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年度	109	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	國立中正大學	申請系所	地球與環境科學系
專案主持人	許昺慕	職等/職稱	教授
專案中文名稱	含氯污染場址之微生物代謝分析與評估
中文關鍵字	四氯乙烯,生物復育,自然衰減法,生物添加法,生物促進法,次世代定序技術,本土菌株(群)
專案英文名稱	Microbial Metabolism Analysis and Assessment from Chlorine-Contaminated Sites
英文關鍵字	Perchloroethene (PCE), Bioremediation, Natural attenuation, Bioaugmentation, Biostimulation, Next Generation Sequencing (NGS), Loacl bacteria
執行金額	850,000元
執行期間	2020/1/1 至 2020/12/31
計畫中文摘要	國內土壤及地下水受揮發性有機物污染事件頻傳，又以含氯揮發性有機溶劑之地下水污染事件所佔的比例最大，其中四氯乙烯(PCE)和三氯乙烯(TCE)為最普遍且常用之含氯有機物溶劑。本研究團隊擬定執行六年期生物復育整治計畫，最終目標有下列三項:(1)評估自然衰減所需期程(2)使用本土菌株添加，進行生物添加法，配合生物炭包覆等技術開發，強化添加物傳輸與作用(3)使用基質改良，並利用高通量檢驗技術評估生物促進法之成效，使得透過土壤基質改良並配合水體灌溉等技術評估，進而有效且簡易的改善地下水汙染問題，克服過往受限於水文條件且水體非菌體優勢生長之困難點，其最終目的在於提供國內有機氯化物汙染整治之系統化輸出。評估現地生物復育技術可行性其中最重要條件之一是場址本身須具有可降解污染物之微生物，任何單一微生物是無法完成所有的代謝反應進而降解環境污染物。但透過不同的細菌群落，可共同交互執行所有的代謝反應，完成生物降解。因此，快速、深入分析菌相變動及可能的生物降解代謝途徑為現地生物復育的關鍵步驟，亦是探討自然衰減的重要指標之一。故本研究第一年規劃受氯溶劑污染的土壤和地下水場址進行代表性樣品的採樣，利用高通量分子檢驗技術進行菌群分析，並透過特殊菌種與代謝基因的分子生物檢測，包含Dehalococcoides sp. 16S rRNA、entC gene、entE gene、vcrA gene、pmoA gene及mmoX gene等了解汙染場址之微生物群聚結果、建立本土菌株(群)資料庫，並探討代謝途徑並與現場化學分析結果比較其相關性。 本年度計畫針對兩處汙染廠址共採樣28個地下水樣本及17個土壤樣本。含氯化合物分析結果顯示有6個樣本有含氯相關汙染物超過管制標準。兩類脫鹵菌屬及五個功能基因檢測結果顯示entE gene在所有樣本中檢出率最高，其次是vcrA gene。Ecoplate結果顯示不同深度土壤樣本菌群對於碳代謝能力有顯著關係。NGS結果顯示使用不同序列資料庫在菌屬會有顯著差異，而NGS結果顯示土壤中帶有甲烷營養菌、乙烯營養菌、脫鹵菌及其他含氯代謝可能菌屬。綜整而論，在未整治場址本身就具有脫鹵菌及相關代謝基因，故現地菌種篩選及現地基質改良等是未來整治工法之關鍵，本年度成果可做為未來評估含氯污染場址生物整治成效及後續菌種篩選之依據。
計畫英文摘要	Many domestic cases regarding soil and groundwater contaminated by volatile organic compounds have been reported. Perchloroethene (PCE) and trichloroethene (TCE) are the most pervasive chlorinated solvents in these events. The six-year project will be proposed for bioremediation and three aims will be included for this project, e.g. (1) To assess the efficacy for natural attenuation; (2) To isolate the indigenous bacteria for bioaugmentation; (3) To assess the efficacy of biostimulation by high-throughput metagenomic technologies. The final vision will provide a systematic output in situ bioremediation for local chloride contaminated area. One of the most key points for assessing the viability of bioremediation technology is that there are microorganisms with the potential to degrade contaminants. Any single microorganism is unable to complete all the metabolisms to degrade contaminants, and only complex bacterial communities can perform all of the metabolic pathways to biodegrade contaminants. Therefore, the key step of bioremediation technology is to predominate bacterial communities’ variation and metabolic pathways, and bacterial communities are also the indicators for natural attenuation. In the first year, representative samples from a soil and groundwater site contaminated with chlorinated solvents will be collected by sequential analysis. We hope to comprehensively understand the bacterial community variations and special bacteria speices through PCR-based technologies and NGS for detecting the presence of Dehalococcoides sp. 16S rRNA, entCgene, entE gene, vcrA gene, pmoA gene, mmoX gene and bacterial communities. A total of 28 underground water samples and 17 soil samples were collected from two chloride contamination sites in this study. The chemical analyzing results showed that six samples with chloride were exceeded the "Groundwater and Soil Pollution Control Standards". The PCR outcome of functional gene and Dehalococcoides sp. showed the highest detection rate was entE gene and then was vcrA gene. Ecoplate results indicated the carbon metabolism capacity levels were associated with its depth of samples. The soil samples contained the methanotrophic bacteria, ethylene-trophic bacteria, Dehalococcoides sp., and other chlorine metabolic bacteria from 16s amplicons surveys. In summary, there were Dehalococcoides sp. and other chlorine metabolic bacteria presented in the non-treated contamination sites indicating isolation and medium improvement would approach for bioremediation. The results of this year can be considered as a model to evaluate the effectiveness of biological remediation in chlorine-contaminated sites.