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研究生:施玉賢
研究生(外文):SHIH, YU-SIAN
論文名稱:應用分子生物技術分析受氯乙烯類污染物污染場址菌相及脫氯功能性基因定量之研究
論文名稱(外文):Microbial Community Analysis and Dechlorination Functional Genes Quantification of the Chlorinated Ethylenes Contaminated Site Using Molecular Biotechnology Tools
指導教授:楊茱芳
指導教授(外文):YANG, CHU-FANG
口試委員:莊順興張簡水紋
口試委員(外文):CHUANG, SHUN-HSINGCHANG CHIEN, SHUI-WEN
口試日期:2020-09-28
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:環境與安全衛生工程系
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:109
語文別:中文
論文頁數:92
中文關鍵詞:氯乙烯類污染物即時定量聚合酶鏈鎖反應次世代定序死活菌數分析還原脫氯基因脫氯還原降解潛力
外文關鍵詞:Chlorinated ethenesReal-time quantitative PCRNext generation sequencingLive/dead bacteriaAnaerobic reductive dechlorinationDehalococcoides
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氯乙烯類化合物廣泛應用於乾洗及金屬除油,但因不當處置或意外滲漏,使氯乙烯類化合物污染土壤與地下水體,又因氯乙烯類污染物具高毒性,對環境與民眾健康構成嚴重威脅。基於環境友善與經濟效益,生物復育是目前推崇的整治方法之一,但採用生物復育策略之前,須對污染場址微生物菌相,甚至目標污染物之降解功能性基因與菌株有一定的掌握,以瞭解場址是否具有生物整治潛勢。
高氯數氯乙烯類化合物的降解主要以厭氧還原脫氯為主,著名菌屬Dehalococcoides不同菌種具有不同還原脫氯功能性基因(vcrA、bvcA與tceA),本研究針對污染場址的菌屬Dehalococcoides暨其還原脫氯功能性基因進行分析與定量;另使用次世代定序技術分析場址枯、豐水期菌相是否產生差異,特別是還原脫氯及產氫菌群之變化;並使用螢光染劑Acridine orange (AO)結合流式細胞儀分析污染場址微生物之死活菌數,最後彙整各項環境水質參數與生物性數據,分別以權重法與統計分析瞭解場址之生物還原脫氯潛勢以及環境因子與菌屬Dehalococcoides暨其還原脫氯功能性基因間的相關性。
豐水期上、中及下游監測井不同採樣深度皆可穩定測得各脫鹵球菌功能性基因,豐水期還原脫氯功能性基因濃度大多較枯水期增加10倍;豐水期多樣性明顯低於枯水期,兩時期皆可測得產氫與脫氯菌群;兩時期之污染物濃度變動並不顯著,但豐水期活菌比例整體高於枯水期活菌比例;枯水期基因DHC與溫度成正相關,基因tceA與溫度亦呈正相關;豐水期之基因DHC與溫度成正相關,顯示溫度為脫鹵球菌生長之重要因子;枯、豐水期之脫鹵球菌及其功能性基因濃度,皆未達文獻提及可快速降解污染物之基因濃度,顯示場址雖存有脫氯菌群,但其濃度不足以有效降解氯乙烯類污染物。

Chlorinated ethenes (CEs) are widely used in dry cleaning and metal degreasing. However, due to improper disposal or accidental leakage, CEs contaminate soil and groundwater. CEs are highly toxic and pose a serious threat to the environment and human health. Based on environment friendly technology and economic benefits, bioremediation is currently one of the recommended remediation methods. However, before adopting bioremediation, it is important to investigate bacterial community, and even the functional genes involved in target pollutants biodegradation to understand the potential of bioremediation.
The biodegradation of CEs with high chlorine content is mainly based on anaerobic reductive dechlorination. Different strains of the well-known genus Dehalococcoides have different reductive dechlorination functional genes (vcrA, bvcA, and tceA). To evaluate the potential of bioremediation of the CEs contaminated site, this study analyzed and quantified the genus Dehalococcoides and its reductive dechlorination functional genes. The next-generation sequencing technology was also used to realize bacterial community of the contaminated site in the dry and wet seasons. Moreover, flow cytometry combined with acridine orange was applied to count dead and live bacteria numbers in the contaminated site. Finally, the collected groundwater quality parameters and bioinformatic data were used to assess anaerobic reductive dechlorination potential and the correlation between environmental factors and the reductive dechlorination functional genes of genus Dehalococcoides by weighting and statistical analysis, respectively.
The results indicated that reductive dechlorination functional genes could be stably detected at different depths in the upper, middle and downstream monitoring wells during the wet season. The genes concentrations in the wet season were mostly 10 times higher than those in the dry season. Bacterial diversity in dry season was higher than that in wet season. H2 production bacteria and dechlorination bacteria could be detected in both seasons. TCE and DCE concentrations of dry and wet seasons had no significant difference, but the proportion of live bacteria in the wet season was higher than that in the dry season. In wet and dry seasons, TCE and cDCE concentrations were positively correlated. Gene DHC was positively correlated with temperature, indicating that temperature was an important parameter affecting Dehalococcoides growth. Although genus Dehalococcoides and its functional genes in dry and wet seasons could be detected, their concentrations was not high enough to effectively degrade CEs.

摘要 i
Abstract ii
目錄 iv
表目錄 vi
圖目錄 viii
第一章、前言 1
1.1 研究緣起 1
1.2 研究目的 2
第二章、文獻回顧 3
2.1 氯乙烯類污染物特性與危害性 3
2.2 氯乙烯類污染物生物降解機制 6
2.3 降解氯乙烯類污染物之微生物—脫鹵球菌屬Dehalococcoides 7
2.3.1 還原脫氯功能性基因 8
2.3.2 影響脫鹵球菌降解氯乙烯類污染物之因子 10
2.4 受氯乙烯類污染物污染場址之微生物分析 13
2.4.1 微生物採樣 13
2.4.2 微生物分析技術 14
2.5 污染場址脫氯還原菌群生物降解潛力指標 20
2.6 環境因子統計分析應用 23
2.6.1 變異數分析 23
2.6.2 皮爾遜相關係數 24
第三章、材料與方法 26
3.1 實驗架構 26
3.2 實驗藥品與材料 27
3.3 實驗儀器設備 28
3.4 流式細胞儀菌數分析 28
3.5 微生物total genomic DNA萃取 30
3.6 聚合酶連鎖反應定性脫鹵球菌功能性基因 31
3.7定量聚合酶連鎖反應 33
3.8次世代序列菌相分析 38
3.9統計分析應用 38
3.9.1 變異數分析 38
3.9.2 相關性分析 39
第四章、結果與討論 40
4.1 污染場址概況及污染濃度 40
4.2 污染場址不同區域、時期之脫鹵球菌功能性基因分析 45
4.2.1 枯水期樣品脫鹵球菌與其功能性基因分析 45
4.2.2 豐水期樣品脫鹵球菌與其功能性基因分析 46
4.2.3 枯、豐水期樣品脫鹵球菌與其功能性基因趨勢變化討論 48
4.3 污染場址樣品不同監測井、時期之次世代定序分析結果 49
4.3.1 枯、豐水期各監測井不同深度之Alpha Indices統計表 49
4.3.2 枯、豐水期樣品占比前十大菌相分析 51
4.3.3 枯、豐水期脫氯菌群與產氫菌群變化 55
4.4 污染場址樣品不同監測井、時期之死活菌數分析 60
4.4.1 枯水期死活菌數分析 60
4.4.2 豐水期死活菌數分析 60
4.4.3 枯、豐水期死活菌數綜合討論 61
4.5 污染場址脫氯還原菌群降解潛力 63
4.6 枯、豐水期間脫鹵球菌暨其功能性基因與污染濃度暨水質參數之相關性分析 67
4.6.1 污染場址環境參數敘述統計分析 67
4.6.2 枯、豐水期不同監測井脫鹵球菌暨其功能性基因之變異數分析 69
4.6.3 枯、豐水期脫鹵球菌暨功能性基因與各項環境因子相關分析 70
第五章、結論與建議 74
5.1 結論 74
5.2 建議 75
參考文獻 76


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