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年度	111	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	國立中山大學	申請系所	環境工程研究所
專案主持人	張耿崚	職等/職稱	副教授
專案中文名稱	以次世代定序技術分析結合生物碳與微生物降解三氯乙烯之菌群結構與功能
中文關鍵字	生物碳；循環經濟；次世代定序；三氯乙烯；土壤污染；生物整治
專案英文名稱	Analysis of the bacterial community structure and function for combining biochar and microbial degradation of trichloroethylene by next-generation sequencing technology.
英文關鍵字	Biochar, Circular Economy, Next-Generation Sequencing, Trichloroethylene, Soil contamination, Bioremediation
執行金額	840,000元
執行期間	2022/7/1 至 2023/6/30
計畫中文摘要	三氯乙烯(Trichloroethylene, TCE)是一種廣泛存在於土壤和地下水的污染物，尤其是在嚴重污染的工業場址中。在受污染的土壤及地下水中，三氯乙烯可以在還原脫氯或有氧共代謝後進行生物降解，生物法具有效率高、成本低、完全降解和無二次污染等優點，成為研究人員積極研究的TCE整治技術之一。將農業廢棄物回收再利用製備為生物碳是一種基於循環經濟的永續再利用方法，生物碳具有獨特的性能，如比表面積較大、pH緩衝能力佳和離子交換能力高，也能改善土壤微生物的生存環境，提高土壤微生物代謝活動的強度、增加菌群豐富度及多樣性與參與微生物的胞外電子傳遞過程。使其成為一種經濟高效且環保的吸附劑及生物載體。因此，本計畫通過 Illumina MiSeq平台使用次世代測序技術，在添加兩種生物碳後，對土壤中的微生物菌群落進行採樣，分析微生物降解TCE過程中對微生物的多樣性以及結構、功能的影響，並採集現地污染土壤樣品，探討TCE之處理效率。研究結果顯示，薄膜界面探測器(MIP)搭配VOC氣體偵測器可得知污染深度大多在深層污染區約6.5 m~10.5 m之間，本研究設井深度為9 m之坋土質砂層。並分別添加2%的竹子生物碳及椰子殼生物碳作為對照組。FTIR結果顯示生物碳含有大量羥基、羧基、羰基等含氧官能團，可提升土壤的陽離子交換能力(CEC)，也可能間接影響微生物電子傳遞的效率。ESEM與BET結果顯示椰子殼生物碳及竹子生物碳孔隙較為規則與均勻分布，且椰子殼生物碳比表面積較高，故本研究採用椰子殼生物碳及竹子生物碳兩種生物碳作為添加材料。本研究在添加椰子殼生物碳之井口土壤中，含氯污染物之轉化趨勢較為完整。透過16S分析結果得知，微生物群落結構之十大優勢菌屬，分別為 Cupriavidus, Sphingomonas, Pseudocyanosarcina, Microbacterium, Pseudarthrobacter, Bradyrhizobium, Afipia, Stutzerimonas, Acinetobacter and Massilia ，且Cupriavidus與 Sphingomonas占有比例非常高。本研究也顯示在菌群已穩定的土壤中添加低比例的生物碳不一定會明顯影響土壤中的細菌多樣性。在本研究的場址中，pH值對菌群的影響能力高於含水率與電導度，其中也顯示pH值和含水率呈現正相關，pH值、電導度和含水率呈現負相關。
計畫英文摘要	Trichloroethylene (TCE) is a pollutant widely present in soil and groundwater, especially in heavily polluted industrial sites. Trichloroethylene can be biodegraded in contaminated soil and groundwater after reductive dechlorination or aerobic co-metabolism. The biological method has the advantages of high efficiency, low cost, complete degradation, and no secondary pollution, and it has become an active research field for researchers. Recycling agricultural waste into biochar is a sustainable reuse method based on a circular economy. Biochar has unique properties such as a large specific surface area, good pH buffering capacity, and high ion exchange capacity, which can also improve the living environment of microorganisms, the intensity of soil microbial metabolic activities, increase the richness and diversity of bacterial flora, and participate in the extracellular electron transfer process of microorganisms. This makes it a cost-effective and environmentally friendly adsorbent and biological carrier. Therefore, this project uses the next-generation sequencing technology through the Illumina MiSeq platform to sample the microbial community in the soil after adding two kinds of biochar and analyze the impact of changes in various operating parameters on the microorganisms in the process of microbial degradation of TCE. To collect on-site contaminated soil samples and discuss the treatment efficiency of the TCE. Preliminary experimental results show that the membrane interface probe (MIP) combined with the VOC gas detector can know that the pollution depth is mostly between 6.5 m and 10.5 m in the deep pollution area. In this study, the depth of the well is 9 m in the earthy sand layer. And add 2% bamboo biochar and coconut shell biochar, respectively, as the control group. FTIR results show that biochar contains many oxygen-containing functional groups such as hydroxyl, carboxyl, and carbonyl, which can improve the cation exchange capacity (CEC) of soil and may also indirectly affect the efficiency of microbial electron transfer. The results of ESEM and BET showed that the pores of coconut shell biochar and bamboo biochar were relatively regular and evenly distributed. The specific surface area of coconut shell biochar was relatively high. Therefore, coconut shell biochar and bamboo biochar were used as additive materials in this study. In this study, the transformation trend of chlorine pollutants in the wellhead soil added with coconut shell biochar is relatively complete. The results of 16S analysis showed that the top 10 dominant genera in the microbial community structure were Cupriavidus, Sphingomonas, Pseudocyanosarcina, Microbacterium, Pseudarthrobacter, Bradyrhizobium, Afipia, Stutzerimonas, Acinetobacter and Massilia. Moreover, Cupriavidus and Sphingomonas account for a very high proportion. This study also showed that adding a low proportion of biochar to the soil with stabilized bacterial flora does not necessarily significantly affect the bacterial diversity in the soil. In the site of this study, the influence of pH value on the flora is higher than that of water content and electrical conductivity, which also shows that pH value and water content are positively correlated, and pH value, electrical conductivity, and water content are negatively correlated.