本研究在厭氧條件下，探討一株從台灣南部電鍍工廠廢水分離之氰化物分解菌Klebsiella oxytoca SYSU-011代謝處理四氰化鎳（tetracyanonickelate）之情況。實驗結果得知，菌株在不同組成成份之培養基( Burk培養基與NFG培養基 )中，對四氰化鎳之降解有不同之效率，經過30天培養，可以發現K. oxytoca在Burk培養基對四氰化鎳之降解效率55﹪較NFG培養基33﹪佳，故在後續之實驗中，選用 Burk培養基作為菌株生長之基質。在Burk培養基下，以瞭解添加不同氮源（硝酸根離子、亞硝酸根離子與銨根離子）對菌株代謝四氰化鎳之影響，研究結果發現硝酸根離子濃度在1 mM下對四氰化鎳有較佳之降解效率（66﹪）；研究結果亦顯示 5 mM及10 mM亞硝酸根離子則會對菌株造成抑制，而銨離子濃度之添加（5 mM及10 mM），並不會對菌株生長有抑制；因此，添加不同氮源會對四氫化鎳之降解有不同程度之影響。在不同碳源（果糖、葡萄糖、醋酸及乙醇），可發現葡萄糖與果糖可當作菌株生長之碳源，但醋酸與乙醇則否。研究結果除了對K. oxytoca在厭氧下分解四氰化鎳之機制有更進一步的瞭解外，亦可提供未來在利用K. oxytoca處理含四氰化鎳廢水所需之重要參考資料。

Tetracyanonickelate (K2(Ni(CN)4), TCN) is one of the most toxic organics to living organisms. In this study, Klebsiella oxytoca (K. oxytoca) SYSU-011 (a cyanide- degrading bacterium), which was isolated from the wastewater of a metal-plating plant, was shown to be able to biodegrade TCN under anaerobic conditions. Two different media (Burk and NFG) were used to grow K. oxytoca. Results indicate that higher TCN biodegradation rate was observed when Burk medium was used as the growth media for K. oxytoca. In the nitrogen source addition experiment, TCN degradation was inhibited by the addition of nitrite. In the carbon source addition experiment, TCN degradation was enhanced by the addition of glucose and fructose. These findings would be helpful in designing a practical in situ or on-site treatment system inoculated with K. oxytoca for the treatment of TCN-containing wastewater.

謝誌 I
摘要 II
英文摘要 III
目錄 IV
圖目錄 VII
表目錄 IX
第一章 前言 1
1.1、氰化物與四氰化鎳之來源與特性 1
1.2、氰化物廢水處理法 3
1.2.1、化學處理法 4
1.2.2、生物處理法 5
1.3、厭氧生物處理法 7
1.3.1、厭氧生物處理方式 7
1.3.2、產能代謝 8
1.3.3、有機物之厭氧分解 9
1.3.4、氰化物之厭氧分解 10
1.4、菌株K. oxytoca SYSU-011與四氰化鎳簡介 11
第二章 研究目的 13
第三章 材料與方法 14
3.1、實驗材料 14
3.1.1、菌種 14
3.1.2、化學藥品 14
3.1.3、培養基 14
3.2、實驗步驟 15
3.2.1、K. oxytoca之培養 15
3.2.2、NFG培養基與Burk培養基之厭氧批次實驗成份及流程 16
3.2.3、不同氮源之厭氧批次實驗成份及流程 16
3.2.4、不同碳源之厭氧批次實驗成份及流程 17
3.3、分析方法 18
3.3.1、細胞質量之測量 18
3.3.2、生長曲線 18
3.3.3、四氰化鎳濃度測定 18
3.3.4、硝酸根離子濃度測定 19
3.3.5、亞硝酸根離子濃度測定 19
第四章 結果與討論 20
4.1、在不同培養基下，K. oxytoca降解四氰化鎳之效率比較 20
4.2、不同氮源對K. oxytoca降解四氰化鎳效率之影響 21
4.2.1、硝酸根離子 22
4.2.2、亞硝酸根離子 23
4.2.3、銨離子 24
4.3、不同碳源對K. oxytoca降解四氰化鎳效率之影響 25
4.4、pH值對K. oxytoca降解四氰化鎳之影響 27
第五章 結論 29
第六章 圖表 31
第七章 參考文獻 41
附錄 50

Adjei, M.D., Ohta, Y., 2000. Factors affecting the Biodegradation of cyanide by Burkholderia cepacia strain C-3. Journal of Bioscience and Bioengineering. 89(3): 274-277.

Bae, H.-S., Cho, Y.-G., Oh, S.-E., Kim I.-S., 2002. Anaerobic degradation of pyrrolidine and piperidine coupled with nitrate reduction. Chemosphere. 48: 329-334.

Barclay, M., Tett, V.A., Knowles, C.J., 1998. Metabolism and enzymology of cyanide/metallocyanide biodegradation by Fusarium solani neutral and acidic conditions. Enzyme and Microbial Technology. 23: 321-330.

Barclay, M., Hart, A., Knowles, C.J., Meeussen, J.C.L., Tett, V.A., 1998. Biodegradation of metal cyanides by mixed and pure culture of fungi. Enzyme and Microbial Technology. 23: 223-231.

Barclay, M., Hart, A., Knowles, C.J., Meeussen, J.C.L., Tett, V.A., 1998. Biodegradation of metal cyanides by mixed and pure culture of fungi. Enzyme Microbial Technology. 22: 223-231.

Chena, S.-C., Liu, J.-K., 1999. The respiratory responses to cyanide of a cyanide-resistant K. oxytoca bacterial strain. FEMS Microbiology Letters. 175: 37-43.

Claisse, O., Aline, L.-F., 2000. Assimilation of glycerol by a strain of Lactobacillus collinoides isolated from cider. Food Microbiology. 17: 513-519.

Fallon, R.-D., Cooper, D.-A., Speece, R., Henson, M., 1991. Anaerobic biodegradation of cyanide under methanogenic conditions. Applied and Environmental Microbiology. 57: 1656-1662.

Goldberg, I., Nadler, V. and Hochma, A., 1987. Mechanism of nitrogenase switchoff by oxygen. Jounral of Bacteriology 169: 874-879

Gijzen, H.J., Bernal, E. and Ferrer, H., 2000. Cyanide toxicity and cyanide degradation in anaerobic wastewater treatment. Water Reserch. 34(9): 2447-2454.

Hela, Z., Marc, L., Sami, Sayadi., 2002. Degradation of 4-chlorophenol by the white rot fungus Phanerochaete chrysosporium in free and immobilized cultures. Bioresource Technology. 84: 145-150.

Hill, S., 1976a. Influence of atmospheric oxygen concentration on acetylene reduction and efficiency on nitrogen fixation in intact Klebsiella pneumoniae. Journal of General Microbiology. 93: 335-345.

Hill, S., Turner, G. L. and Bergersen, F. J., 1984. Synthesis and activity of nitrogenase in Klebsiella pneumoniae exposed to low concentrations of oxygen. Journal of General Microbiology 130: 1061-1067.

Hope, K.M. and Knowles, C.J., 1991. The anaerobic utilisation of cyanide in the presence of sugars by microbial cultures can involve an abiotic process. FEMS Microbiology Letters. 80: 217-220.

Juan, S.A., Richmond, M.G., Nagappan, O., Kunz, D.A., 1990. Degradation of the Metal-Cyano Complex Tetracyanonickelate (Ⅱ) by Cyanide-Utilizing Bacterial Isolates. Applied and Environmental Microbiology. 56: 3664-3670

Kao, C.M., Liu, J.K., Lou, H.R., Lin, C.S., Chen, S.C., 2003. Biotrasformation of cyanide to methane and ammonia by Klebsiella oxytoca. Chemosphere. 50: 1055-1061.

Kelly, M., 1968. The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitrogenase preparation from Azotobacter chroococcum and the effects of inhibitors. The Biochemical Journal 107(1): 1-6.

Knowles, C.J., 1976. Microorganisms and cyanide. Bacteriological Reviews 40: 652-680.

Krieg, N.R., Holt, J.G., 1984. Bergey’s manual of systematic bacteriology volume 1. Williams and Wilkins. 408-420.

Kwon, H.K., Woo, S.H., Park, J.M., 2002. Degradation of tetracyanonickelate（Ⅱ）by Cryptococcus humicolus MCN2. FEMS Microbiology Letters. 214: 211-216.

Lattuati, A., Metzger, P., Acquaviva, M., Bertrand, J.-C., Largeau, C., 2002. n-Alkane degradation by Marinobacter hydrocarbonoclasticus strain SP 17: long chain β-hydroxy acids as indicators of bacterial activity. Organic Geochemistry 33: 37-45.

Liu, C.-K., Wu, Y.-W., Hsu, C.-H., 1992. Physiological adaptability of a cyanide-utilizing Klebsiella oxytoca strain. Proceedings of the National Science Council, R.O.C. 16: 188-193.

Liu, C.-K., Liu, C.-H., Lin, C.-S., 1997. The role of nitrogenase in a cyanide-degrading Klebsiella oxytoca strain. Proceedings of the National Science Council, R.O.C. 21: 37-42.

Mester, K.G., Kosson, D.S., 1996. Anaerobic biodegradation of toluene under denitrifying conditions in contaminated groundwater and soil. Journal of Hazardous Materials. 45: 219-232.

Mosher, J.B. and Figueroa, L., 1996. Biological oxidation of cyanide: a viable treatment option for the minerals processing industry. Minerals Engineering. 9(5): 573-581.

Nester, E.W., Roberts, C.E., Nester, M.T., 1995. Metabolism: The generation of energy and synthesis of small molecules. Microbiology. Wm. C. Brown Communications, Inc.

Nicholas, V.-C., Darryl, R.-N., Trevor, D., 1997. Aerobic biodeagradation of hexahydro-1,3,5-trinitro-1,3,5,-triazine（RDS）as a nitrogen source by a Rhodococcus sp., strain DN22. Soil Biology and Biochemistry, 30: 1159-1167.

Ogutveren, U.B., Toru, E., Koparal, S., 1998. Removal of cyanide by anodic oxidation for wastewater treatment. Water Research 33(8): 1851-1856.

Olivier, C., Aline L.-F., 2000. Assimilation of glycerol by a strain of Lactobacillus collinoides isolated from cider. Food Microbiology. 17:513-519.

Palmer, S.A.K., Breton, M.A., Nunno, T.J., Sullivan, D.M., Surprenant N.F., 1988. Metal/Cyanide Containing Wastes: Treatment Technologies. Noyes Data Crop, Park Ridge, NJ.

Patil, Y.B., Paknikar, K.M., 1999. Removal and recovery of metal cyanides using a combination of biosorption and biodegradation processes. Biotechnology letters. 21: 913-919.

Pereira, P.T., Arrabaca, J.D., Amaral-Collaco, M.T., 1996. Isolation, Selection and Characterization of a Cyanide-Degrading Fungus from an Industrial Effluent. International Biodeterioration & Biodegradation. 45-52.

Raina, M.M., Ian, L.P., Charles, P.G., 2000. Environmental microbiology. Academic Press. 331-339.

Rollinson G., Jones R., Meadows M.P., Knowlese, D.J., 1987. The growth of a cyanide-utilizing of Pseudomonas fluorescens in liquid culture on nickel cyanide as a source of nitrogen. FEMS Microbiology Letters. 40: 199-205.

Song, Y.C., Piak, B.C., Shin, H.S., La, S.J., 1998. Influence of electron donor and toxic materials on the activity of sulfate reducing bacteria for the treatment of electroplating wastewater. Water Science and Technology. 38(4-5): 187-194.

Taherzadeh, M.J., Gustafsson, L., Niklasson, C., Liden, G., 2000. Inhibition effects of furfural on aerobic cultivation of Saccharomyces cerevisiae growing on ethanol and/or acetic acid. Journal of Bioscience and Bioengineering. 90(4): 374-380.

Thomas, B., Per, A., Frank, L., Bo, S., 2003. Biodegradation of chlorinated solvents in a water unsaturated topsoil. Chemosphere. 51: 143-152.

Wojciech, H., Magdalena, P.-J., Mieczyslaw, B., Roman, M., 2000. Nitrite as agent selecting anaerobic phenoldegradating microflora in petroleum refining sediments. Water Research 34: 1354-1358.

Wong, T.Y., 1993. Effects of calcium on sugar transport in Azotobacter vinelandii. Applied and Environmental Microbiology. 59: 89-92.

Yanase, H., Sakamoto, A., Okamoto, K., Kita, K., Sato, Y., 2000. Degradation of metal-cyano complex tetracyanonickelate (Ⅱ) by Fusarium oxysporum N-10. Applied Biochemistry and Biotechnology 53: 328-334.

王進琦、王西華, 1992, 微生物學實驗, 藝軒圖書出版社印行。

行政院環境保護署環境保護人員訓練所, 2001, 水污染防治法規。

行政院環境保護署環境檢驗所, 1995, 水中陰離子檢測方法-離子層析法, NIEA W415.50T。

行政院環境保護署環境檢驗所, 2002, 水中亞硝酸鹽氮檢測方法-分光光度計法, NIEA W418.51C。

林志杰, 2001, Klebsiella oxytoca對四氰化鎳之分解能力探討, 國立中山大學生命科學研究所碩士論文。

林秋裕, 1995, 環境工程微生物學, 國彰出版社。

林宏懋, 1999, 在葡萄糖或半乳糖培養下Azotobacter vinelandii 突變株TN20電子傳遞鍊途徑的探討, 國立中山大學生命科學研究所碩士論文。

林宗新, 1996, 中小電鍍業含氰廢液集中處理可行性之探討, 工業污染防治, 第57期，p.34-41。

林彥穎、蔡青松、劉文佐, 2000, 生物除磷系統中以葡萄糖為基質的活性污泥厭氧代謝行為之研究, 第12屆環工年會論文集。

孫連鴻、盧至人、邱應志, 1998, 主要基質醋酸鈉濃度對厭氧共代謝1,1,1-三氯乙烷之效應, 第10屆環工年會論文集。

黃啟裕、何盈蒼、陳乃慈, 2001, 好氧脫硝菌Acinetobacter baumannii進行同時硝化與脫硝反應（Simultaneous nitrification and denitrification）之探討與研究, 第13屆環工年會論文集。

經濟部工業局, 1995, 廢水生物處理技術。

劉家漢, 1994, 氰化物利用菌Klebsiella oxytoca之氰分解生理探討, 國立中山大學生命科學研究所碩士論文。

蔡國鈞, 2000, 細說氰化物, 毒化物分析, 第144期, p. 5-6。

蔡啟堂, 2002, 五氯酚分解菌之生理特性探討, 國立中山大學生命科學研究所碩士論文。