臺灣博碩士論文加值系統

三硝基甲苯（Trinitrotoluene，TNT）又名黃色炸藥，廣泛使用於工業及軍事活動上。環境中可在兵工廠或是戰爭遺址中偵測到高含量的TNT，可能是由於炸藥的使用而釋放到環境中造成汙染。使用後之硝基苯環廢棄物會藉由滲透作用汙染土壤及地下水，進而影響土壤、水中生物及人體，三硝基甲苯亦被列為可能的致癌物。三硝基甲苯因苯環立體化學結構穩定，以化學或物理手段處理其廢棄物需耗費大量成本，且效果不彰，亦可能產生硝基副產物或其他有毒物質釋放至環境中，因此價格相對低廉且不產生額外有毒代謝產物的生物處理程序往往視為處理環境污染物之最佳手段。
本研究首先以不經培養方式，利用隨機選殖（random coloning）與變性梯度電泳（Denaturing gradient gel electrophoresis，DGGE）兩種方式探討遭TNT 污染之土壤的微生物相（Microbiota）。同時進一步利用富集培養（enrichment culture）技術，由三硝基甲苯污染之土壤分離含有可利用三硝基甲苯作為唯一碳、氮源生長之本土菌株，並針對所挑選之優勢菌株進一步探討。純化兩株以三硝基甲苯作為生長唯一碳氮源之菌株，使用16S rRNA 基因序列分析比較，分別屬於假單胞菌屬之菌種Pseudomonas putida strain TP1及Pseudomonas aeruginosa strain TP6。
本研究除了探討此兩株菌株在含三硝基甲苯為唯一之碳源、氮源的限定培養基生長情形外，也利用高效率液相層析儀（ High Pressure Liquid Chromatography，HPLC ）對其生長時TNT 降解之狀況以及可能之中間代謝產物之分析。並建立甲基苯環化合物（ Benzene、Toluene、Xylene，BTX ）之HPLC分析方法，增加TNT代謝途徑之推斷。
本研究參考與TNT生物降解有關之文獻後，進一步進行靜息細胞（resting cell ）對於降解TNT能力之探討；以及設計其他限定培養基，觀測純化之優勢菌株於不同限定培養基中對於硝基苯環化合物之降解能力；更探討醱酵槽對於菌株降解能力的影響，此結果可用以未來現場整治菌株之放大培養依據。
本研究之成果將有助於釐清為生物降解三硝基甲苯所產生之中間代謝產物與其代謝機制。

The 2,4,6-trinitrotoluene (TNT） is a secondary explosive widely used both for military and civil purposes all over the world. As a result, residual TNT has been detected as an environmental pollutant both in soil and groundwater. The source of TNT contamination could be due to the release of TNT to the environment from manufacturing or demilitarization facilities but also after munitions firing/detonation or leakage from explosive remnants of war. The presence of TNT in the soil and groundwater has been associated with adverse impacts on biological activities and human communities. Therefore, control and remediation of TNT-containing products in soil and groundwater are critical issues in environmental protection.
We used the random cloning approach and DGGE (Denaturing gradient gel electrophoresis） to investigate microbiota in soil contaminated with trinitrotoluene. TNT was also used as sole sources of carbon, energy and nitrogen for the enrichment cultures in order to isolate TNT degrading bacteria. Several strains of microorganisms from TNT contaminated soil using enrichment culture techniques. Identifications of these isolates were determined by 16S rRNA gene comparison. The isolates including species belong to Pseudomonas putida and Pseudomonas aeruginosa. Two strains (Pseudomonas putida strain TP1 and Pseudomonas aeruginosa TP6） were selected for further examination. Both strains showed growth ability on medium with TNT as sole carbon, energy and nitrogen sources. Both strains also showed apparent ability for degradation of TNT as determined by HPLC. Both strains could degrade more than 95% (initial concentration of TNT was about 100 ppm） of TNT after 25 days incubation. These strains may be valuable in the development of bioremediation of TNT by indigenous microorganisms.

目 錄
書名頁 i
論文指導教授推薦函 ii
論文口試委員會審定書 iii
授權書 iv
中文摘要 viii
英文摘要 x
誌謝 xii
目錄 xiv
表目錄 xviii
圖目錄 xix
第一章、緒論 1
1.1三硝基甲苯 (2,4,6-Trinitrotoluene)與環境汙染 1
1.1.1三硝基甲苯性質簡介 1
1.1.2三硝基甲苯的應用 1
1.1.3環境汙染 1
1.2生物整治 2
1.2.1土壤堆肥和生物泥漿 2
1.2.2植物 2
1.2.3微生物 2
1.3土壤生物相 10
1.4實驗架構 11
第二章、材料與方法 12
2.1藥品與試劑 12
2.2儀器 14
2.3利用隨機選殖法觀察TNT汙染土之菌相 15
2.3.1土壤中微生物總DNA之萃取 15
2.3.2以PCR技術選殖16S rRNA基因 18
2.3.3以限制酵素（Restriction Enzyme）切位初步分類菌株 18
2.4利用DGGE觀察高TNT汙染土之菌相分布情形 20
2.4.1儀器介紹 20
2.4.2高TNT汙染土壤中染色體DNA純化 20
2.4.3引子設計 20
2.4.5瓊脂膠體電泳 22
2.4.6 DGGE製膠 23
2.4.7 DGGE電泳分析 23
2.4.8 Ethidium Bromide染色 24
2.4.9 Dolphin-DOC電泳照膠系統 24
2.4.10 切膠純化 24
2.4.11 樣品放大與序列分析（Sequence determination） 25
2.5分離與鑑定以TNT為唯一碳、氮源之本土微生物 26
2.5.1液態培養基的製備 26
2.5.2固態培養基的製備 26
2.5.3分離高汙染土中TNT利用菌 26
2.5.4 16S rRNA基因定序與序列比對TNT利用之本土微生物 27
2.6 HPLC用於TNT殘留量分析測試 28
2.6.1儀器介紹 28
2.6.2 標準品之檢量線配製 28
2.6.3 菌株以TNT作為生長唯一碳、氮源之TNT降解能力 29
2.6.4 靜息細胞降解TNT能力測試 29
2.6.5細胞萃取液之菌株降解TNT能力探討 31
2.6.6 菌株以TNT作為生長唯一氮源之TNT降解能力 32
2.7建立HPLC用於偵測BTX之分析方法 34
2.7.1 HPLC分析條件與方法 34
2.7.2 BTX檢量線配置 34
2.7.3 於培養後樣品中萃取BTX 35
2.8於批次醱酵實驗之菌株降解TNT能力探討 36
2.8.1 醱酵槽儀器簡介 36
2.8.2 批次醱酵實驗 36
第三章、結果與討論 38
3.1利用隨機選殖篩選TNT汙染土中之可能菌株 38
3.2利用DGGE觀察TNT汙染土壤之菌相分佈 38
3.3具降解TNT菌株之序列鑑定與親源樹 44
3.4菌株以TNT作為生長唯一碳、氮源生長曲線與降解TNT殘餘量測試 46
3.5靜息細胞降解TNT測試 51
3.6細胞萃取液降解TNT測試 55
3.7菌株以TNT作為生長唯一氮源生長曲線與降解TNT殘餘量測 試 57
3.8 HPLC用於偵測BTX之不同分析條件之測試結果與BTX檢量 線 60
3.7.1甲醇:水=70:30，波長254 nm之HPLC測試 60
3.7.2甲醇:水=70:30，波長210 nm之HPLC測試 63
3.7.3氰甲烷:水=65:35，波長254nm之HPLC測試 64
3.7.4 BTX檢量線 67
3.9批次醱酵實驗結果 68
第四章、結論 70
參考文獻 72
附錄 86



 
表 目 錄
表 1、有氧環境下降解TNT的各種微生物 4
表 2、無氧環境下降解TNT的各種微生物 8
表 3、1X TAE buffer 配方(per liter) 23
表 4、40 %–80 %變性梯度膠體配置比例 23
表 5、PBS buffer 配方（per liter） 30
表 6、T101 ~ T120質體DNA之定序結果 40
表 7、T236~T259質體DNA之定序結果 41
表 8、T260~T283質體DNA之定序結果 42
表 9、DGGE各band對應之菌株 43
表 10、利用富集培養篩選純化之TNT降解菌株一覽表 44
表 11、各波峰滯留時間所相對應之化學物質 47
表 12、V3region及V6 / V9 region之primer比較 86
表 13、好氧環境下各培養基及菌種比較 88



 
圖 目 錄
圖 1、三硝基甲苯結構圖 1
圖 2、菌株在好氧情況下之TNT降解途徑 33
圖 3、T101 ~ T120質體DNA於限制酶反應後之電泳圖 40
圖 4、T236~T259質體DNA於限制酶反應後之電泳圖 41
圖 5、T260~T283質體DNA於限制酶反應後之電泳圖 42
圖 6、兩種不同土壤之V3及V6/V9 region於DGGE之電泳結果 43
圖 7、菌株TP1與TP6 16S rRNA基因序列與其它相關菌株之16S rRNA基因序列比對之親緣樹狀圖 45
圖 8、標準品於培養基中之HPLC波峰圖 46
圖 9、菌株於37 °C培養條件下，利用三硝基甲苯作為唯一碳源及氮源之生長曲線與TNT殘留量圖 48
圖 10、菌株於32 °C培養條件下，利用三硝基甲苯作為唯一碳源及氮源之生長曲線與TNT殘留量圖 48
圖 11、菌株於37 °C培養條件下，培養1天後之TNT殘餘量分析圖 49
圖 12、菌株於37 °C培養條件下，培養7天後之TNT殘餘量分析圖 49
圖 13、菌株於37 °C培養條件下，培養22天後之TNT殘餘量分析圖 50
圖 14、濃縮100 mL TNT培養液後回溶至10 mL之含有100 ppm TNT 的1X PBS buffer之靜息細胞降解TNT殘餘量分析圖 52
圖 15、濃縮200 mL TNT培養液後，控制OD600≒0.30之靜息細胞降解TNT殘餘量分析圖 53
圖 16、回溶 LB培養菌液，控制OD600≒0.30之靜息細胞降解TNT殘餘量分析圖 53
圖 17、回溶 TNT medium培養菌液，控制OD600≒0.30之靜息細胞降解TNT殘餘量分析圖 54
圖 18、經法式破碎儀破碎之細胞萃取液降解TNT殘餘量分析圖 55
圖 19、經超音波破碎儀破碎之細胞萃取液降解TNT殘餘量分析 56
圖 20、菌株於32 °C培養條件下，利用三硝基甲苯作為唯一氮源之生長曲線與TNT殘留量圖 57
圖 21、菌株於32 °C的 1% glucose + TNT medium中，培養1天後之TNT殘餘量分析圖 58
圖 22、菌株於32 °C的 1% glucose + TNT medium中，培養7天後之TNT殘餘量分析圖 59
圖 23、菌株於32 °C的 1% glucose + TNT medium中，培養22天後之TNT殘餘量分析圖 59
圖 24、100 ppm 苯於之HPLC分析結果（沖提液甲醇:水=70:30，吸收波長254 nm） 61
圖 25、100 ppm 甲苯之HPLC分析結果（沖提液甲醇:水=70:30，吸收波長254 nm） 61
圖 26、 100 ppm 鄰-二甲苯之HPLC分析結果（沖提液甲醇:水=70:30，吸收波長254 nm） 62
圖 27、 100 ppm 對-二甲苯之HPLC分析結果（沖提液甲醇:水=70:30，吸收波長254 nm） 62
圖 28、 100 ppm BTX混合物之HPLC分析結果（沖提液甲醇:水=70:30，吸收波長254 nm） 63
圖 29、10 ppm 苯之HPLC分析結果（沖提液甲醇:水=70:30，吸收波長210 nm） 64
圖 30、10 ppm 苯之HPLC分析結果（沖提液氰甲烷:水=65:35，吸收波長254 nm） 64
圖 31、10 ppm 甲苯之HPLC分析結果（沖提液氰甲烷:水=65:35，吸收波長254 nm） 65
圖 32、10 ppm 鄰-二甲苯之HPLC分析結果（沖提液氰甲烷:水=65:35，吸收波長254 nm） 65
圖 33、10 ppm 對-二甲苯之HPLC分析結果（沖提液氰甲烷:水=65:35，吸收波長254 nm） 66
圖 34、 10 ppm BTX混合物之HPLC分析結果（沖提液氰甲烷:水=65:35，吸收波長254 nm） 66
圖 35、菌株TP1於醱酵槽實驗中之生長曲線及TNT降解情形 68
圖 36、菌株TP1於醱酵槽實驗中之生長曲線及TNT降解情形 69
圖 37、EPA 8330A標準品檢量線 95
圖 38、菌株利用TNT作為生長之碳、氮源，降解TNT後所產生之代謝產物化學結構圖 95
圖 39、菌株利用TNT作為生長之氮源，降解TNT後所產生之代謝產物化學結構圖 96
圖 40、苯之檢量線 96
圖 41、甲苯之檢量線 97
圖 42、鄰-二甲苯之檢量線 97
圖 43、對-二甲苯之檢量線 98

Alvarez, M. A., Kitts, C. L., Unkefer, P. J., & Botsford, J. L. (1995). Pseudomonas aeruginosa strain MA01 aerobically metabolizes the aminodinitrotoluenes produced by 2, 4, 6-trinitrotoluene nitro group reduction.Canadian journal of microbiology, 41(11), 984-991.

Ayoub, K., van Hullebusch, E. D., Cassir, M., & Bermond, A. (2010). Application of advanced oxidation processes for TNT removal: a review. Journal of hazardous materials, 178(1), 10-28.

Bae, B., Autenrieth, R.L., Bonner, J.S. (1995) Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene, p. 231–238. In R. E. Hinchee, R. E. Hoeppel, and D. B. Anderson (ed.), Bioremediation of recalcitrant organics. Battelle Press, Columbus, Ohio.

Bahrami, A., Mahjub, H., Sadeghian, M., & Golbabaei, F. (2011). Determination of Benzene, Toluene and Xylene (BTX) Concentrations in Air Using HPLC Developed Method Compared to Gas Chromatography. International Journal of Occupational Hygiene, 3(1).

Balázs, M., Rónavári, A., Németh, A., Bihari, Z., Rutkai, E., Bartos, P., ... & Szvetnik, A. (2013) Effect of DNA polymerases on PCR-DGGE patterns. International Biodeterioration and Biodegradation 84, 244-249.
Baldwin, B. R., Nakatsu, C. H., & Nies, L. (2003). Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR. Applied and Environmental Microbiology, 69(6), 3350-3358.

Barreto-Rodrigues, M., Silva, F. T., & Paiva, T. C. (2009). Optimization of Brazilian TNT industry wastewater treatment using combined zero-valent iron and fenton processes. Journal of hazardous materials, 168(2), 1065-1069.

Behrends, L. L., & Almond, R. (1999, November). Phytoremediation of explosives-contaminated ground water using constructed wetlands. In Wetlands & Remediation: An International Conference (pp. 375-381).

Blears, M. J., De Grandis, S. A., Lee, H., & Trevors, J. T. (1998). Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications. Journal of Industrial Microbiology and Biotechnology, 21(3), 99-114.

Boopathy, R., & Kulpa, C. F. (1992). Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacteriumDesulfovibrio sp.(B strain) isolated from an anaerobic digester. Current microbiology, 25(4), 235-241.

Botstein, D., White, R. L., Skolnick, M., & Davis, R. W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms.American journal of human genetics, 32(3), 314.

Brosius, J., Palmer, M. L., Kennedy, P. J., & Noller, H. F. (1978). Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.Proceedings of the National Academy of Sciences, 75(10), 4801-4805.

Bumpus, J. A., & Tatarko, M. (1994). Biodegradation of 2, 4, 6-trinitrotoluene by Phanerochaete chrysosporium: Identification of initial degradation products and the discovery of a TNT metabolite that inhibits lignin peroxidases. Current microbiology, 28(3), 185-190.

Bradley, P. M., & Chapelle, F. H. (1995). Factors affecting microbial 2, 4, 6-trinitrotoluene mineralization in contaminated soil. Environmental science & technology, 29(3), 802-806.

Chen, W. S., Juan, C. N., & Wei, K. M. (2007). Decomposition of dinitrotoluene isomers and 2, 4, 6-trinitrotoluene in spent acid from toluene nitration process by ozonation and photo-ozonation. Journal of hazardous materials, 147(1), 97-104.

Claus, H., Bausinger, T., Lehmler, I., Perret, N., Fels, G., Dehner, U., Preuß, J., König, H. (2007) Transformation of 2,4,6-trinitrotoluene (TNT) by Raoultella terrigena. Biodegradation 18(1), 27-35.
Copley, S.D. (2009) Evolution of efficient pathways for degradation of anthropogenic chemicals. Nature Chemical Biology 5 (8), 559-566.

Cruz-Uribe, O., Rorrer, G.L. (2006) Uptake and biotransformation of 2,4,6-trinitrotoluene (TNT) by microplantlet suspension culture of the marine red macroalga Portieria hornemannii. Biotechnology and Bioengineering 93(3), 401-412.

Dewhirst, F.E., Chien, C-C., Paster, B.J., Ericson, R.L., Schauer, D.B., Orcutt, R.P., Fox, J.G.. (1999) Phylogeny of a Group of Murine Defined Microbiota (Altered Schaedler Flora). Applied and Environmental Microbiology 65, 3287-3292.

Donnelly, K.C., Chen, J.C., Huebner, H.J., Brown, K.W., Autenrieth, R.L., Bonner, J.S. (1997) Utility of four strains of white-rot fungi for the detoxification of 2,4,6-trinitrotoluene in liquid culture. Environmental Toxicology and Chemistry 16(6), 1105-1110.

Duque, E., Haidour, A., Godoy, F., Ramos, J.L. (1993) Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6- trinitrotoluene. Journal of Bacteriology 175(8), 2278-2283.

Drzyzga, O., Bruns-Nagel, D., Gorontzy, T., Blotevogel, K.-H., Gemsa, D., Von Löw, E. (1998) Mass balance studies with 14C-labeled 2,4,6-trinitrotoluene (TNT) mediated by an anaerobic Desulfovibrio species and an aerobic Serratia species. Current Microbiology 37(6), 380-386.

Ederer, M.M., Lewis, T.A., Crawford, R.L. (1997) 2,4,6-Trinitrotoluene (TNT) transformation by clostridia isolated from a munition-fed bioreactor: Comparison with non-adapted bacteria. Journal of Industrial Microbiology and Biotechnology 18(2-3), 82-88.

Esteve-Núñez, A., Caballero, A., Ramos, J.L. (2001) Biological degradation of 2,4,6-trinitrotoluene. Microbiology and Molecular Biology Reviews 65(3), 335-352.

Esteve-Nuñez, A., Lucchesi, G., Philipp, B., Schink, B., Ramos, J.L. (2000) Respiration of 2,4,6-trinitrotoluene by Pseudomonas sp. strain JLR11. Journal of Bacteriology 182(5), 1352-1355.

Esteve-Núñez, A., Ramos, J.L. (1998) Metabolism of 2,4,6-trinitrotoluene by Pseudomonas sp. JLR11. Environmental Science and Technology 32 (23), 3802-3808.

Fiorella, P.D., Spain, J.C. (1997) Transformation of 2,4,6-trinitrotoluene by Pseudomonas pseudoalcaligenes JS52. Applied and Environmental Microbiology 63(5), 2007-2015.

French, C.E., Nicklin, S., Bruce, N.C. (1998) Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase. Applied and Environmental Microbiology 64(8), 2864-2868

González‐Pérez, M., Van Dillewijn, P., Wittich, R. M., Ramos, J. L. (2007). Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth. Environmental microbiology, 9(6), 1535-1540.

Gilbert, E.E. (1980). TNT. In: Kaye, S.M., Herman, H.L.(Eds.), Encyclopedia of Explosives and Related Items, US Army Armament Research and Development Command, Dover, NJ, pp. T235–T287.

Gilcrease, P. C., Murphy, V. G. (1995). Bioconversion of 2, 4-diamino-6-nitrotoluene to a novel metabolite under anoxic and aerobic conditions. Applied and environmental microbiology, 61(12), 4209-4214.

HaÏdour, A., & Ramos, J. L. (1996). Identification of products resulting from the biological reduction of 2, 4, 6-trinitrotoluene, 2, 4-dinitrotoluene, and 2, 6-dinitrotoluene by Pseudomonas sp. Environmental science & technology, 30(7), 2365-2370.

Hawari, J., Halasz, A., Beaudet, S., Paquet, L., Ampleman, G., Thiboutot, S. (1999). Biotransformation of 2, 4, 6-Trinitrotoluene with Phanerochaete chrysosporium in Agitated Cultures at pH 4.5. Applied and environmental microbiology, 65(7), 2977-2986.

Hodgson, J., Rho, D., Guiot, S. R., Ampleman, G., Thiboutot, S., & Hawari, J. (2000). Tween 80 enhanced TNT mineralization by Phanerochaete chrysosporium. Canadian journal of microbiology, 46(2), 110-118.

Hughes, J. B., Wang, C. Y., Bhadra, R., Richardson, A., Bennett, G. N., Rudolph, F. B. (1998). Reduction of 2, 4, 6‐trinitrotoluene by Clostridium acetobutylicum through hydroxylamino‐nitrotoluene intermediates.Environmental toxicology and chemistry, 17(3), 343-348.

Jones, A.M., Greer, C.W., Ampleman, G., Thiboutot, S., Lavigne, J., Hawari, J. (1995) Biodegradability of selected highly energetic pollutants under aerobic conditions, p. 251–257. In E. Hinchee, R. E. Hoeppel, and D.B. Anderson (ed.), Bioremediation of recalcitrant organics. Battelle Press, Columbus, Ohio.

Kalafut, T., Wales, M.E., Rastogi, V.K., Naumova, R.P., Zaripova, S.K., Wild, J.R. (1998) Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria. Current Microbiology 36(1), 45-54.

Kao, C.M., Chen, S.C., Liu, J.K., Wang, Y.S. (2001) Application of microbial enumeration technique to evaluate the occurrence of natural bioremediation. Water Research 35, 1951-1960.

Kao, C.M., Prosser, J. (1999) Intrinsic bioremediation of trichloroethene and chlorobenzene: field and laboratory studies. Journal of Hazardous Materials 69, 67-79.
Khan, T.A., Bhadra, R., Hughes, J. (1997) Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum. Journal of Industrial Microbiology and Biotechnology 18 (2-3), 198-203.

Lewis, T.A., Ederer, M.M., Crawford, R.L., Crawford, D.L. (1997) Microbial transformation of 2,4,6-trinitrotoluene. Journal of Industrial Microbiology and Biotechnology 18(2-3), 89-96.

Lewis, T.A., Newcombe, D.A., Crawford, R.L. (2004) Bioremediation of soils contaminated with explosives. Journal of Environmental Management 70(4), 291-307.

Lopez, I., Ruiz-Larrea, F., Cocolin, L., Orr, E., Phister, T., Marshall, M., ... & Mills, D. A. (2003). Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis. Applied and Environmental Microbiology, 69(11), 6801-6807.

Martin, J. L., Comfort, S. D., Shea, P. J., Drijber, R. A., & Kokjohn, T. A. (1997). Denitration of 2, 4, 6-trinitrotoluene by Pseudomonas savastanoi.Canadian journal of microbiology, 43(5), 447-455.


McCormick, N.G., Feeherry, F.E., Levinson, H.S. (1976). Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds. Applied and Environmental Microbiology 31(6), 949-958.

Michels, J., Gottschalk, G. (1994) Inhibition of the lignin peroxidase of Phanerochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene. Applied and Environmental Microbiology 60(1), 187-194.

Michels, J., Gottschalk, G. (1995) Pathway of 2,4,6-trinitrotoluene (TNT) degradation by Phanerochaete chrysosporium, p.135–149. In J. C. Spain (ed.), Biodegradation of nitroaromatic compounds. Plenum Press, New York, N.Y.

Montpas, S., Samson, J., Langlois, É., Lei, J., Piché, Y., Chênevert, R. (1997) Degradation of 2,4,6-trinitrotoluene by Serratia marcescens. Biotechnology Letters 19 (3), 291-294.

Muyzer, G., De Waal, E.C., Uitterlinden, A.G. (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes encoding for 16S rRNA. Applied Environmental Microbioogy 59(3), 695-700.

Nakatsu, C. H., Torsvik, V., Øvreås, L. (2000). Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Science Society of America Journal, 64(4), 1382-1388.

Naumova, R.P., Selivanovskaia, S.I., Mingatina, F.A. (1988) Possibilities for the deep bacterial destruction of 2,4,6-trinitrotoluene. Mikrobiologiya 57(2), 218-222.

Nirenberg, H.I. (1981) A simplified method for identifying Fusarium spp. occurring in wheat. Canadian Journal of Botany 59, 1599-1609.

Nirenberg, H.I. (1990) Recent advances in the taxonomy of Fusarium. Studies in Mycology. (32), 91-101.

Nishino, S. F., Paoli, G. C., Spain, J. C. (2000). Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2, 6-dinitrotoluene.Applied and Environmental Microbiology, 66(5), 2139-2147.

Nyanhongo, G.S., Aichernig, N., Ortner, M., Steiner, W., Guebitz, G.M. (2009) Incorporation of 2,4,6-trinitrotoluene (TNT) transforming bacteria into explosive formulations. Journal of Hazardous Materials 165 (1-3), 285-290.

Oh, K.-H., Kim, Y.-J. (1998) Degradation of explosive 2,4,6-trinitrotoluene by s-Triazine degrading bacterium isolated from contaminated soil. Bulletin of Environmental Contamination and Toxicology 61(6), 702-708.
Pak, J.W., Knoke, K.L., Noguera, D.R., Fox, B.G., Chambliss, G.H. (2000) Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C. Applied and Environmental Microbiology 66 (11), 4742-4750.

Park, C., Kim, T.-H., Kim, S., Kim, S.-W., Lee, J., Kim, S.-H. (2003) Optimization for biodegradation of 2,4,6-trinitrotoluene (TNT) by Pseudomonas putida. Journal of Bioscience and Bioengineering 95 (6), 567-571.

Preuss, A., Fimpel, J., Diekert, G. (1993) Anaerobic transformation of 2,4,6-trinitrotoluene (TNT). Archives of Microbiology 159(4), 345-353.

Rahal, A.G., Moussa, L.A. (2011) Degradation of 2,4,6-trinitrotoluene (TNT) by soil bacteria isolated from TNT contaminated soil. Australian Journal of Basic and Applied Sciences 5(2), 8-17.

Robertson, B. K., Jjemba, P. K. (2005). Enhanced bioavailability of sorbed 2, 4, 6-trinitrotoluene (TNT) by a bacterial consortium. Chemosphere, 58(3), 263-270.

Ro, K.S., Venugopal, A., Adrian, D.D., Constant, D., Qaisi, K., Valsaraj, K.T., Thibodeaux, L.J., Roy, D., (1996) Solubility of 2,4,6-trinitrotoluene (TNT) in water. Journal of Chemical and Engineerind Data 41(4), 758–761.
Schackmann, A., Müller, R. (1991) Reduction of nitroaromatic compounds by different Pseudomonas species under aerobic conditions. Applied Microbiology and Biotechnology 34(6), 809-813.

Scheibner, K., Hofrichter, M., Fritsche, W. (1997a) Mineralization of 2-amino-4,6-dinitrotoluene by manganese peroxidase of the white-rot fungus Nematoloma frowardii. Biotechnology Letters 19(9), 835-839.

Scheibner, K., Hofrichter, M., Herre, A., Michels, J., Fritsche, W. (1997b) Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene. Applied Microbiology and Biotechnology 47(4), 452-457.

Shim, C.Y., Crawford, D.L. (1995) Biodegradation of trinitrotoluene (TNT) by a strain of Clostridium bifermentans, p. 57–69. In R. E. Hinchee, J. Fredrickson, and B. C. Alleman (ed.), Bioaugmentation for site remediation. Battelle Press, Columbus, Ohio.

Spain, J. C. (1995). Biodegradation of nitroaromatic compounds. Annual Reviews in Microbiology, 49(1), 523-555.

Spanggord, R.J., Stewart, K.R., Riccio, E.S. (1995) Mutagenicity of tetranitroazoxytoluenes: A preliminary screening in Salmonella typhimurium strains TA100 and TA100NR. Mutation Research - Environmental Mutagenesis and Related Subjects 335(3), 207-211.

Stenuit, B., Eyers, L., Rozenberg, R., Habib-Jiwan, J. L., Agathos, S. N. (2006). Aerobic growth of Escherichia coli with 2, 4, 6-trinitrotoluene (TNT) as the sole nitrogen source and evidence of TNT denitration by whole cells and cell-free extracts. Applied and environmental microbiology, 72(12), 7945-7948.

Traxler, R. W., Wood, E., Delaney, J. M. (1974). Bacterial degradation of alpha-TNT. Developments in industrial microbiology 16, 71-76.

USEPA, Method 8330A, Nitroaromatic and Nitramines by High Performance Liquid Chromatography（HPLC）, 2007

Vanderberg, L. A., Perry, J. J., & Unkefer, P. J. (1995). Catabolism of 2, 4, 6-trinitrotoluene by Mycobacterium vaccae. Applied Microbiology and Biotechnology, 43(5), 937-945.

Vorbeck, C., Lenke, H., Fischer, P., & Knackmuss, H. J. (1994). Identification of a hydride-Meisenheimer complex as a metabolite of 2, 4, 6-trinitrotoluene by a Mycobacterium strain. Journal of bacteriology, 176(3), 932-934.

Vorbeck, C., Lenke, H., Fischer, P., Spain, J. C., Knackmuss, H. J. (1998). Initial reductive reactions in aerobic microbial metabolism of 2, 4, 6-trinitrotoluene. Applied and environmental microbiology, 64(1), 246-252..

Widrig, D. L., Boopathy, R., Manning, J. F. (1997). Bioremediation of TNT‐contaminated soil: A laboratory study. Environmental toxicology and chemistry,16(6), 1141-1148.
Won, W. D., DiSALVO, L. H., Ng, J. A. M. E. S. (1976). Toxicity and mutagenicity of 2, 4,-6-trinitrotoluene and its microbial metabolites. Applied and Environmental Microbiology, 31(4), 576-580.

Yardin, G., Chiron, S. (2006). Photo–Fenton treatment of TNT contaminated soil extract solutions obtained by soil flushing with cyclodextrin. Chemosphere,62(9), 1395-1402.

行政院環境保護署環境檢驗所，火炸藥物質檢測方法－高效液相層析儀/紫外光偵測器法 NIEA M804，（2012）

許麗秋，尿中BTEX與其代謝產物關係之研究，中國醫藥學院環境醫學研究所碩士論文，(2000)