汞甲基化與去甲基化試驗，並藉由qPCR相對定量及metagenomics次世代定序技術，分析主要操控甲基汞生成的厭氧微生物族群。所得的結果顯示不同培養條件下的甲基汞去除率相差無幾，代表孔隙水中甲基汞的累積程度主要還是取決於甲基汞的生成潛勢，且由化學分析與汞甲基化基因的量化也可再次確認主導汞甲基化作用的菌群的確以硫酸鹽還原菌為主。qPCR的結果也表明Delta-proteobacteria為主要的汞甲基化菌群；Metagenomics的分析結果指出Geobacter為普遍存在且物種豐富度最高的汞甲基化菌屬。綜合上述結果，可得知本研究所選定的水田場址主要的汞甲基化菌群為Deltaproteobacteria中的硫酸鹽還原菌與鐵還原菌。此結果或許可作為往後水稻系統中汞污染相關預防、管理與整治工作相關策略的擬定基礎。;Recent studies have shown that in addition to intake of piscivorous fish, rice consumption is another critical route of human expose to methylmercury (MeHg), the most toxic form of mercury (Hg) in the environment. Nonetheless, there is still a paucity of data on the biogeochemical mechanisms that control the formation (in the rhizosphere), uptake (by root), and eventual accumulation of MeHg (in rice grain) in the paddy ecosystem. To gain an in-depth understanding of this undesirable environmental process, in 2014 we began our investigation and initiated fieldwork at rice paddies that were proximal to the coal-fired power station in Taichung. Preliminary results of microcosm incubations (of root soil samples) suggested that sulfate-reducing bacteria (SRB) might be the primary Hg methylators at our study sites. However, because the incubation tests were conducted with synthetic media instead of pore water, there was a potential fraud in our methodology that might have resulted in a bias in our observations. Further, a detail look at the microbial community structure has not yet carried out. In light of this, here we aimed at rectifying our former protocols of microcosm incubations to confirm the role of SRB as the principal Hg methylating guild. More importantly, this study incorporated certain advanced molecular biology techniques including real-time polymerase chain reactions (qPCR), metagenomics, as well as the next-generation sequencing (NGS) into this inquiry, hoping to obtain a complementary interpretation of methylation results at the cellular level. Results from root soil/pore water incubations assayed with Hg methylation & demethylation confirmed that SRB indeed were the major Hg-methylating guild in the rhizosphere of our study sites. Relative quantification of the hgcA level by qPCR also indicated that Deltaproteobacteria was the principal Hg-methylators at the class level, consistent with the aforementioned role of SRB. In addition, our data suggested that iron-reducers and methylotrophic- and hydrogentrophic-methanogens, while not prominent, might as well play a certain role in MeHg production in paddies. However, metagenomic analysis of 16S rRNA genes showed that Geobacter was the most abundant genus in all samples, suggesting that there are a significant amount of unknown Hg-methylating microbes inhabiting in paddies that await to be identified. On the basis of all these results, time-course experiments focusing on RT-PCR and RT-qPCR of mRNA transcribed from the hgcAB gene cluster are warranted for the future study to pinpoint Hg-methylators at the species level. Ultimately, information gain from this type of investigations may entail us to devise more efficient and sounder remediation strategies to deal with Hg contamination issues in farmland.
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