四氯乙烯(tetrachloroethene，PCE)、三氯乙烯(trichloroethene，TCE)、二氯乙烯(dichloroethene，DCE)是地下水中常見的氯烯類污染物，早期因被不當的排放至地下水中，導致地下水遭受環境污染。在多氯化合物處理中，厭氧處理主要原理是利用能呼吸有機鹵化物的細菌，(organohalide-respiring bacteria，OHRB) 來降解地下水中的污染物，但在處理中可以發現地下水中存在PCE與TCE可能會有脫氯不完全的情況，造成主要中間污染物的累積，而cDCE 與VC揮發性高、移動性強容易隨著地下水流至土壤滲流層中。好氧直接代謝反應中，微生物能直接利多烯類污染物當做能量來源，有學者發現有的混菌能在好氧的環境下將DCE 直接代謝，而能將 DCE直接降解的純菌目前已知只有Polaromonas sp. JS666(Coleman, Mattes et al. 2002)。 Polaromonas sp. JS666可以將cDCE降解成對環境無害的二氧化碳，Polaromonas sp. JS666 菌落外表呈現黃色，革蘭氏染色呈現陰性，無運動能力，適合生長pH值為7.2，在溫度 20-25度時菌株活性較高，但是超過30度下會完全失去活性。 
此研究的實驗總共分為四部分：(1)降解效能測試，目的是測試JS666 對二氯乙烯的分解能力，(2)水中溶氧測試，目的是找出降解cDCE的最佳的溶氧條件(3)乙醇馴化實驗，目的是嘗試誘發JS666 降解基因，(4)模擬地下水中環境測試，目的是模擬地下水中JS666 在有其他原生污染物下cDCE的降解能力。
降解效能測試中，JS666 的降解效率與菌株濃度呈現相關性，能在9.1天內將 0.5 mM 的 cDCE 降解至低於第二類地下水 cDCE 的管制標準。在控制組中，cDCE 濃度幾乎無顯著變化且JS666培養瓶內的氯離子濃度變化與理論cDCE 降解所應產生的氯離子量接近(劑量比為 1:0.98)。這些結果證實內 cDCE 的損失是由JS666的生物降解反應所導致。水中溶氧測試中時間為期間一個月可以看到五種不同條件的溶氧批次接只有些微的下降，從0.6 mM cDCE 下降到0.4 mM，在各濃度中均沒有明線的差異。乙醇馴化實驗中，有植種JS666的實驗組(JS666)經過7天的時間二氯乙烯濃度從0.79 mM下降到0.54 mM，DJS666濃度由0.8mM降低至0.53 mM，三種濃度均有些微的下降但彼此的差異不大。在模擬地下水中實驗中發現JS666在降解 cDCE的過程中可同時分解瓶中TCE(0.57 mM-0.28 mM)，在17.6天後將瓶中TCE濃度下降50.7%。
此研究成果對於cDCE 提供了實驗室的直接好氧生物降解資訊，期望未來對於模廠或實場整治時之實際應用能提供有用之參考。

Tetrachloroethene (PCE), trichloroethene (TCE), and dichloroethene (DCE) are common chlorinated pollutants in groundwater. In the early days, they were improperly discharged into groundwater, causing groundwater pollution. In the treatment of polychlorinated compounds, the main principle of anaerobic treatment is to use bacteria that can breathe organic halides (organohalide-respiring bacteria, OHRB) to degrade pollutants in groundwater. However, during the treatment, it can be found that the dechlorination of PCE and TCE in groundwater is incomplete, resulting in the accumulation of major intermediate pollutants. cDCE and VC are highly volatile and mobile, and can easily flow into the soil seepage layer with groundwater. In aerobic direct metabolic reactions, microorganisms can directly use polyethylene pollutants as an energy source. Studies have found that some mixed bacteria can directly metabolize cDCE in an aerobic environment, while the only pure bacteria known to directly degrade cDCE is Polaromonas sp. JS666 (Coleman, Mattes et al. 2002). Polaromonas sp. JS666 can degrade cDCE into carbon dioxide that is harmless to the environment. The colony of Polaromonas sp. JS666 is yellow in appearance, Gram-negative, and non-motile. The pH value suitable for growth is 7.2. The strain is more active at a temperature of 20-25 degrees, but it will completely lose its activity at temperatures above 30 degrees.

The experiment was divided into four parts: (1) degradation efficiency test, the purpose of which was to test the ability of JS666 to decompose dichloroethylene; (2) dissolved oxygen test in water, the purpose of which was to find the best dissolved oxygen conditions for degrading cDCE; (3) ethanol acclimation experiment, the purpose of which was to induce the degradation gene of JS666; and (4) simulated groundwater environment test, the purpose of which was to simulate the ability of JS666 to degrade cDCE in the presence of other native pollutants in groundwater.

In the degradation efficiency test, the degradation efficiency of JS666 was correlated with the strain concentration, and it could degrade 0.5 mM cDCE to below the regulatory standard for Class II groundwater cDCE within 9.1 days. In the control group, the cDCE concentration had almost no significant change and the chloride ion concentration in the JS666 culture bottle was close to the amount of chloride ions that should be produced by the theoretical cDCE degradation (dose ratio of 1:0.98). These results confirmed that the loss of internal cDCE was caused by the biodegradation reaction of JS666. In the dissolved oxygen test in water over a period of one month, it can be seen that the dissolved oxygen in the five different batches has only a slight decrease, from 0.6 mM cDCE to 0.4 mM, and there is no obvious difference in each concentration. In the ethanol acclimation experiment, the concentration of dichloroethylene in the experimental group planted with JS666 (JS666) decreased from 0.79 mM to 0.54 mM after 7 days, and the concentration of DJS666 decreased from 0.8 mM to 0.53 mM. All three concentrations decreased slightly but the differences between them were not large. In the simulated groundwater experiment, it was found that JS666 could decompose TCE (0.57 mM-0.28 mM) in the bottle while degrading cDCE, and the TCE concentration in the bottle was reduced by 50.7% after 17.6 days.
This study provides direct laboratory information on the aerobic biodegradation of cDCE, with the expectation of serving as a useful reference for future applications in pilot-scale or full-scale remediation.

目錄
摘要	摘-I
ABSTRACT	摘-III
目錄	I
圖目錄	IV
表目錄	VII
第一章 緒論	1
1.1研究起源	1
1.2 研究目的	2
第二章 文獻回顧	3
2.1 地下水中氯烯類污染概論	3
2.1.1 二氯乙烯物理化學特性	3
2.1.2地下水中現地復育技術	4
2.1.3監測式自然降解法	4
2.1.4生物刺激法	4
2.1.5生物添加法	5
2.2 生物復育法	5
2.2.1 厭氧脫氯法	5
2.2.2 好氧降解法	7
2.2.3 Polaromonas sp. JS666 介紹	9
第三章 實驗方法	13
3.1 Polaromonas SP. JS666培養	14
3.1.1冷凍乾燥菌株活化	14
3.1.2 固態與液態培養	14
3.1.3 菌株生長曲線	17
3.1.4 無機鹽培養液	19
3.1.5 菌株保存	22
3.2 Polaromonas SP. JS666鑑定	22
3.2.1革蘭氏染色法	22
3.2.2微生物塗抹法	24
3.2.3核酸萃取及菌種鑑定	25
3.3分析方法	28
3.3.1 GC-MS分析設定	28
3.3.2多氯化合物分析方法	34
3.3.3氯離子濃度分析	38
3.4 Polaromonas SP. JS666降解實驗	39
第四章 結果與討論	40
4.1 Polaromonas SP. JS666鑑定	40
4.1.1外觀確認	40
4.1.2 核酸萃取與定序	45
4.1.3分解菌分子生物偵測方法建立	50
4.2 Polaromonas SP. JS666降解CDCE實驗	53
4.2.1降解效能測試	53
4.2.2水中溶氧測試	58
4.2.3乙醇馴化實驗	60
4.2.4 模擬地下水中環境測試	62
第五章 結論與建議	68
5.1 結論	68
5.2 建議	69
參考文獻	70

 

圖目錄
圖2.1.1.1 二氯乙烯(cDCE)藥品包裝	4
圖2.2.1.1 地下水中生物降解途徑(Dolinová, Štrojsová et al. 2017)	6
圖2.2.2.1 目前已知能降解含氯乙烯類化合物能力的微生物及其分解基質與反應產物。橢圓形符號代表氯乙烯(vinyl chloride，VC)或二氯乙烯(dichloroethylene，DCE)同化(assimilating)分解菌，正方形符號帶表好氧共代謝分解菌，三角形符號代表厭氧脫氯細菌。微生物可利用的含氯乙烯類基質及其最終產物則註記在各類符號之後(Mattes, Alexander et al. 2010)	8
圖2.2.3.1 Polaromonas sp. JS666可能降解路徑(Nishino, Shin et al. 2013)	12
圖3 實驗方法流程圖	13
圖3.1.1.1 Polaromonas sp. JS666 凍菌乾粉	14
圖3.1.2.1 胰蛋白大豆培養基(trptic soy broth soybean-casein digest medium，TSB)	15
圖3.1.5.1 菌株-80oC冷凍保存照片	22
圖3.2.2.1 Polaromonas sp. JS666 微生物塗抹法結果照	24
圖3.2.3.1 (a) primer R藥品照片	27
圖3.2.3.2 (b) primer F藥品照片	27
圖3.1.1.1 (a) SCAN模式TCE離子碎片質譜圖，(b) SIM模式TCE離子碎片質譜圖，(c) SCAN模式TCE與資料庫比對圖，(d) TCE資料庫搜尋圖	30
圖3.1.1.3 (a) GC-MS TCE線性檢量線，(b) GC-MS cDCE線性檢量線	34
圖3.3.1.1 分析多氯烯類污染物所使用之氣相層析質譜儀(GC-MS)	36
圖3.3.1.2 GC-MS 質譜儀軟體參數設定之條件	37
圖3.3.2.1氯離子濃度分析所使用之離子層析儀(Dionex Aquion RFIC)	38
圖3.4.1 Polaromonas sp. JS666降解 cDCE實驗流程圖	39
圖4.1.1.1 (a)使用顯微鏡放大100倍觀測之Polaromonas sp. JS666，(b)使用顯微鏡放大400倍觀測之Polaromonas sp. JS666	42
圖4.1.1.2 菌落劃碟法塗盤照	43
圖4.1.1.3  菌落塗抹法塗盤照	43
圖4.1.1.4 菌落塗盤照	44
圖4.1.2.1 Polaromonas sp. JS666染色體核酸萃取結果	45
圖4.1.2.2 Polaromonas sp. JS666 16S rRNA 基因序列比對結果	47
圖4.1.3.1 Polaromonas sp. JS666 即時聚合酶連鎖反應(qPCR)擴增CMO基因片段之結果	51
圖4.1.3.2 Polaromonas sp. JS666即時聚合酶連鎖反應(qPCR)之檢量線，及溶解曲線	52
圖4.2.1.2 Polaromonas sp. JS666降解 cDCE實驗(1)	54
圖4.2.1.3 Polaromonas sp. JS666降解 cDCE實驗(2)	55
圖4.2.1.4 Polaromonas sp. JS666降解 cDCE實驗(3)	56
圖4.2.1.5 Polaromonas sp. JS666降解 cDCE實驗(4)	57
圖4.2.2.1 Polaromonas sp. JS666水中溶氧實驗	59
圖4.2.3.2 Polaromonas sp. JS666乙醇馴化實驗	61
圖4.2.4.1 Polaromonas sp. JS666模擬下水中環境測試(a) cDCE濃度變化，(b) TCE濃度變化，(c) PCE濃度變化	65
圖4.2.4.2 cDCE TCE JS666 瓶中多氯化合物濃度變化圖	66
圖4.2.4.3 cDCE PCE JS666 瓶中多氯化合物濃度變化圖	66
圖4.2.4.4 cDCE TCE PCE JS666 瓶中多氯化合物濃度變化圖	67
圖4.2.4.5 控制組瓶中多氯化合物濃度變化圖	67

 

表目錄
表2.1.1 地下水中多氯化合物基本特性整理	3
表3.1.4.1無基鹽培養液(mineral salt medium , MSM)之配方	20
表3.1.4.2(a) 儲備濃縮液MSM stock solution-1 之配方	21
表3.1.4.2(b) 儲備濃縮液MSM stock solution-2 之配方	21
表3.3.1.1 GCMS 條件參數彙整	36
表4.3.1.1 Polaromonas sp. JS666降解 cDCE實驗(3)，JS666 瓶中氯離子濃度變化換算表	56
表4.4.4.1 模擬地下水中環境測試之瓶中內容物整理表	62

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