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年度	110	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	國立臺灣大學	申請系所	生物環境系統工程學系
專案主持人	蕭友晉	職等/職稱	助理教授
專案中文名稱	以穩定同位素探針與次世代定序技術解析降解含氯有機物之微生物族群結構與活性
中文關鍵字	穩定同位素探針技術, 次世代定序技術, 生物降解含氯有機物
專案英文名稱	Using DNA Stable-Isotope Probing and Next Generation Sequencing to determine the Active Chloro-organics Degrading Microbial Structure in the Chlorinated Contaminated Soils
英文關鍵字	Precision Bioremediation, DNA Stable-Isotope Probing, Next Generation Sequencing, Microbial Remidiation, Chloro-Organics
執行金額	980,000元
執行期間	2021/1/1 至 2021/12/31
計畫中文摘要	含氯的有機物通常都具有致癌性，對於人體、土壤與地下水等生態，皆有潛在的接觸及污染風險。截至109年8月資料顯示，國內土壤及地下水受含氯有機污染物公告之列管場址為16處，共有12處整治場址、4處控制場址，顯示目前在土壤及地下水含氯有機污染場址之管控與整治是十分重要的。現今土壤污染整治主要多以化學整治法為主，然而近年來國內政府已開始重視，國際間所提出的精準生物整治 (precision bioremediation)，應用於土壤及地下水污染。DNA-穩定同位素探針(DNA Stable Isotope Probing; DNA-SIP)技術為一種環境微生物學者用來判斷環境特定活性微生物之新穎技術。其透過微生物代謝同位素有機物過程將同位素標記於生物體DNA中，以在複雜的環境中迅速分離具有特定功能性的活性微生物族群。DNA-SIP 的技術應用於難分解有機物(含氯有機物、多環芳香族碳氫化合物)的生物整治上，在近兩年出現了許多的研究案例。以DNA-SIP技術結合現地(in situ)的生物整治技術，將能快速提供活性功能性微生物的族群結構資訊與污染物代謝的直接證據。 有鑒於此，本計畫期望能將環境微生物領域中所使用的 DNA-SIP 技術引入土壤污染整治，藉由透過DNA-SIP搭配次世代定序的技術整合，期望能更快速的探究土壤中難分解有機物污染物降解之相關功能微生物類群。在本階段的計畫，以桃園地區某含氯有機污染土壤為模擬的對象，評估結合 DNA-SIP 於其場址主要之污染物－1,2-二氯苯的精準生物整治可行性。受限於經費所致，本階段研究乃針對 DNA-SIP 時驗所需添加之同位素碳源進行前期培養實驗。並以透過實驗室模擬厭氧情境，評估降解二氯苯之可能微生物所需要之碳源種類以及是否需要其他營養鹽。研究使用六種不同的營養鹽集碳源的配置，包含僅添加二氯苯、添加二氯苯與二氧化碳、添加二氯苯與甲烷、前述三種組合分別加上氮磷營養鹽、以及對照組一組。另外好氧培養實驗則以添加二氯苯及氮磷營養鹽一組進行實驗。 研究結果顯示，在厭氧環境條件下，環境中可能存在許多具有降解二氯苯活性但目前尚未被培養成功之厭氧微生物占了約80%的相對豐富度。而在已知具降解活性之厭氧微生物則主要以脫氮菌族群，如：Pseudomonas、Nocardioides以及Anaeromyxobacter 等為主，此外另有部分產甲烷菌可能具有降解二氯苯之潛力。對於脫氮菌族群可能利用二氯苯作為其生長所需碳源，因此未來若要透過 DNASIP 技術標記降解二氯苯之活性微生物，則須優先考慮同位素13C之二氯苯進行標記，另外亦可考慮使用同位素二氧化碳，然而其成功率可能較低。
計畫英文摘要	Chloro-organics are usually carcinogenic and have high toxic risks to human body, soil, groundwater, and ecology system. As of August 2020, the Taiwanese government has subjected 16 places as chloro-organics contaminated sites, among which 12 sites are announced as remediation sites and four sites as control sites. Thus, the management and remediation of contaminated sites are critical. The concept of precision bioremediation of soil and groundwater pollution have been widely proposed around the globe. DNA-Stable Isotope Probing (DNA-SIP) technology has been used to identify the active functional microbial communities of desired in various studies. This technique has also been introduced to identify and evaluate the microbes that can decompose recalcitrant organics such as chlorinated organics and polycyclic aromatic hydrocarbons. With using DNA-SIP and next-generation sequencing (NGS), scientists will get comprehensive and detailed information on the activities and compositions of microorganisms having abilities in degrading the chloro-organics. Thus, the ultimately goal of this study is to determine whether DNA-SIP and NGS technologies can be utilized for future biological remediation projects. This project will simulate the conditions of a chloro-organics contaminated site in Taoyuan in laboratory. The site is mainly contaminated by 1,2-dichlorobenzene (1,2-DCB) with concentration of 4,070 mg/Kg. Because of the limited funding, in this current research project, we identified the carbon sources for the microorganisms that are responsible to the 1,2-DCB decomposition through a series of laboratory incubation experiments. The results showed that more than 80% of the microbial communities may be able to decompose the 1,2-DCB while yet been classified. For the rest 20% of the microbial communities, Pseudomonas, Nocardioides and Anaeromyxobacter appeared to have high potential in decomposing the 1,2-DCB. Moreover, the carbon source that were utilized by these microbes was likely to be 1,2-DCB. In addition, some methanogens may also be able to reduce the 1,2-DCB as well while they may likely use CO2 as their C source. Thus, 13C amended 1,2-DCB and/or 13CO2 should be considered for future DNA-SIP experiments to label the active 1,2-DCB utilizing microbes in the field.