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年度	112	專案性質	實驗性質
專案類別	研究專案	研究主題	整治
申請機構	國立雲林科技大學	申請系所	環境與安全衛生工程系
專案主持人	楊茱芳	職等/職稱	教授
專案中文名稱	氯乙烯類污染物之好氧共代謝管柱試驗輔以分子生物技術監測
中文關鍵字	氯乙烯類污染物,好氧共代謝,管柱試驗,分子生物技術
專案英文名稱	Study on aerobic co-metabolism of chlorinated ethylenes using column experiment and molecular-biological investigation
英文關鍵字	Vinyl chloride pollutants, aerobic co-metabolism, column test, molecular biology technology
執行金額	1,025,000元
執行期間	2024/1/1 至 2024/12/31
計畫中文摘要	整治國內受氯乙烯類污染物污染之土壤及地下水時，生物整治技術多採用厭氧還原脫氯機制，因其為序列式的脫氯作用，容易導致低氯數氯乙烯類污染物的累積與擴散，而好氧共代謝可克服厭氧脫氯不完全，並縮短整治時程，好氧共代謝機制仰賴菌株利用主要代謝基質(如甲烷、甲苯及酚等)所表現的低專一性酵素，同時造成氯乙烯類化合物結構上的改變而達到分解污染物之目的。 甲烷氧化菌群為適合好氧共代謝的菌群，因為作為主要基質的甲烷相較於其他好氧共代謝菌群的基質而言較不具毒性，且甲烷氧化菌群廣泛存在於自然環境中；甲烷氧化菌產生的甲烷單氧氧化酵素分成兩種形式，分別為位於細胞質中之溶解性甲烷單氧氧化酵素(soluble methane monooxygenase, sMMO)，以及分布於細胞膜的膜結合性甲烷單氧氧化酵素(particulate methane monooxygenase, pMMO)；可藉由好氧共代謝降解的氯乙烯類污染物包括三氯乙烯、1,1-二氯乙烯、順-1, 2二氯乙烯、反-1, 2二氯乙烯及氯乙烯。含水層生物整治的成效取決於微生物的組成，分子生物技術相較於傳統微生物培養可提供更完整的菌相資訊，亦可定量特定污染物降解基因的數量，協助評估好氧共代謝的成效，乃至於整治策略的改善建議。 本計畫規劃採用好氧共代謝的整治手段，遴選具有潛在暴露風險的場址作為好氧共代謝之甲烷氧化菌群來源，馴養現地的甲烷氧化菌群，藉由管柱試驗模擬受氯乙烯類污染的地下水層，建立整治操作參數並調查場址菌相及管柱內部甲烷氧化菌群分布與結構。甲烷氧化菌群取自受三氯乙烯污染場址內的兩口監測井(MW02與B00583)，以及場址外下游一處監測井(B00422)地下水，由於污染場址地下水pH偏酸，故於馴養甲烷氧化菌群時，分成中性與酸性條件，並以三氯乙烯濃度逐步提升方式馴養，研究成果指出，源自監測井MW02的菌群至多能降解0.3 mg/L三氯乙烯，其餘兩菌群(B00422與B00583)，無論以酸性或中性條件馴養，皆能降解場址最高三氯乙烯污染濃度(0.4 mg/L)；從馴養菌群可檢測有甲烷氧化菌三種功能性基因mmoX、pmoA與mxaF的存在，且基因量隨三氯乙烯馴養濃度而變化，菌相分析亦可分析有甲烷氧化菌屬(Methylobacterium-Methylorubrum、Methylocystis和Methylomonas)與甲基氧化菌屬(Methylobacillus、Methyloversatilis、Methyloparacoccus和Methylophilus)，顯示馴養手段對於甲烷氧化菌的強化與增量是必要的；管柱實驗進流不同三氯乙烯濃度結果顯示，當管柱進流0.05和0.2 mg/L三氯乙烯時，管柱B相對於管柱A與管柱C穩定，進流0.4 mg/L三氯乙烯時，以管柱C有較佳的三氯乙烯降解；當進流流量由0.06 mL/min降低至0.02 mL/min，三管柱的三氯乙烯降解皆變差；當頂空甲烷濃度分別為100%與40%時，管柱C在100%甲烷濃度的三氯乙烯降解表現佳，而管柱B則在40%甲烷時三氯乙烯降解表現較佳；將進流地下水pH調降至pH 5時，額外植種馴養菌群的管柱B較管柱C有更好的三氯乙烯降解表現。
計畫英文摘要	Anaerobic reductive dechlorination mechanism is extensively applied for bioremediating soil and groundwater contaminated with chloroethylenes (CEs). However, its dechlorination rate decreases as the increase of chlorine atoms is removed, leading to the accumulation and dispersion of low-chlorine CEs. To address this issue, the aerobic co-metabolism mechanism provides another optional strategy. Contrary to the anaerobic reductive dechlorination mechanism, the CE removal rate increases as the increase of chlorine atoms is removed. Aerobic co-metabolism involves the induction of specific enzymes by providing primary metabolic substrates (such as methane, toluene, and phenol) to primary substrates metabolic bacteria, and the induced enzyme with low-specificity can combine with target contaminants (such as CEs) to facilitate their biodegradation. Methane-oxidizing bacteria are ideal for aerobic co-metabolism because of their extensive existence and nontoxic substrate. Methane-oxidizing bacteria express two types of methane monooxygenase (MMO) and are soluble methane monooxygenase (sMMO), found in the cytoplasm, and particulate methane monooxygenase (pMMO), distributed in the cell membrane. Both MMOs can cometabolize trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-dichloro-ethylene (cis-1,2-DCE), trans-1,2- dichloroethylene (trans-1,2-DCE), and vinyl chloride (VC). The success of bioremediation in the aquifer depends on the microbial composition. Molecular biology techniques provide higher resolution toward microbial communities than traditional microbiological incubation. Moreover, the number of specific pollutant degradation genes can also be quantitatively analyzed. The gathered information can be applied to evaluate aerobic cometabolism performance and parameter suggestion. This proposal aimed to apply aerobic co-metabolism as a bioremediation approach. At first, the suitable CEs contaminated sites were selected as the sources of target methanotrophic bacteria. After acclimation, the enriched methanotrophic bacteria were used for column experiments to simulate CEs aerobic cometabolic degradation in the aquifer. In addition, molecular biology techniques such as real-time quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS) are used for MMO gene quantification and microbial community analysis, respectively. The methane-oxidizing bacteria were collected from two monitoring wells (MW02 and B00583) in the TCE-contaminated site and the groundwater from a monitoring well (B00422) downstream of the contaminated site. Since the pH of the groundwater in the contaminated site was acidic. Therefore, the methane-oxidizing bacteria are enriched under neutral and acidic conditions, and TCE concentration increases step by step. The research results indicated that the microbial community from monitoring well MW02 could degrade 0.3 mg/L TCE after enrichment. The other two microbial communities (B00422 and B00583), whether enriched under acidic or neutral conditions, could degrade 0.4 mg/L TCE. Three functional genes (mmoX, pmoA, and mxaF) involving methane utilization could be detected from the enriched microbial community, and the gene quantity varied with the TCE concentration. Besides, methanotrophic genera, such as Methylobacterium-Methylorubrum, Methylocystis, and Methylomonas, and methylotrophic genera, such as Methylobacillus, Methyloversatilis, Methyloparacoccus, and Methylophilus, could be detected from various enrichment phases, indicating that enrichment was necessary. The column experiment results demonstrated that when three columns were fed with 0.05 and 0.2 mg/L TCE, Column B was stable relative to Columns A and C. When the influent was 0.4 mg/L TCE, Column C had better TCE degradation performance. When the flow rate of the groundwater decreased from 0.06 mL/min to 0.02 mL/min, the TCE degradation of the three columns became unsatisfactory. When the headspace methane concentration was 100%, Column C had better performance in the TCE degradation. While Column B had the better performance in TCE degradation at 40% methane in the headspace. When the pH of the feeding groundwater was adjusted to pH 5, Column B, which was additionally inoculated with the enriched consortium, had better TCE degradation performance than Column C.