含氯有機溶劑為土壤及地下水中常見之重質非水相溶液(dense non-aqueous phase liquids, DNAPL)污染物，而三氯乙烯(trichloroethylene, TCE)則為最具代表性之含氯有機物。本研究以TCE為目標污染物，研發處理DNAPL污染在不飽和層土壤及飽和層地下水之整治技術。過去針對DNAPL之生物降解需長期注入營養基質，但基質之注入往往造成地下水阻塞及酸化問題，而地下水中常存在之硫酸鹽經還原作用生成硫化物，除了產生毒性物質，亦造成現地異味問題，綜觀所言，現地土壤地下水污染整治衍生之議題包含基質傳輸效果不佳、地下水質酸化及操作維護費用增加。因此，本研究利用植物油(蓖麻油)中碘值低特性替代食用油品(大豆油)，並於基質中搭配緩衝溶液檸檬酸和磷酸氫二鈉結合發展一套具有緩衝能力及高流動性之釋碳基質，進行微生物批次降解試驗，瞭解TCE降解效率及酸化控制能力。此外，本研究結合[polymerase chain reaction (PCR) 及 denaturing gradient gel electrophoresis (DGGE)]分子生物技術，瞭解試驗中微生物菌群變化與豐富之程度。由基質穩定性試驗結果顯示，合成之釋碳基質在72小時內穩定性良好，且經由動態光散射儀(dynamic light scattering, DLS)觀察油滴粒徑，平均粒徑為299 nm，界達電位為-13.3 mV。微生物降解試驗結果顯示，各組別之降解效率以緩衝釋碳基質組為最佳，在140天試驗中可穩定降解93%之高濃度TCE且pH值控制在7.1(中性)，具有緩衝能力之效果。藉由一階反應動力學模擬試驗後期基質之TCE殘留率，添加緩衝釋碳基質組之降解速率和其他基質組別相較下為佳，降解速率常數為1.93×10-2 day-1，由此可知，添加緩衝溶液來維持水中pH環境有助於TCE持續降解發生。藉由DGGE菌相分析結果顯示，釋碳基質組別菌群豐富度高，並以微生物定量分析結果顯示，第0天之Dehalococcoides spp. (DHC)菌數約為2.33 × 102 gene copies/g，第120天DHC菌數約為4.99 × 104 gene copies/g，此結果亦顯示添加具有緩衝釋碳基質可有效刺激脫氯菌生長。

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 this study, trichloroethylene (TCE) was used as the target compound for the feasibility and pilot-scale studies. 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. In this study, a carbon-releasing substrate [mainly vegetable oil (castor oil) (with a low iodine value)] with pH buffer capacity (with citric acid and sodium hydrogen phosphate as buffering system) was developed. The microcosm study was performed to evaluate the feasibility of using the developed substrate to enahcne the reuctive dechlorination of TCE. In addition, molecular biology techniques [polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] were used to identify the dominant microcial sepcies and variations in microbial diversity, which can contribute to the contaminant biodegradation. The results show that the developed emulsified substrate could maintain a stable condition for a 72-h period. The average diameter and zeta potential of the oil globule was 299 nm and -13.3 mv, respectively, analyzed by the dynamic light scattering (DLS). Results from the microcosm study show that approximately 93% of the TCE can be removed and the pH was maintained neutral after 140 days of operation, and the first-order decay rate for TCE was 1.93 × 10-2 1/d. Results from the real-time PCR analyses show that the populations of Dehalococcoides spp. (DHC) increased from 2.33 × 102 on day 0 to 4.99 × 104 gene copies/g on day 120. The results indicate that the developed pH buffer carbon-releasing substrate was able to enhance the growth of DHC and also caused the increase in anaerobic TCE dechlorination rate.

誌謝 i
中文摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 x
第一章 前言 1
1.1研究緣起 1
1.2研究目的 2
第二章 文獻回顧 4
2.1地下水污染 4
2.1.1 地下水污染種類 4
2.1.2含氯脂肪族碳氫化合物污染概況 5
2.1.3三氯乙烯之特性及對人體之危害 9
2.1.4三氯乙烯之傳輸機制 12
2.2 土讓及地下水整治技術 13
2.2.1物理及化學整治技術 13
2.2.2生物復育技術 14
2.2.3現地生物整治技術(In situ bioremediation) 16
2.2.4三氯乙烯厭氧還原脫氯機制 16
2.3 透水性反應牆(Permeable reactive barrier) 18
2.4三氯乙烯之生物反應機制 21
2.4.1 三氯乙烯之好氧生物處理 21
2.4.2三氯乙烯之厭氧生物處理 23
2.5不同油品及基本特性 28
2.5.1油品的介紹及特性 28
2.5.2界面活性劑的種類 31
2.5.3地下水中的pH值和緩衝藥劑的探討 33
2.6分子生物技術於污染整治上的重要性與應用 38
第三章 實驗設備與方法 40
3.1研究流程 40
3.2實驗材料與設備 42
3.2.1實驗材料 42
3.2.2實驗設備 42
3.2.3供試土壤來源 43
3.2.4供試地下水來源 43
3.3乳化型基質之製備 44
3.4釋碳基質之製備 46
3.5流通性試驗 47
3.6污染物批次實驗 49
3.7實驗分析方法 52
3.7.1成分分析 52
3.8分子生物菌相分析 54
3.8.1土壤樣品微生物DNA萃取 54
3.8.2聚合酶連鎖反應(Polymerase Chain Reaction，PCR) 54
3.8.3變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis，DGGE) 57
3.8.4 Mixed DNA cloning及定序 58
3.8.5 即時定量PCR(realtime-PCR) 59
第四章 結果與討論 62
4.1釋碳基質製備與性能之研究 62
4.1.1單一界面活性劑對於蓖麻油乳化之影響 62
4.1.2混合型界面活性劑對蓖麻油乳化之影響 65
4.1.3選擇最佳乳化合成方式 67
4.2 釋碳基質基本特性分析 69
4.3基質流通性試驗評估 71
4.4不同基質對生物可利用性評估 74
4.4.1 pH值酸化試驗 74
4.4.2 不同pH緩衝溶液控制之比較 76
4.5 微生物批次實驗 79
4.5.1 自然降解三氯乙烯 80
4.5.2 添加乳化型基質與釋碳基質降解三氯乙烯 81
4.5.3 緩衝釋碳基質降解三氯乙烯 84
4.5.5各基質水質基本參數分析 85
4.5.6各基質副產物降解效率比較 90
4.5.7 綜合討論 94
4.6三氯乙烯之降解速率常數 95
4.7 微生物菌相分析 99
4.7.1 DGGE菌相分析 99
4.7.2 q-PCR定量分析 (qPCR) 101
第五章 結論與建議 104
5.1結論 104
5.2建議 106
參考文獻 107

AFCEE. (2007) “Protocol for In Situ Bioremediation of Chlorinated Solvents Using Edible Oil”, Prepared by Solutions IES, Inc., Terra Systems, Inc., and Parsons Infrastructure & Technology Group, Inc. Final. October. (available at http://www.afcee.brooks.af.mil/products/techtrans/).
Aulenta, F., Fuoco, M., Canosa, A., Papini, M.P., Majone, M. (2008) “Use of poly-beta-hydroxy-butyrate as a slow-release electron donor for the microbial reductive dechlorination of TCE. “Water Science Technology 57, 921-925.
Azizian, M.F., Marshall, I.P.G., Behrens, S., Spormann, A.M., Semprini, L. (2010) “Comparison of lactate, formate, and propionate as hydrogen donors for the reductive dehalogenation of trichloroethene in a continuous-flow column”, Journal of Contaminant Hydrology, 113(1-4), 77-92.
Bernardino Virdis, Korneel Rabaey, René A. Rozendal, Zhiguo Yuan, Jürg Keller, “Simultaneous nitrification, denitrification and carbon removal in microbial fuel cells”, (2010), 44, 2970-2980.
Borden, R.C. (2011), “pH and Aquifer Remediation”, Ground Water Processionals of North Carolina. December 8, 2011, Raleigh, NC.
Borden, R.C., & Rodriguez, B.X. (2006) “Evaluation of slow release substrates for anaerobic bioremediation”, Bioremediation Journal, 10(1-2), 59-69.
Bruce, M. and Henry, P.G. (2010) “Biostimulation for Anaerobic Bioremediation of Chlorinated Solvents”, Environmental Remediation Technology, 357-423.
Chivers, T and Philip J.W. (2013), “Ubiquitous trisulfur radical anion: fundamentals and application s in materials science, electrochemistry, analytical chemistry and geochemistry, Chem. Soc. Rev., 42, 5996-6005.
Chomsurin, C., Kajorntraidej, J., Luangmuang, K. (2008) “Bioremediation of trichloroethylene contaminated groundwater using anaerobic process”, Water Science and Technology, 58(11), 2127-2132.
Comas, D. I., Wagner, J. R. and Tomas, M. C. (2006) “Creaming stability of oil in water (O/W) emulsions: Influence of pH on soybean protein-lecithin interaction”, Food Hydrocolloids, 20(7), 990-996.
Delgado, A.G., Parameswaran1, P., Devyn, F.W., Halden, R.U. and Rosa, K.B. (2012) “Role of bicarbonate as a pH buffer and electron sink in microbial dechlorination of chloroethenes”, Microbial Cell Factories, 11(1), 128.
Duhamel, M., & Edwards, E.A. (2006) “Microbial composition of chlorinated ethene-degrading cultures dominated by Dehalococcoides”, FEMS microbiology ecology, 58(3), 538-549.
E.P. Minet, C. O’Carroll, D. Rooney, C. Breslin, C.P. McCarthy, L. Gallagher, K.G. Richards. (2013), “Slow delivery of a nitrification inhibitor (dicyandiamide) to soil using a biodegradable hydrogel of chitosan”, 93, 2854-2858.
Eaddy A. (2008). Scale-up and characterization of an enrichment culture for bioaugmentation of the P-area chlorinated ethene plume at the Savannah River site. MS Thesis. Clemson University, Clemson, SC, USA.
Ferguson, J.F. and Pietari, J.M.H. (2000) “Anaerobic transformations and bioremediation of chlorinated solvents”,107,209-215.
Frascari, D., Zanaroli, G., Danko, A.S. (2015), “In situ aerobic cometabolism of chlorinated solvents: A review. Journal of hazardous materials”, 283, 382-399.
Ginzinger, D.G., (2002) “Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream”, Experimental hematology 30(6), 503-512.
Guerrero-Barajas, C., García-Peña, E.I. (2010) “Evaluation of enrichments of sulfate reducing bacteria from pristine hydrothermal vents sediments as potential inoculum for reducing trichloroethylene (TCE)”, World Journal of Microbiology and Biotechnology, 26(1), 21-28.
Ha, C., Kim, N., Park, H., Kwon, S.Y., Lee, H.S., Hong, U.J., Park, S., Kim, S., Kim, Y. (2010) “Natural gradient drift tests for assessing the feasibility of in situ aerobic cometabolism of trichloroethylene and evaluating the microbial community change”, Water Air Soil Poll, 17, 35-42.
Harkness, M., & Fisher, A. (2013), “Use of emulsified vegetable oil to support bioremediation of TCE DNAPL in soil columns”, Journal of contaminant hydrology, 151, 16-33.
He, Y.T., Wilson, J.T., Wilkin, R.T. (2010) “Impact of iron sulfide transformation on trichloroethylene degradation”, Geochimica et Cosmochimica Acta, 74(7), 2025-2039.
Helena Nadais, Marta Barbosa, Isabel Capela, Luis Arroja, Christian G. Ramos, Andre Grilo, Silvia A. Sousa, Jorge H. Leitao. (2011), “Enhancing wastewater degradation and biogas production by intermittent operation of UASB reactors.” Energy Volume 36, Issue 4,Pages 2164-2168.
Hiortdahl, K. M., & Borden, R. C. (2013),“Enhanced Reductive Dechlorination of Tetrachloroethene Dense Nonaqueous Phase Liquid with EVO and Mg(OH)2”, Environmental science & technology, 48(1), 624-631.
ITRC (The Interstate Technology & Regulatory Council). (2008), “In Situ Bioremediation of Chlorinated Ethene DNAPL Source Zones”.
Karasulu, H.Y. (2008). “Microemulsions as novel drug carriers: the formation, stability, applications and toxicity”, Informa Healthcare, 5(1), 119-135.
Kouznetsova, I., Mao, X., Robinson, C., Barry, D. A., Gerhard, J. I., & McCarty, P. L. (2010), “Biological reduction of chlorinated solvents: Batch-scale geochemical modeling”, Advances in Water Resources, 33(9), 969-986.
Kralova, I. and Sjoblom, J. (2009) “Surfactants Used in Food Industry: A Review”, Journal of Dispersion Science and Technology, 30, 1361-1383.
Kuo, Y.C., Wang, S.Y., Chang, Y.M., Chen, S.H. & Kao, C.M., (2014), “Control of trichloroethylene plume migration using a biobarrier system: a field-scale study”, Water Science & Technology, 69(10), 2074-2078.
Lacroix, E., Brovelli, A., Maillard, J., Rohrbach-Brandt, E., Barry, D. A., & Holliger, C. (2014),“Use of silicate minerals for long-term pH control during reductive dechlorination of high tetrachloroethene concentrations in continuous flow-through columns”, Science of the Total Environment, 482, 23-35.
Lee, Y. C., Kwon, T. S., Yang, J. S., & Yang, J. W. (2007), “Remediation of groundwater contaminated with DNAPLs by biodegradable oil emulsion”, Journal of hazardous materials, 140(1), 340-345.
Liang S. H., Kuo Y. C., Chen S. H., Chen C. Y., Kao C. M. (2013) “Development of a slow polycolloid-releasing substrate(SPRS) biobarrier to remediate TCE-contaminated aquifers”, Journal of Hazardous Materials Volume 254-255, Pages 107-155.
Liang, S.H., Kao, C.M., Kuo, Y.C., Chen, K.F. (2011) Application of persulfate-releasing barrier to remediate MTBE and benzene contaminated groundwater. J Hazard Mater 185, 1162-1168.
Löffler, F. E., Jun Yan, K. M., Ritalahti, L. A., Elizabeth, A. E., Konstantinos, T. K., Jochen, A. M., Heather Fullerton, Stephen, H. Z., & Alfred, M. S., (2012), “Dehalococcoides mccartyi gen. nov., sp. nov., obligate organohalide-respiring anaerobic bacteria, relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidetes classis nov., within the phylum Chloroflexi, “International Journal of System Evolutionary Microbiology, ijs.0.034926-0; published ahead of print April 27, 2012, doi:10.1099/ijs.0.034926-0
Long, C. M., and Borden, R. C. (2006), “Enhanced reductive dechlorination in columns treated with edible oil emulsion”, Journal of contaminant hydrology, 87(1), 54-72.
Lowe SE, Jain MK, Zeikus JG. (1993), Biology, ecology, and biotechnological applications of anaerobic-bacteria adapted to environmental stresses in temperature, pH, salinity, or substrates. Microbiol Rev 57:451-509.
McCarty PL, Chu M-Y, Kitanidis P. (2007), “Electron donor and pH relationships for biologically enhanced dissolution of chlorinated solvent DNAPL in groundwater”. Eur J Soil Biol 43:276-282.
Miyata, R. (2010) “Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp.using alternately binding probe competitive polymerase chain reaction”, Molecular and Cellular Probes, 24, 131-137.
Mudliar, S., Giri, B., Padoley, K., Satpute, D., Dixit, R., Bhatt, P., Pandey, R., Juwarkar, A. and Vaidya, A. (2010), “Bioreactors for treatment of VOCs and odours-A review”, Journal of Environmental Management, 91, 1039-1054.
Ozturk, Z., Katsenovich, Y., Tansel, B., Laha, S., Moos, L., Allen, M. (2009) “Enhancement of TCE attenuation in soils by natural amendments”, Soil and Sediment Contamination, 18(1), 1-13.
Philips, J., Muylder, R.V., Springae, D. and Smolder, E. (2012) “Electron donor limitations reduce microbial enhanced trichloroethene DNAPL dissolution: A flux-based analysis using diffusion-cells”, Chemosphere, In Press, Corrected Proof-Note to users.
Prabhakar Pant, Sudhakar Pant.(2010) “A review: Advances in microbial remediation of trichloroethylene (TCE)”, 22,116-126.
Rivett, M.O., Wealthall, G.P., Dearden, R.A., McAlary, T.A. (2011). “Review of unsaturated-zone transport and attenuation of volatile organic compound (VOC) plumes leached from shallow source zones.” Journal of contaminant hydrology, 123(3), 130-156.
Robinson, C., & Barry, D. A. (2009), “Design tool for estimation of buffer requirement for enhanced reductive dechlorination of chlorinated solvents in groundwater”, Environmental Modelling & Software, 24(11), 1332-1338.
Robinson, C., Barry, D. A., McCarty, P. L., Gerhard, J. I., & Kouznetsova, I. (2009), “pH control for enhanced reductive bioremediation of chlorinated solvent source zones”, Science of the Total Environment, 407(16), 4560-4573.
Rowlands, D. (2004), “Development of Optimal pH for Degradation of Chlorinated Solvents by the KB-1™ Anaerobic Bacterial Culture. PhD Thesis, University of Guelph”, Guelph, Ontario, Canada.
Sa´ra Re´ve´sza,Rita Siposa,Aniko´ Kendeb, Tama´s Rikkerb, Csaba Romsicsa,E´va Me´sza´rosa, Anita Mohra, Andra´s Ta´ncsicsa, Ka´roly Ma´rialigeti,(2006) ” Bacterial community changes in TCE biodegradation detected in microcosm experiments”, 58, 239-247.
Sabalowsky, A. R., & Semprini, L. (2010), “Trichloroethene and cis‐1, 2‐dichloroethene concentration‐dependent toxicity model simulates anaerobic dechlorination at high concentrations: I. batch‐fed reactors”, Biotechnology and bioengineering, 107(3), 529-539.
Scheutz, C., Durannt, N. D., Hansen, M. H. and Bjerg, P. L. (2011), “Natural and enhanced anaerobic degradation of 1,1,1-trichloroethane and its degradation products in the subsurface-A critical review”, Water Reasearch Volume, 45(9), 2701-2723.
SERDP and ESTCP (Remediation Technology Monograph Series). (2010), “In Situ Remediation of Chlorinated Solvent Plumes”.
SERDP and ESTCP (Remediation Technology Monograph Series). (2014), “Chlorinated Solvent Source Zone Remediation”
Sheu, Y. T., Chen, S. C., Chien, C. C., Chen, C. C. and Kao, C. M. (2015), “Application of a long-lasting colloidal substrate with pH and hydrogen sulfide control capabilities to remediate TCE-contaminated groundwater”, Journal of Hazardous Materials, 284, 222-232.
Silvia Comba, Davide Dalmazzo, Ezio Santagata, Rajandrea Sethi,” Rheological characterization of xanthan suspensions of nanoscale iron for injection in porous media”, (2011), 185, 598-605.
Smulke W., Zdarta A., Luczak M., Krawczyk P., Jesionowski T., Kaczorek E., (2016), “Sqpindus saponios’ impact on hydrocarbon biodegradation by bacteria strains shor- and long-term contact with pollutant”,Colloids and Surfaces B: Biointerfaces, Volume 142 , Pages 207-213.
Su, C., Puls, R.W., Krug, T.A., Watling, M.T., O'Hara, S.K., Quinn, J.W. and Ruiz, N.E. (2014) “A two and half-year-performance evaluation of a field test on treatment of source zone tetrachloroethene and its chlorinated daughter products using emulsified zero valent iron nanoparticles”, Water Research, 46(16), 5071-5084.
Sung, Y., Fletcher, K.F., Ritalaliti, K.M., Apkarian, R.P., Ramos-Hernandez, N., Sanford, R.A., Mesbah, N.M., Loffler, F.E. (2006) Geobacter lovleyi sp nov strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium, Application Environmental Microbiology 72, 2775-2782.
Tsai, T.T., Liu, J.K., Chang, Y.M., Chen, K.F., Kao, C.M. (2014) “Application of polycolloid-releasing substrate to remediate trichloroethylene-contaminated groundwater: A pilot-scale study”, Journal of Hazardous Materials, 268, 92-101.
Ushikubo,F.Y. and Cunha, R.L. (2014) “Stability mechanisms of liquid water-in-oil emulsions”, Food Hydrocolloids, 34, 145-153.
Vainberg S, Steffan RJ, Rogers R, Ladaa T, Pohlmann D, Leigh D.(2006), Production and application of large-scale cultures for bioaugmentation. Proceedings, 5th International Conference on Remediation of Chlorinated and Recalcitrant Compounds, Monterey, CA, USA. May 22–25. Paper A-50.
Wei, Z., Seo, Y. (2010) “Trichloroethylene (TCE) adsorption using sustainable organic mulch”, 181,147-153.
Wooley, J. C., Godzik, A. and Friedberg, I., (2010) “A primer on metagenomics”, PLoS computational biology, 6(2), e1000667.
Xiaolei Zhang , Song Yan , R.D. Tyagi a, R.Y. Surampall.(2011) ” Review Synthesis of nanoparticles by microorganisms and their application in enhancing microbiological reaction rates”, 82, 489-494.
Y.C. Kuo , S.F. Cheng , P.W.G. Liu , H.Y. Chiou & C.M. Kao (2012) Application of enhanced bioremediation for TCE-contaminated groundwater: a pilot-scale study, Desalination and Water Treatment, 41:1-3, 364-371, DOI: 10.1080/19443994.2012.664742
Yates, M.D., Kiely, P.D., Call, D.F., Rismani-Yazdi, H., Bibby, K., Peccia, J., Regan, J.M. and Logan,B.E. (2012) “Convergent development of anodic bacterial communities in microbial fuel cells”, The ISME Journal 6, 2002-2013.
Zhang, T., Fang, H.H.P., (2006) “Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples”, Applied Microbiology and Biotechnology 70(3), 281-289.
Zhong, L., Truex, M. J., Kananizadeh, N., Li, Y., Lea, A. S. and Yan, X., (2015) “Delivery of vegetable oil suspensions in a shear thinning fluid for enhanced bioremediation”, Journal of contaminant hydrology, 175, 17-25.
行政院環保署土壤及地下整治網，2015，國內土壤及地下水場址列管情形，(http://sgw.epa.gov.tw/public/0401.asp)。
行政院環境保護署，2004，土壤及地下水污染整治技術手冊。
行政院環境保護署，2008，土壤及地下水受比水重非水相液體污染場址整治技術選取系統設計要點與注意事項參考手冊。
行政院環境保護署，2011，土壤污染管制標準，環署土字第1000008495號令。
行政院環境保護署，2013，地下水污染管制標準，環署土字第1000010141號令。
行政院環境保護署，2014，列管毒性化學物質及其運作管理事項。
行政院環境保護署，2014，飲用水水質標準，環署毒字第 1030001229 號令。
林財富，2008，土壤與地下水污染整治：原理與應用，中華民國環境工程學會。
張永宜，(2007)，「乳化奈米級零價鐵處理水溶液中之三氯乙烯」，國立中山大學環境工程研究所碩士論文。
陳逸明，2010，以乳化型基質處理受三氯乙烯污染之地下水，國立中山大學環境工程研究所。
劉嘉庭，2011，利用乳化型釋碳基質提昇三氯乙烯污染地水之生物降解效率：現地模場試驗，國立中山大學環境工程研究所碩士論文。
簡華逸，2010，應用現地生物整治技術去除三氯乙烯污染之地下水，國立中山大學環境工程研究所博士論文。