5-1 結論
本研究以 HYL-QT1(Ralstonia sp. P-10) 作為實驗菌種應用批次培養法 進行微生態系試驗，主要目的為(1) 探討 HYL-QT1(Ralstonia sp. P-10) 以 甲苯為基質好氧共代謝三氯乙烯的功能性，(2) 求取 HYL-QT1(Ralstonia sp.
P-10) 微生態系中好氧共代謝三氯乙烯的生物降解效率及一階反應速率常 數，(3) 執行甲苯基質多次注入試驗，探討甲苯存在生物處理系統中的有效 反應時間對三氯乙烯生物降解效率的影響。综合本研究所得之結論如下：
(1) 批次試驗結果證實中，HYL-QT1(Ralstonia sp. P-10) 能將微生態系中甲 苯濃度由 7.7 ± 0.2 mg/L 降解至儀器偵測極限 (1.1 µg/L)以下，移除率 為 100%。同時，由甲苯添加試驗組及未添加甲苯試驗組間 13% 三氯 乙烯移除率的差異證實 HYL-QT1(Ralstonia sp. P-10) 以甲苯為主要基 質好氧共代謝三氯乙烯的功能性。
(2) 在單一培養期程的反應時間內 (4 天)，甲苯被 HYL-QT1(Ralstonia sp.
在時，三氯乙烯的移除非常顯著；當甲苯完全耗盡時，只要殘餘的氧 化酵素尚未完全失去活性，三氯乙烯生物共代謝現象仍會持續發生，
只是降解速率減緩許多。
(3) 根據批次培養試驗的結果，擬合一階反應動力參數方程式，在基質甲苯 分三針及九針注入時，三氯乙烯的一階反應動力參數分別為
0.22/day、0.14/day 及 0.12/day。在本研究中，甲苯存在的有效反應期 間，三氯乙烯的一階反應速率參數範圍為 0.16 ± 0.05/day。
(4) 根據甲苯基質多次注入試驗結果顯示，當甲苯存在微生態系中的有效反 應時間由數小時增加至 3 天，三氯乙烯的生物降解效率由 21% 提升 到 33%，溶氧消耗及 OD605 明顯提升及菌落數也由 106 增加到 108 CFU/mL。因此，藉由延長基質甲苯存在生物處理系統中的有效反應時 間，可使 HYL-QT1(Ralstonia sp. P-10) 代謝活性提升，進而有效增加 三氯乙烯生物共代謝效率。
5-2 建議
本研究探討單一菌種 HYL-QT1(Ralstonia sp. P-10) 以甲苯為基質好氧 共代謝三氯乙烯的可行性，並深入研究甲苯存在時間的長短對於三氯乙烯 移除率的影響。以下針對本研究結果提出幾點建議，作為後續研究之參考。
(1) 本研究以 HYL-QT1(Ralstonia sp. P-10) 進行批次培養試驗時，因為考 慮到現地的環境條件，對於會影響生物降解的因子(如酸鹼值、溫度及 導電度等)僅僅只有監測其變化。建議未來可以增加環境因子變化對於 Ralstonia sp. P-10 好氧共代謝三氯乙烯的影響，配合實驗設計找出最
佳操作條件。
(2) 本研究已證實 HYL-QT1(Ralstonia sp. P-10) 可以甲苯為主要基質好氧 共代謝三氯乙烯，但對於分解甲苯時所產生的甲苯氧化酵素類型尚未 明確。建議未來可以更深入研究甲苯氧化酵素類型。
(3) 由甲苯基質多次注入試驗結果證實，基質存在於生物反應系統中的時 間越長，三氯乙烯的移除效率越高。而基質不存在於反應系統後，三 氯乙烯的生物降解效率幾乎沒有發生。建議由 Ralstonia sp. P-10 在試
率常數，應用到現地受三氯乙烯污染場址，提供給國內土壤及地下水 污染整治技術作為參考。
參考文獻
土壤中酸鹼值測定方法。行政院環境保護署。NIEA S410.60C, 1995。
土壤及地下水污染整治網。行政院環境保護署。http://sgw.epa.gov.tw/public/
水中揮發性有機化合物檢測方法-吹氣捕捉/氣相層析質譜儀。行政院環境保護 署。NIEA W785.53B, 2003。
水中總菌落數檢測方法-塗抹法。行政院環境保護署。NIEA E203.54B, 2005。
王銀波、吳繼光、黃衍龍、趙雲慶，「汙染土壤中囊叢枝菌根菌族群數及重金
環境微生物檢測通則 I-細菌。行政院環境保護署。NIEA E101.01C, 1999。
劉文得，「抑制性基質之生物降解動力學：鹽度影響與參數可檢定性」，博士論
鄭顯榮、吳文娟，「土壤污染防治基本政策與推動方向」，第二屆土壤污染防治 研討會論文集，21-36，1990。
蔡文田、邱伸彥，「蒸氣脫脂用含氯溶劑之特性管制和污染預防」，工業污染防
治，第 41 期，145-160，1992。
Alexander M. “Biodegradation and Bioremediation＂. Second edition, 1994.
Ball BR, Edwards MD. Air stripping VOCs from groundwater: process design considerations. Environ. Prog. 11(1):39-48, 1992.
Bouwer EJ, Rittmann BE, McCarty PL. Anaerobic degradation of halogenated 1- and 2-carbon organic compounds. Environ. Sci. Technol. 15(5): 596-599, 1981.
Criddle CS. The kinetics of cometabolism. Biotechnol. Bioeng. 41: 1048-1056, 1993.
Dabrock B, Riedel J, Bertram J, Gottschalk G. Isopropyl-benzen(cumene) - a new substrate for the isolation of trichloroethylene - degrading bacteria. Arch.
Microbiol. 158: 9-13, 1992.
Dorigo U, Volatier L, Humbert JF. Molecular approaches to the assessment of biodiversity in aquatic microbial communities. Wat. Res. 39(11): 2207-2218, 2005.
Eguchi M, Kitagawa M, Suzuki Y, Nakamuara M, Kawai T, Okamura K, Sasaki S, Miyake Y. A field evaluation of in situ biodegradation of trichloroethylene through methane injection. Wat. Res. 35(9): 2145-2152, 2001.
Fan S, Scow KM. Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil. Appl. Environ. Microbiol. 59(6):1911-1918, 1993.
Fogel MM, Taddeo AR, Fogel S. Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture. Appl. Environ. Microbiol. 51(4):720-724, 1986.
Folsom BR, Chapman PJ, Pritchard PH. Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrate.
Appl. Environ. Microbiol. 56(5): 1279-1285, 1990.
Fuller ME, Scow KM, Lau S, Ferris H. Trichloroethylene and toluene effects on the structure and function of the soil community. Soil. Biol. Biochem.
29(1):75-89, 1997.
Futamata H, Harayama S, Watanabe K. Diversity in kinetics of
trichloroethylene-degrading activities exhibited by phenol-degrading bacteria.
Appl. Microbiol. Biotech. 55(2): 248-253, 2001.
Gavaskar AR. Design and construction techniques for permeable reactive barriers.
J. Hazard. Mater. 68(1): 41-71, 1999.
Han YL, Kuo MC Tom , Tseng IC, Lu CJ. Semicontinuous microcosm study of aerobic cometabolism of trichloroethylene using toluene. Accepted by J.
Hazard. Mater., 2007.
Heald S, Jenkins RO. Trichloroethylene removal and oxidation toxicity mediated by toluene dioxygenase of Pseudomonas putida. Appl. Environ. Microbiol.
1993a.
Hopkins GD, Semprini L, McCarty PL. Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing
microorganisms. Appl. Environ. Microbiol. 59(7): 2277-2285, 1993b.
Hopkins GD, McCarty PL. Filed evaluation of in situ aerobic cometabolism of trichloroethylene and three dichloroethylene isomers using phenol and toluene as the primary substrates. Environ. Sci. Technol. 29(6): 1628-1637, 1995.
Hubert C, Shen Y, Voordouw G. Changes in soil microbial community composition induced by cometabolism of toluene and trichloroethylene.
Biodegradation 16: 11-22, 2005.
Ishida H, Nakamura K. Trichloroethylene degradation by Ralstonia sp. KN1-10A constitutively expressing phenol hydroxylase: transformation products, NADH limitation, and product toxicity. J. Biosci. Bioengine. 89(5): 438-445，2000.
Jenal-Wanner U, MaCarty PL. Development and evaluation of semicontinuous slurry microcosms to simulate in situ biodegradation of trichloroethylene in contaminated aquifers. Environ. Sci. Technol. 31(10): 2915-2922, 1997.
Kelly CJ, Bienkowski PR, Saylar GS. Kinetic analysis of a tod-lux bacterial reporter of toluene degradation and trichloroethylene cometabolism.
Biotechnol. Bioeng. 69 (3): 256-265, 2000.
Kleopfer RD, Easley DM, Haas BB, Jr., Deihl TG, Jackson DE, Wurrey CJ.
Anaerobic degradation of trichloroethylene in soil. Environ. Sci. Technol.
Leahy JG, Byrne AM, Olsen RH. Comparison of factors influencing
trichloroethylene degradation by toluene-oxidizing bacteria. Appl. Environ.
Microbiol. 62(3): 825-833, 1996.
Lee CY, Liu WD. The effect of salinity conditions on kinetics of trichloroethylene biodegradation by toluene-oxidizing cultures. J. Hazard. Mater. B137,
541-549, 2006.
Little CD, Palumbo AV, Herbes SE, Lidstrom ME, Tyndall RL, Gilmer PJ.
Trichloroethylene biodegradation by a methane-oxidizing bacterium. Appl.
Environ. Microbiol. 54(4): 951-956, 1988.
Lu CJ, Lee CM, Chung MS. The comparison of trichloroethylene removal rates by methane- and aromatic-utilizing microorganisms. Wat. Sci. Technol. 38(7):
19-24, 1998.
Lu CJ, Lee MS. The effect of toluene on the cometabolic thrichloroethylene removal from soil. Journal of the Chinese Institute of Environmental Engineering 13(1): 7-15, 2003.
Maier RM, Pepper IL, Gerba CP. Environmental Microbiology. Academic Press, San Diego, CA, USA, 2000.
Mars AE, Prins GT, Wietzes P, Koning WD, Janssen DB. Effect of
trichloroethylene on the competitive behavior of toluene-degrading bacteria.
Appl. Environ. Microbiol. 64(1): 208-215, 1998.
McCarty PL, Goltz MN, Hopkins GD, Dolan ME, Allan JP, Kawakami BT, Carrothers TJ. Full-scale evalution of in situ cometabolic degradation of
Mu DY. and Scow KM. Effect of trichloroethylene and toluene concentrations on TCE and toluene biodegradation and the population density of TCE and
toluene degraders in soil. Appl. Environ. Microbiol. 60(7): 2661-2665, 1994.
Nakamura K, Ishida H, Iizumi T. Constitutive trichloroethylene degradation led by tac promoter chromosomally integrated upstream of phenol hydroxylase genes of Ralstonia sp. KN1 and its nucleotide sequence analysis. J. Biosci.
Bioeng. 89(1): 47-54, 2000.
Nelson MJK, Montgomery SO, O’Neill EJ, Pritchard PH. Aerobic metabolism of trichloroethylene by a bacterial isolate. Appl. Environ. Microbiol. 52(2):
383-384, 1986.
Nelson MJK, Montgomery SO, Mahaffey WR, Pritchard PH. Biodegradation of trichloroethylene and involvement of an aromatic biodegradative pathway.
Appl. Environ. Microbiol. 53(5): 949-954, 1987.
Nelson MJK, Montgomery SO, Pritchard PH. Trichloroethylene metabolism by microorganisms that degrade aromatic compounds. Appl. Environ. Microbiol.
54(2): 604-606, 1988.
Oldenhuis R, Vink RLJM, Janssen DB, Witholt B. Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. Appl. Environ. Microbiol. 55(11):
2819-2826, 1989.
Rabideau AJ, Blayden JM, Ganguly C. Field performance of air-sparging system
biodegration of chlorinated ethenes: Part 1, methodology and field site characterization. Ground Water 28(4): 591-604, 1990.
Schaumburg FD. Banning trichloroethylene: responsible reaction or overkill?
Environ. Sci. Technol. 24(1): 17-22, 1990.
Schroth MH, Oostrom M, Wietsma TW, Istok JD. In-situ oxidation of trichloroethene by permanganate: effects on porous medium hydraulic properties. J. Contam. Hydrol. 50(1-2): 79-98, 2001.
Semprini L, Roberts PV, Hopkins GD, McCarty PL. A field evaluation of in-situ biodegration of chlorinated ethenes: Part 2, results of biostimulation and biotransformation experiments. G. Wat. 28(5): 715-727, 1990.
Semprini L, Hopkins GD, Roberts PV, Grbic-Galic D, McCarty PL. A field evaluation of in-situ biodegration of chlorinated ethenes: Part 3, studies of competitive inhibition. G. Wat. 29(2): 239-250, 1991.
Shields MS, Montgomery SO, Cuskey TM, Chapman PJ, Pritchard PH. Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene. Appl. Environ. Microbiol. 57(7): 1935-1941, 1991.
Speitel GE, McLay DS. Biofilm reactors for treatment of gas streams containing chlorinated solvents. J. Envir. Engrg. 119(4): 658-678, 1993.
Vannelli T, Logan M, Arciero DM, Hooper SR. Degradation of halogenated aliphatic compounds by the ammonia-oxidizing bacterium Nitrosomonas europaea. Appl. Environ. Microbiol. 56(4): 1169-1171, 1990.
Vogel TM, McCarty PL. Biotransformation of tetrachloroethylene to
54(7): 1703-1708, 1988.
Wackett LP, Householder SR. Toxicity of thrichloroethylene to Pseudomonas putida F1 is mediated by toluene dioxygenase. Appl. Environ. Microbiol.
55(10): 2723-2725, 1989.
Wilson JT, Wilson BH. Biotransformation of trichloroethylene in soil. Appl.
Environ. Microbiol. 49(1): 242-243, 1985.
Yang Y, McCarty PL. Biologically enhanced dissolution of tetrachloroethene DNAPL. Environ. Sci. Technol. 34(14): 2979-2984, 2000.
Zylstra GJ, Wackett LP, Gibson DT. Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.
Appl. Environ. Microbiol. 55(12): 3162-3166, 1998.