以宏觀基因體分析新穎 Candidatus Dehalobacterium strain DLY  降解二氯甲烷機制

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丁律妤(Lu-Yu Ding) 查詢紙本館藏

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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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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 二氯甲烷
★ Dehalobacterium
★ mec gene cassette
★ Wood-Ljungdahl 途徑
★ 宏觀基因體

關鍵字(英)

論文目次

國立中央大學圖書館學位論文授權書 i
國家圖書館學位論文延後公開申請書 ii
國立中央大學碩士班研究生論文指導教授推薦書 iii
國立中央大學碩士班研究生論文口試委員審定書 iv
致謝 v
摘要 vi
Abstract vii
目錄 viii
圖目錄 xi
表目錄 xii
第壹章緒論（Introduction） 1
1.2 微生物生物整治 2
1.2.1 生物修復法應用於含氯有機物污染場址 2
1.2.2 厭氧菌群應用於含氯有機物污染場址 3
1.3 二氯甲烷(Dichloromethane, methylene chloride） 3
1.3.1 二氯甲烷對於環境及人體的危害與影響 3
1.3.2 二氯甲烷之型態、污染來源 4
1.3.3 二氯甲烷降解相關菌株 5
1.3.4 厭氧二氯甲烷降解細菌之代謝及降解路徑 6
第貳章實驗目的和實驗架構（Purpose and Architecture） 8
第參章實驗材料與方法（Materials and Methods） 10
3.1 實驗材料 10
3.1.1 常用藥品與試劑 10
3.2 實驗方法 12
3.2.1 厭氧降解二氯甲烷之菌群來源與繼代培養 12
3.2.2 菌株來源、保存與繼代培養(E.coli) 13
3.2.3 氣相層析火焰離子化偵檢器（Gas chromatography-flame ionization detector, GC-FID） 13
3.2.4 氣相層析質譜儀（Gas Chromatography-Mass Spectrometry, GC-MS） 14
3.2.5 細菌Genomic DNA萃取 14
3.2.6 細菌RNA萃取 15
3.2.7 反轉錄作用 16
3.2.8 DNA洋菜膠電泳 16
3.2.9 TSS法之大腸桿菌勝任細胞備製 17
3.2.10 TSS法之大腸桿菌勝任細胞轉型（Transformation） 17
3.2.11 DNA膠體純化 18
3.2.12 質體純化 18
3.2.13 聚合酶連鎖反應（Polymerase chain reaction, PCR） 19
3.2.14 即時聚合酶連鎖反應（Real-time polymerase chain reaction, qPCR） 19
3.2.15 反轉錄即時聚合酶連鎖反應（Reverse transcription-quantitative polymerase chain reaction, RT-qPCR） 20
3.2.16 Colony PCR 20
3.2.17 TA Clone基因選殖 21
3.2.18 醋酸濃度測定 21
3.2.19 次世代全基因定序（Next Generation Sequencing, NGS） 22
3.2.20 Full-length 16S rRNA gene amplicon analysis 23
3.2.21 宏觀基因體學分析(Metagenomic analysis) 24
3.2.22 平均核甘酸相似性Average Nucleotide Identity (ANI) 及平均胺基酸相似性 Average Amino acid Identity(AAI)分析 25
3.2.23 親緣關係樹狀圖分析（Phylogenomic analysis） 25
第肆章實驗結果（Results） 26
4.1 從台灣本土地下水中篩選降解二氯甲烷菌群其降解特性 26
4.2 厭氧降解二氯甲烷菌群GW08之菌相組成 26
4.3 以GC-MS分析13C標定二氯甲烷菌群降解之產物 27
4.4 台灣本土菌群菌種Dehalobacterium之宏觀基因體學分析 28
4.5 以RT-qPCR分析Ca. Dehalobacterium strain DLY降解二氯甲烷之代謝基因表現 29
第伍章討論（Discussion） 31
5.1 探討菌群GW08降解二氯甲烷之特性及代謝產物分析 31
5.1.1 探討菌群GW08培養條件及特性 31
5.1.2 以GC-MS分析菌群GW08降解二氯甲烷之代謝產物 32
5.2 探討菌群GW08菌相 33
5.2.1 菌群GW08中Dehalobacterium菌種佔比變化 33
5.2.2 菌群GW08菌相其他菌種組成及功能分析 33
5.2.3利用共現網絡（co-occurrence network）分析菌群中細菌間相互關係 34
5.3 Candidatus Dehalobacterium strain DLY菌種鑑定、命名 35
5.4 探討Candidatus Dehalobacterium strain DLY全基因特徵及代謝非氯化物之物質之可能性 36
5.5 探討Candidatus Dehalobacterium strain DLY降解二氯甲烷之mec gene cassette基因表現 40
第六章結論（Conclusion） 41
參考文獻（Reference） 42
圖表 49
附加資料（Supplementary File） 64

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指導教授

陳師慶

審核日期

2023-7-20

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