以宏觀基因體分析新穎 Candidatus Dehalobacterium strain DLY 降解二氯甲烷機制;The mechanisms of novel Candidatus Dehalobacterium strain DLY for degrading dichloromethane with metagenomic analysis

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题名:	以宏觀基因體分析新穎 Candidatus Dehalobacterium strain DLY 降解二氯甲烷機制;The mechanisms of novel Candidatus Dehalobacterium strain DLY for degrading dichloromethane with metagenomic analysis
作者:	丁律妤;Ding, Lu-Yu
贡献者:	生命科學系
关键词:	二氯甲烷;Dehalobacterium;mec gene cassette;Wood-Ljungdahl 途徑;宏觀基因體
日期:	2023-07-20
上传时间:	2024-09-19 15:20:04 (UTC+8)
出版者:	國立中央大學
摘要:	全球主要的污染物為含氯脂肪族碳氫化合物（Chlorinated aliphatic hydrocarbons, CAHs），因 CAHs 為重質非水溶相液體（Dense Non-aqueoes Phase Liquid, DNAPL），一旦進入地下水層則易形成污染團累積底層，造成難以根除的困境，二氯甲烷（Dichloromethane, DCM）污染議題在近年逐漸受到重視，而在國際上討論厭氧降解二氯甲烷菌株相對少。本實驗目的致力於培養及篩選台灣本土厭氧降解二氯甲烷菌群，以二氯甲烷做為單一碳源進行增殖培養，發現菌屬 Dehalobacterium 成為主要優勢菌，在菌群 GW08 中佔比高達 81.2%。在 13 C 標記二氯甲烷進行培養實驗結果，以氣相層析質譜儀分析後確立代謝產物主要為二氧化碳（Carbon dioxide）及醋酸（Acetate），展現二氯甲烷降解透過 Wood-Ljungdahl pathway。以宏觀基因體進行分析（Metagenomic analysis），在平均核甘酸相似性及平均胺基酸相似性結果表明此株細菌為新穎菌株並命名為 Candidatus Dehalobacterium strain DLY。以反轉錄即時定量聚合酶連鎖反應（RT-qPCR）分析可能參與降解二氯甲烷 mec gene cassette 表現，結果中 10 個 mec 基因皆有顯著性增加，確立 mec gene cassette 參與將二氯甲烷與四氫葉酸（Tetrahydrofolate）形成5, 10-methylene-tetrahydrofolate，並透過 Wood-Ljungdahl pathway 產生二氧化碳及醋酸。本研究針對 Candidatus Dehalobacterium strain DLY 之降解途徑、產物、基因體及基因表現，建立降解二氯甲烷系統，提供以生物修復法整治二氯甲烷污染場址之策略。;Chlorinated aliphatic hydrocarbons (CAHs) are the major pollutants globally. Due to CAHs are Dense Non-aqueous Phase Liquid (DNAPL), they can accumulate in the lower layers of contaminated groundwater, making their removal challenging. The issue of dichloromethane (DCM) pollution has gained attention in recent years, although there has been limited research on anaerobic DCM-degrading bacteria strains internationally. The bacteria consortia of DCM-contaminated groundwater were sampled. After the enrichment of this consortium using DCM as the sole carbon source in the defined medium, we found that the Dehalobacterium genus accounted for 81.2% of the community. To delineate the by-product of DCM by bacterial consortia, we proved that carbon dioxide and acetate were major by-product using gas chromatography-mass spectrometry (GC-MS) analysis. The stable carbon isotope experiments provided further evidence that the organism utilized carbon from DCM through the Wood- Ljungdahl pathway. Metagenomic analysis was conducted. With the Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) analysis, Candidatus Dehalobacterium strain DLY, one novel bacterium was confirmed. The discovery of the mec gene cassette in the genome of Candidatus Dehalobacterium strain DLY suggests its potential involvement in the degradation of DCM. Using RT-qPCR analysis, it was found that all 10 mec genes showed a significant increase. The involvement of the mec gene cassette in the degradation of DCM, leading to the formation of 5, 10-methylene-tetrahydrofolate and the subsequent production of carbon dioxide and acetate via the Wood-Ljungdahl pathway has been established. This study focused on the degradation pathways, products, genome, and gene expression of Candidatus Dehalobacterium strain DLY in the context of DCM degradation. A comprehensive understanding of these aspects was obtained, leading to the establishment of a DCM degradation system. This research provides a strategy for utilizing bioremediation methods to address dichloromethane-contaminated sites.
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