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年度	106	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	西灣講座教授
專案中文名稱	以高分子聚合物γ-PGA處理受含氯有機物污染地下水之現地模場試驗
中文關鍵字	三氯乙烯, γ-PGA, 現地生物復育,次世代定序,綠色整治
專案英文名稱	Application of poly-γ-glutamic acid to enhance the in situ bioremediation of chlorinated-solvent contaminated groundwater: a pilot-scale study
英文關鍵字	trichloroethylene, γ-PGA, In-situ bioremediation, next generation sequencing, green remediation
執行金額	1,300,000元
執行期間	2017/1/9 至 2017/11/30
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，因此生物整治技術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，但基質之注入往往造成地下水阻塞及酸化問題。聚麩胺酸(gamma-polyglutamic 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。本計畫主要之目的為評估以γ-PGA為基質，以整治受TCE污染地下水之可行性。本計畫工作項目包括(1)γ-PGA基本特性分析，以評估γ-PGA 之物化和生物特性；(2)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；(3)管柱試驗以模擬現地應用之成效並以管柱試驗獲得現地應用之設計參數；及(4)現地模場試驗，以獲得實場應用之成果並驗證實驗室數據。本計畫完成γ-PGA基本特性分析、厭氧批次生物降解試驗、管柱試驗以及模場試驗場址。γ-PGA基本特性分析之結果顯示γ-PGA之二個主要粒徑包含437 nm 及5,423 nm，因此在奈米到微米之間。此粒徑大小可使γ-PGA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。此外，γ-PGA之介達電位為-36 mV，此負電位可使γ-PGA向下游傳輸，不會被主要代負電的土壤顆粒吸附在注入點附近。厭氧微生物批次實驗結果顯示，實驗中以γ-PGA為主要基質時，TCE具有最佳之降解效率。TCE濃度由初始濃度之4.23 mg/L在42天內可持續穩定降解至0.26 mg/L，反應至84天濃度剩餘0.01 mg/L，降解效率高達99%，已低於地下水污染管制標準0.05 mg/L。然而，實驗中以乳化油為主要基質時，pH在第28天後迅速下降至5.95，pH值並在84天後降為5.66，呈現酸化之情形。而γ-PGA組之pH值在第84天仍在7.32，維持良好之中性環境。γ-PGA之總有機碳(total organic carbon, TOC)初始濃度為1,977 mg/L，在第84天後降為量646 mg/L，生物利用率為67.3%。乳化油組之TOC初始濃度為841 mg/L，在第84天後為492 mg/L 生物利用率為41%。此外，由變性梯度膠體電泳(denaturing gradient gel electrophoresis, DGGE)菌相分析結果可知，γ-PGA可使環境中之菌相群集豐富度增加，且各組別以即時定量聚合酶連鎖反應(real-time polymerase chain reaction, real-time PCR)分析初始脫氯菌(Dehalococcoides spp., DHC)菌量約為104 gene copies/g soil，γ-PGA組於84天增加到1.49×106 gene copies/g，生長效果較乳化油於84天之9.4×104 gene copies/g soil為佳。由管柱試驗結果得知，添加γ-PGA於管柱中做為碳源時，反應期間並未監測到出流水質酸化問題，pH值可維持在中性(6.5-7.5之間)，而氧化還原電位(oxidation-reduction potential, ORP)亦因γ-PGA之注入，使環境由氧化態轉變為還原態(ORP為-142 mV)，顯示γ-PGA可有效做為微生物碳源。管柱試驗結果顯示管柱出流水TCE濃度於操作40天後，可由初始之1.51 mg/L降為0.05 mg/L以下，出流端3初期DHC菌量在約103 gene copies/g，於120天增加到1.02×106 gene copies/g，由批次及管柱實驗結果可知，添加γ-PGA基質可促進現地脫氯菌生長，並能加強現地厭氧還原脫氯效果。現地模場評估結果顯示，本場址地下水TCE濃度介於0.04至0.15 mg/L之間，水力傳導係數為7.269×10-4 cm/s，地下水流速為41.2 cm/day，地下水流向由西南向東北流動。W1上游井做為γ-PGA灌注井，W3、W4、W5三口下游井做為監測井，現地試驗於注藥後W1井TCE濃度11周內為0.089 mg/L改善0.007 mg/L。監測井結果顯示，W3井TCE濃度5周內由0.149 mg/L改善至0.035 mg/L。另，初期各井脫氯菌量在約103 gene copies/L，經灌注基質後各井位均有變化趨勢，後期第75天脫氯菌量維持在1.1×105~4.6×107 gene copies/L；W2井無灌注基質，脫氯菌量介於1.67×103~4.52×103 gene copies/L。實驗結果證實上游注藥井降解效果顯著，亦形成透水性生物整治牆，符合現地、被動及生物整治概念之綠色整治技術。由基質成本計算，相較其他須添加緩衝溶液基質，以每公斤NT 30至50元γ-PGA釋碳基質較為優勢。本計畫執行成果顯示：(1)以γ-PGA為基質，可加速TCE之還原脫氯效率，並縮短整治期程；(2) γ-PGA基質黏度低，可提升藥劑傳輸之能力；(3) γ-PGA具有緩衝pH之效果，解決以往生物基質會造成水質酸化之問題，維持良好之中性環境；(4) γ-PGA可有效促進厭氧分解反應，維持厭氧環境和菌種豐富度；(5)γ-PGA釋碳基質可避免其他釋碳基質造成地下水體pH值酸化問題，亦較其他基質具有較佳之傳輸性及可維持高濃度之生物可利用碳性質。實場數據顯示γ-PGA可有效促進現場TCE之生物降解，使γ-PGA之基質系統成為一種更具經濟效益及環境友善之綠色整治工法。
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 DNAPL (dense-non-aqueous phase liquid) 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 objective of this proposed study is to evaluate the feasibility of applying the poly-(γ-glutamic acid) (γ-PGA) as the primary substrate to enhance the TCE reductive dechlorination. The 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 one-year project includes four major tasks: (1) analysis of basic physical, chemical, and biological characteristics of γ-PGA, (2) performance of anaerobic microcosm experiments, (3) conduction of anaerobic column experiments, and (4) in situ pilot-scale study. This project results from the particle size distribution of γ-PGA show that two major diameters of γ-PGA were observed, which were around 437 nm and 5,423 nm. Thus, the diameter of γ-PGA globule was in nano to micro-scale ranges. These two different size ranges of γ-PGA globule allowed it to distribute evenly in soil particles. The average zeta potentials of γ-PGA was -36 mV. This result implies that γ-PGA globule was in a reduced state, and the reduced zeta potential would result in the increase in the repulsion and friction force between γ-PGA globule and soil particles. This would promote γ-PGA to migrate to farther downgradient area, and thus, the influence zone can be enlarged. The microcosm study was conducted to evaluate the feasibility and effectiveness of using γ-PGA for the enhancement of TCE dechlorination. Results show that the addition of γ-PGA created anaerobic conditions and also caused a rapid increase in the total organic carbon concentration (about 1,977 mg/L). This led to a more thorough biodegradation of TCE through reductive dechlorinating mechanisms. Up to 99% of TCE (with initial concentration of 4.23 mg/L) was degraded after 84 days of operation. Acidification was prevented due to the release of amine functional groups could react with water and form ammonia, which resulted in alkaline conditions due to its basic characteristic. Thus, pH was maintained in neutral conditions, which benefited the growth of Dehalococcoides spp. (DHC). Increase in DHC population indicates that the supplement of γ-PGA enhanced the growth of DHC (increased from 1×104 to 1.49×106 gene copies/g soil) after 84 days of operation, and thus, the TCE dechlorination rate was enhanced. Microbial analyses reveal that dechlorinating bacteria existed in soils, which contributed to TCE dechlorination. The increased ethene concentrations indicate the complete TCE dechlorination with γ-PGA addition. The column experiments was performed to evaluate the effectiveness of using the γ-PGA as the primary substrates for TCE control and biodegradation. Results from the column study show that the TCE concentration in the column effluent dropped from the initial concentration of 1.51 mg/L to below 0.05 mg/L after 40 days of operation. TCE biodegradation byproducts (dichloroethylene and vinyl chloride) were also observed. The results indicate that the addition of γ-PGA could effectively enhance the TCE dechlorination. The produced byproducts were further degraded and accumulation of byproducts was not observed. In this study, Increase in DHC population indicates that the supplement of effluent 3 enhanced the growth of DHC (increased from 1×103 to 1.02×106 gene copies/g ) after 120 days of operation, and thus, a DNAPL-contaminated site (Yun-Kun 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 0.04 to 0.15 mg/L. The site hydraulic conductivity and groundwater flow velocity are 7.269×10-4 cm/s and 62.8 cm/d, respectively. The groundwater flows to the northeastern direction. One upgradient well and three downgradient wells 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. Results from the contaminated site show that the TCE concentration in the degradation dropped from the initial concentration of 0.089 mg/L to below 0.007 mg/L after 11 weeks of operation in W1 wells Perfusion matrix, Results from the Monitoring wellsstudy show that the TCE concentration in the degradation dropped from the initial concentration of 0.149 mg/L to below 0.035 mg/L after 5 weeks of operation in W3 wells. In addition, the initial amount of each well is about 103 gene copies/L, Increase in DHC population indicates that the supplement of γ-PGA enhanced the growth of DHC (increased from 1.1×105 to 4.6×107 gene copies/L )after 75 days of operation, and thus, W2 wells without perfusion matrix, DHC between 1.67×103 to 4.52×103 gene copies/L . The results show that the degradation effect of upstream injection is significant and the formation of permeable biological treatment wall is realized. The technology of conforming to the concept of local, passive and biological remediation includes the local biological rehabilitation combined with the permeable reaction wall. Calculated from the matrix cost, compared to other need to add buffer solution matrix, γ-PGA to NT 30 to 50 yuan per kilogram carbon matrix is more advantage. The major conclusions include the following: (1) γ-PGA can be applied as a primary substrate and create a reduced condition, which enhances the in situ reductive dechlorination of TCE, (2) γ-PGA has a low globule diameter, which allows it to migrate to the farther downgradient without causing a clogging problem, (3) γ-PGA has a pH buffering capacity, which prevents the groundwater acidification problem after supplement, (4) γ-PGA could be used as the carbon source and improve the microbial diversity, and (5) γ-PGA carbon-depleting matrix can avoid the problem of acidification of groundwater by other carbon-releasing matrix, and it has better transportability and high bio-usable carbon properties than other nature. Results of this study will aid in designing an in situ bioremediation system containing γ-PGA substrate for remedial application. The proposed treatment scheme would be expected to provide a more cost-effective alternative to remediate chlorinated-solvent contaminated aquifers.