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年度	108	專案性質	實驗性質
專案類別	模場試驗	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	高志明	職等/職稱	教授
專案中文名稱	以聚麩胺酸基質生物復育受含氯有機物污染地下水：基質開發及現地模場試驗
中文關鍵字	三氯乙烯、γ-PGA、現地生物復育、次世代定序
專案英文名稱	Application of poly-γ-glutamic acid to enhance the in stiu bioremediation of chlorinated-solvent contaminated groundwater: matrix developmenta and pilot-scale study
英文關鍵字	trichloroethylene; γ-PGA; In-situ bioremediation; next generation sequencing
執行金額	1,200,000元
執行期間	2017/1/10 至 2018/11/30
計畫中文摘要	本計畫以三氯乙烯(trichloroethylene, TCE)為目標污染物，研究發展處理重質非水相溶液(dense non-aqueous phase liquids, DNAPL)污染地下水之整治技術。由於DNAPL污染場址之整治是屬於長期性的工作，選擇生物整治工法是較為經濟可行的整治方式。惟DNAPL之生物降解需長期注入主要基質，但基質注入往往造成地下水阻塞及酸化問題。聚麩胺酸(poly-γ-glutamic acid, γ-PGA)是一種經由生化作用合成之高分子聚合物，由於具有保濕性、無毒性、金屬螯合性、生物可分解性及生體相容性等特性，已在各工業領域上有廣泛之運用性。因此γ-PGA具有發展做為加強含氯有機物污染地下水之厭氧生物整治基質之優勢和潛力。本計畫目的為評估以γ-PGA為基質改質性質前後，整治TCE污染地下水之可行性。本計畫為二年工作項目包括(1)以評估γ-PGA改質試驗後基本特性分析與物化和生物特性；(2)改質後基質厭氧批次生物降解試驗，以評估γ-PGA加強TCE還原脫氯之可行性；及(3)以γ-PGA改質前後進行現地模場試驗，以獲得實場應用之成果並驗證實驗室數據。第一年本計畫在改質γ-PGA基本特性分析結果顯示，各組別粒徑均為400 nm~5,500 nm之間，粒徑分布於奈米到微米之間。推估改質組別粒徑大小有效分佈到粗細顆粒間(包括黏土到砂粒)，達到均勻分佈及增加生物可及性目標。改質後組別測量介達電位均為負電位，可使基質有效向下游傳輸，不會被主要帶負電的土壤顆粒吸附在注入點附近。管柱流通性試驗結果顯示γ-PGA基質注入體積約6~7 pore volume (PV)，總有機碳(total organic carbon , TOC)濃度達到平衡趨勢。添加羧甲基纖維素（carboxymethyl cellulose, CMC）之改質組別，約11~13 PV TOC濃度達到平衡趨勢，較γ-PGA高出5 PV貫穿時間，顯示添加羧甲基纖維素之改質可提升其分佈係數(partition coefficient)，增加基質和土壤吸附性。γ-PGA與乳化油混合組別，在注入體積約13~15 PV後，TOC濃度可達到平衡，均較γ-PGA更能達到緩釋TOC效果。以乳化油進行γ-PGA改質，pH則有緩慢降低情形，顯示乳化油之分解性較佳，因其生物降解產生有機酸而使pH降低。TCE生物分解實驗結果顯示，各組別均迅速達到厭氧狀態， TCE在35天降解率均可達77%以上，其中又以乳化型γ-PGA基質組中之TCE降解產率最高(35天達85%TCE去除率)。主要原因為TCE具有與油脂較高親和性，當乳化型基質注入地下水中可有效快速攔阻TCE包附於油滴顆粒中，形成水相與油相間之相轉移作用，此時微生物可利用碳源後迅速增長，而提高污染物降解效率。現地模場評估結果顯示，本場址地下水TCE濃度介於0.439至5.89 mg/L之間，水力傳導係數為3.9×10-3 cm/s，地下水流向由東向西流動，土壤組成份以砂質壤土。107年現地模場試驗中設置1口γ-PGA灌注井(IW)、2口下游監測井(DW1及DW2) 及一口上游監測井(UW)，改質前現地試驗中於IW注入10 Lγ-PGA藥劑並稀釋36倍藥劑量的推進水。108年現地模場試驗再新設3口下游監測井(DW3、DW4及DW5)。於3月與8月灌注藥劑，其注入20 L 改質γ-PGA藥劑並稀釋27倍藥劑量的推進水。本研究現地注藥後1年，於注藥(IW)井TCE濃度為1.423 mg/L改善0.012 mg/L。監測井結果顯示，DW2井（距離注藥井2.5 m）TCE濃度由1.993降至0.014 mg/L，經灌注基質後各井位均有變化趨勢，IW井脫氯菌菌屬維持於106~107 gene copies/L，各井TCE濃度有下降趨勢，而副產物濃度亦有上升情形，地下水未有酸化現象發生。實驗結果顯示上游注藥井降解效果顯著。現地次世代定序結果顯示，本場址於注藥後皆有豐富的Proteiniclasticum 可降解三氯乙烯之菌種，亦發現可進行氮還原、硫酸還原及其他可以消耗環境中氧氣之菌種，快速營造適合脫氯菌生長及還原脫氯之環境。本研究成果顯示：(1)實驗室成果顯示以γ-PGA結合乳化油或CMC作為基質，可有效控制pH，加速TCE之還原脫氯效率縮短整治期程，並有緩釋及長效性效果。(2)以γ-PGA基質具有極佳之傳輸性，並可釋出高濃度TOC，達到還原脫氯目標；(3)改質前後 γ-PGA具有緩衝pH之效果，使pH維持中性，解決過去生物基質分解後造成水質酸化之問題；(4) γ-PGA可有效促進現場TCE之生物降解，使γ-PGA成為一種更具經濟效益及環境友善之綠色基質；(5)推估基質可應用性質，黏壤土類型可使用γ-PGA基質，砂土類、壤土類可使用乳化型γ-PGA或是CMCγ-PGA均較長效性，未來可評估土讓各項參數選擇較適合基質。
計畫英文摘要	Soil and groundwater at many existing and former industrial areas and disposal sites are contaminated by halogenated organic compounds that were released into the environment. When they are released into the subsurface, they tend to adsorb onto the soils and cause the appearance of dense-non-aqueous phase liquid (DNAPL)pool. In situ bioremediation of DNAPLs sites need to inject carbon substrates into the subsurface, which would cause the acidification and odor problems of the subsurface environment. This would deteriorate the groundwater quality and cause the increase in maintenance cost. The poly-γ-glutamic acid (γ-PGA) is a biopolymer synthesized by biochemical processes. Due to its characteristics of moisture resistance, no toxicity, and chelating ability with metals, it has been widely applied by the industry. The objective of this proposed study is to evaluate the feasibility of applying the γ-PGA before and after the modified as the primary substrate to enhance the trichloroethylene (TCE) reductive dechlorination. The two-year project includes three major tasks: (1) analysis of modified basic physical, chemical, and biological characteristics of γ-PGA, (2) performance of anaerobic microcosm experiments, and (3) in situ pilot-scale study. First year results project results from the particle size distribution of modified group show that scope major diameters of modified groupwere observed, which were around 400 nm and 5,500 nm. Thus, the diameter of modified group globule was in nano to micro-scale ranges. These two different size ranges of modified group globule allowed it to distribute evenly in soil particles. The average zeta potentials of modified group was negative potential. This result implies that modified group globule was in a reduced state, and the reduced zeta potential would result in the increase in the repulsion and friction force between modified group globule and soil particles. This would promote modified group to migrate to farther downgradient area, and thus, the influence zone can be enlarged. The column flow test results show that the γ-PGA matrix injection volume is about 6~7 pore volume (PV).In the modified carboxymethyl cellulose (CMC) group, about 11-13 PV were observed. The trend of total organic carbon (TOC) concentration reached equilibrium. Compared with γ-PGA, there was about 5 PV breakthrough time. It is shown that the addition of carboxymethyl cellulose improves the partition coefficient and increases the matrix and soil adsorption. Subsequent observation of γ-PGA and emulsified oil mixed group injection volume of about 13 ~ 15 PV compared to γ-PGA about 7 PV breakthrough time. The sustained release TOC effect is better than γ-PGA. The γ-PGA was modified with emulsified oil, and the pH was slowly lowered, indicating that the decomposition property of the emulsified oil was better. The microcosm study was conducted to evaluate the feasibility and effectiveness of using modified group for the enhancement of TCE dechlorination. Each group quickly reached the anaerobic state, and the degradation rate of TCE in 35 days was over 77%. Among them, the degradation yield of TCE in the emulsified γ-PGA matrix group was the highest (85% TCE removal rate at 35 days). The main reason is that TCE has higher affinity with grease. When emulsified matrix is injected into groundwater, it can effectively block TCE from being attached to oil droplets, forming phase transfer between water phase and oil phase. At this time, microorganisms can use carbon source. After rapid growth, the efficiency of pollutant degradation is efficiency. In this study, a DNAPL-contaminated site (TCE-contaminated site) is selected to apply γ-PGA as the primary substrate for field application. The TCE concentrations in groundwater at the selected site ranged from 0.439 to 5.890 mg/L. The site hydraulic conductivity is around 3.9×10-3 cm/s, and the groundwater flows to the west direction, The soil composition is sandy loam. 107 years one upgradient well(IW) and two downgradient wells(DW1 and DW2) are selected as the substrate injection and monitoring wells, respectively. Moreover, an upgradient well with TCE contamination is used as a control well for comparison. In-situ test, before modifying 10 L γ-PGA agent was injected into the IW and the 36-fold dose of propellant water was diluted. 108 years Add three new downgradient wells(DW3、DW4 and DW5) are selected as the substrate injection and monitoring wells, respectively. In-situ test, infused with matrix in March and August, after modifying 20 L γ-PGA agent was injected into the IW and the 27 fold dose of propellant water was diluted. Results from the contaminated site show that the TCE concentration in the degradation dropped from the initial concentration of 1.423 mg/L to below 0.012 mg/L after 1 year of operation in IW wells Perfusion matrix, results from the monitoring wellsstudy show that the TCE concentration in the degradation dropped from the initial concentration of 1.993 mg/L to below 0.014 mg/L ooperation in DW2 wells (2.5 m from the IW wells). Increase in Dehalococcoides spp.(DHC) population indicates that the supplement of γ-PGA enhanced the growth of DHC. The IW well dechlorinogen was maintained at 106~107 gene copies/L. The TCE concentration of each well decreased, and the by-products showed an upward trend. The water quality parameter pH did not acidificaton. The experimental results confirmed that the degradation of the upstream injection wells was significant, and from groundwater samples will be collected from these wells and analyzed for contaminant concentrations, geochemical indicators, DHC, and microbial diversity. The results of the next-generation sequencing show that the site is rich in Proteiniclasticum degradable trichloroethylene species after injection, and it has also been found that nitrogen reduction, sulfuric acid reduction and other strains that can consume oxygen in the environment can be quickly constructed. Suitable for the growth and dechlorination of dechlorination bacteria. The results of this study show: (1)The results of the laboratory show that γ-PGA combined with emulsified oil or CMC as matrix can effectively control pH, accelerate the reduction and dechlorination efficiency of TCE, and shorten the remediation period, the modified matrix has a slow release and long-lasting effect; (2)The γ-PGA matrix has excellent transmission and can release high concentration TOC to achieve the reductive dechlorination target; (3)Before and after the modificationγ-PGA has the effect of buffering the pH, solving the problem that the biological matrix will cause acidification of water in the past; (4) The γ-PGA can effectively promote the biodegradation of TCE In site. The proposed treatment scheme would be expected to provide a more cost effective alternative to remediate chlorinated-solvent contaminated aquifers; (5) The current estimation of matrix is applicable, the clay loam type can use γ-PGA matrix, sand soil and loam soil can be emulsified γ-PGA or CMCγ-PGA, the parameters can used for long-term effects.In the future, the soil can be evaluated to make the parameters more suitable for the matrix.