台灣多氯乙烯污染生物整治的突破：本土脫鹵球菌的分離、應用與環境監控創新技術研究

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姓名	呂哲瑋(Che-Wei Lu) 查詢紙本館藏	畢業系所	生命科學系
論文名稱	台灣多氯乙烯污染生物整治的突破：本土脫鹵球菌的分離、應用與環境監控創新技術研究 (Breakthrough in the Bioremediation of Chloroethenes Pollution in Taiwan: Isolation, Application and Innovative Environmental Monitoring Techniques of Indigenous Dehalococcoides mccartyi)
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摘要(中)	多氯乙烯污染對全世界的土壤和地下水構成了長期而棘手的挑戰，特別是致癌化合物氯乙烯（Vinyl Chloride, VC）在自然環境中難以被微生物分解，導致VC在地下水中長期累積，使得污染難以徹底清除。本研究成功分離了能夠完全脫氯VC的台灣本土脫鹵球菌(Dehalococcoides mccartyi)菌種CWV2及其菌群，證實了其在降解四氯乙烯及三氯乙烯至乙烯的過程中，不易產生氯乙烯。進一步利用多體學技術（包括基因體、轉錄體、轉譯體以及蛋白體），我們深入探討了其關鍵還原脫氯酵素VcrA的深度分析，確定了其代謝多氯乙烯的分子調控機制，為未來加強台灣多氯乙烯污染整治提供了重要的策略參考。第二部分中，我們將上述研究應用於一個多氯乙烯污染場址，開發了脫鹵球菌結合生物反應牆的創新脫氯膠體整治技術。實地應用證實，此技術能在300天內達到持久的脫氯效果，並將多氯乙烯完全轉化為無毒的乙烯，展示了該生物膠體整治工法在污染攔截上的良好效果。第三部分，針對生物整治過程中地下水微生物群落的即時監控需求，我們開發了環境生物晶片Dehalochip，以快速檢測脫氯菌群的菌相變化及整治效果。該晶片能夠針對氯烯、氯烷、氯甲烷，氯苯等常見的脫氯基因進行靈敏且專一的檢測。通過現地水樣本的分析，我們驗證了其在實地應用中的有效性，確認Dehalochip可作為現地環境監控的可靠工具。
摘要(英)	Chloroethenes (CE) pollution poses a long-term and challenging issue for soil and groundwater in the world, especially since the carcinogenic compound vinyl chloride (VC) is resistant to microbial degradation in the natural environment, leading to prolonged accumulation of VC in groundwater, thus complicating the remediation efforts. This study successfully isolated a novel strain CWV2 of Dehalococcoides mccartyi (Dhc), and its consortia capable of completely dechlorinating VC. It was confirmed that this strain produces little or no vinyl chloride during the degradation of tetrachloroethylene and trichloroethylene to ethene. Using multi-omics technologies (including genomics, transcriptomics, translatomics, and proteomics), we conducted an in-depth analysis of the key reductive dehalogenase enzyme, VcrA, elucidating the molecular regulatory mechanisms involved in CEs metabolism. This offers significant strategic guidance for future efforts to enhance CEs pollution remediation in Taiwan. In the second part of the study, we applied the research at a CE-contaminated site, developing an innovative dechlorination colloidal gel combined with a permeable reactive bio-barrier (PRBB) using Dhc. Field applications confirmed that this technology achieves lasting dechlorination effects within 300 days, completely transforming CEs into non-toxic ethene, thereby demonstrating the effectiveness of this PRBB remediation method in pollution interception. In the third part, addressing the need for real-time monitoring of microbial communities in groundwater during bioremediation, we developed an environmental biochip, Dehalochip, for the rapid detection of shifts in dechlorinating bacterial communities and the effectiveness of remediation. This chip is capable of sensitive and specific detection of common dechlorination genes associated with CEs, chloroalkanes, chloromethanes, and chlorobenzenes. Through analysis of field water samples, we verified its effectiveness in real-world applications, confirming that Dehalochip serves as a reliable tool for in-situ environmental monitoring.
關鍵字(中)	★ 脫鹵球菌 ★ 多氯乙烯 ★ 多體分析 ★ 轉譯體 ★ 透水性生物反應牆 ★ 現地整治 ★ 生物整治 ★ 生物晶片 ★ 生物添加	關鍵字(英)	★ Dehalococcoides mccartyi ★ Chlorinated ethenes ★ Multi-omics analysis ★ Ribo-seq ★ Permeable reactive bio-barriers ★ In-situ treatment ★ Bioremediation ★ Microarray ★ Bioaugmentation
論文目次	Table of Contents 國立中央大學圖書館學位論文授權書 I 論文指導教授推薦書 II 論文口試委員審定書 III Publications IV 致謝 VI 摘要 VII Abstract VIII Table of Contents IX List of Figures XII List of Tables XVII Chapter 1 Multi-omic Profiling of Dehalococcoides mccartyi Strain CWV2 Reveals Mechanisms of Chloroethene Dechlorination and Potential for Groundwater Bioremediation 1 Abstract 2 1. Introduction 3 2. Materials and methods 6 2.1 Groundwater sample collection and isolation of Dhc strain CWV2 from enriched consortia 6 2.2 Determination of dechlorination ability for CWV2 7 2.3 Genomic analysis of CWV2 7 2.4 RNA extraction and transcriptomic analysis 9 2.5 Ribo-seq translatome analysis of CWV2 9 2.6 Protein extraction and BN-PAGE analysis for dechlorination activity 10 2.7 In-gel trypsin digestion and mass spectrometry 12 2.8 Validation by reverse transcription quantitative PCR 12 2.9 Data available 13 3. Results and discussion 13 3.1. Dechlorination of Chloroethenes by Strain CWV2 13 3.2. Insights from the genome sequence of Dhc CWV2 15 3.3. Transcriptome analysis of Dhc CWV2 17 3.4. Ribo-seq analysis for translation of strain CWV2 19 3.5. BN-PAGE dechlorination activity and Proteomic analysis 20 4. Conclusions 21 Chapter 2 Site Study - An innovative permeable reactive bio-barrier to remediate trichloroethene-contaminated groundwater: A field study 23 Abstract 24 1. Introduction 25 2. Materials and methods 27 2.1. Site characteristics 27 2.2. Chemicals and materials 27 2.3. Preparation of silica gel column and bacterial growth conditions 28 2.4. Practical application of PRBB for remediation 29 2.5. Analysis and Sampling of Chemicals in Groundwater 30 2.6. DNA extraction and cell number quantification 31 2.7. 16S rRNA gene amplicon sequencing analysis 32 3. Results and discussion 32 3.1. Groundwater analyses 32 3.2. Effects of PRBB on dechlorination 35 3.3. Effects of PRBB on reductive dehalogenase genes 37 3.4. Effects of PRBB on bacterial community 38 3.5. The effects of subsurface hydrology on complete reductive dechlorination 40 4. Conclusions 42 Chapter 3 Analyzing Dehalochip: A Functional DNA Microarray for Reductive Dichlorination in Chloroethene-Contaminated Sites 43 Abstract 44 1. Introduction 45 2. Materials and methods 47 2.1 Characteristics of the Site 47 2.2 Practical application of bioaugmentation 48 2.3 Microarray features: Genes Targeted and Probe Design 48 2.4 DNA labeling and microarray hybridization 49 2.5 Analysis and Sampling of Chemicals in Groundwater 50 2.6 DNA extraction and cell number quantification 51 3 Result and discussion 52 3.1 Overall Description of Dehalochip Features 52 3.2 Selection of Functional Genes 52 3.3 Evaluation of the Dehalochip sensitivity and specificity 53 3.4 Comparison of Microarray and qPCR data 54 3.5 Application of Dehalochip to The Bioaugmentation site 55 4 Conclusion 57 Reference 58 Figures 79 Tables 95 Supplementary Figures 104 Supplementary Tables 124
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指導教授	陳師慶羅秀容(Ssu-Ching Chen Hsiu-Jung Lo)	審核日期	2024-7-15
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