臺灣博碩士論文加值系統

　　氫氣已被視為21世紀的新能源，氫燃燒之後產生水，因而不會對環境造成污染為乾淨的能源。利用微生物產氫的方式已逐漸受到重視，其中利用異營菌將廢棄物分解產氫，亦可達到資源回收再利用。本研究自稻草堆肥、牛糞堆肥、稻殼和食品及酒廠廢水處理廠共分離出71株菌株，其中27株具有產氫能力，但其差異相當大。其中菌株C008為產氫活性最高的菌株，其氫轉換率為1.43mole H2/mole Glucose，仍低於目前被視為產氫能力最佳的菌株C. butyricum。分離菌株C008經由16S rDNA序列比對的結果，其親源關係與Clostridium tyrobutyricum最為相近。C008菌株屬於發酵產氫菌，在葡萄糖含Yeast extract或Peptone的複合基質其產氫量顯著的高於只含葡萄糖的單一基質。C008菌株在發酵產氫時的產物以乙酸和丁酸為主，在生長初期乙酸和丁酸的比值大於1，且在生長過程呈現二階段的產氫特性。 此外，本研究亦探討溫度、pH、Na+及K+對分離菌株C008和C. butyricum產氫的影響。

　　Hydrogen is a clean and renewable energy, not contribute to the green house effect. Bio-hydrogen production by microorganisms can be divided into two main categories: one is by photosynthetic bacteria and the other is by anaerobic fermentation. The Clostridium, has been well studies for its ability to form endospores and its potential to generate hydrogen. In this research, we focus on the indigenous Clostridia and physiological characteristics of hydrogen production. 71 strains were isolated from composts, rice shell, food and wine wastewater treatment plants, but only 27 strains can produce hydrogen. C008 strain was isolated from compost with optimum growth at 30℃ and pH 7.0. The effects of nutrients such as peptone, yeast extract, and vitamins on growth, fermentation products, and hydrogen production were discussed in this study. The enhance of hydrogen production was achieved by combination of glucose and yeast extract as the growth medium. The hydrogen production potential of Clostridium butyricum and C008 strain was 1.39 and 2.86 mole hydrogen/mole glucose, respectively. The fermentation products of Clostridium butyricum, acetate and butyrate were produced in the ratio of less than 1, however, the ratio was greater than 1 during the 20 hours growth by C008. The transition from the acidiogenic phase to solventogenic phase was not observed during the growth of C008 strain. The effects of sodium and potassium ion on the hydrogen production of Clostridium butyricum and C008 strain was also discussed in this study. The 16S rDNA-based phylogenetic analysis of C008 strain is member of Clostridia cluster I, Clostridium tyrobutyricum.

摘要I
誌謝III
目錄IV
表目錄VI
圖目錄VII

一、前言1
二、文獻回顧2
　　2.1 全球的能源問題2
　　2.2氫氣2
　　　　2.2.1 氫氣是未來能源2
　　　　2.2.2 國外的相關研究工作3
　　　　2.2.3 產氫的方式4
　　2.3 生物產氫6
　　　　2.3.1 為何選擇生物產氫6
　　　　2.3.2 產氫生物及產氫方式6
　　2.4 Clostridium10
　　　　2.4.1 Clostridium的特性10
　　　　2.4.2 影響Clostridium產氫的因素10
　　　　2.4.3 目前遭遇的困難及未來的展望12
三、材料與方法13
　　3.1 梭菌屬產氫菌及培養基13
　　　　3.1.1 菌種來源13
　　　　3.1.2 培養基13
　　　　　　3.1.2.1 優厚培養13
　　　　　　3.1.2.2 生長培養基13
　　3.2 研究方法13
　　　　3.2.1 本土性產氫菌株的分離13
　　　　3.2.2 氫氣的分析17
　　　　3.2.3 揮發酸的分析17
　　　　3.2.4 醇的分析17
　　　　3.2.5 培養基的選擇17
　　　　3.2.6 溫度影響的測定18
　　　　3.2.7 鈉離子影響的測定18
　　　　3.2.8 鉀離子影響的測定18
　　　　3.2.9 生長曲線的測定18
　　　　3.2.10 pH調控對葡萄糖利用的影響19
　　　　3.2.11 產氫效能的測定19
　　　　3.2.12 菌相的觀察20
　　　　3.2.13 DNA的萃取21
　　　　3.2.14 聚合酵素連鎖反應（PCR）21
　　　　3.2.15 變性梯度明膠電泳（DGGE）23
　　3.3 主要儀器24
四、結果26
　　4.1 本土性產氫菌株的分離26
　　4.2 培養基質與溫度對氫氣生成的影響28
　　　　4.2.1 溫度對生物產氫的影響28
　　　　4.2.2 培養基質對生物產氫的影響31
　　　　4.2.3 C008菌株之氫轉換率和產氫活性35
　　4.3 產氫菌之生長與氫氣之形成.37
　　　　4.3.1 分離菌株C008之生長37
　　　　4.3.2 分離菌株R0402之生長40
　　　　4.3.3 C.butyricum之生長.43
　　4.4 調控pH對氫氣的生成的影響.46
　　4.5 定序結果51
五、討論53
六、結論55
七、參考文獻56

施翠盈、許恆維、劉憶芬、曾怡禎 2001 本土性梭菌屬產氫菌株之生理特性。第二六屆廢水處理技術研討會，p.1-82。［NSC-90-2211-E-006-032］

曾怡禎、許恆維、陳佳隆 2000 梭菌屬﹙Clostridium﹚的分離及其產氫活性的影響因子。第二五屆廢水處理技術研討會，p.315-320。［NSC-89-2211-E-006-032］

簡青紅、施翠盈、劉憶芬、曾怡禎 2001 本土性梭菌屬產氫菌株之分離與產氫活性。 微生物學會第35屆年會。

Andreesen, J. R., Bahl, H. and Gottschalk, G. 1989. Introduction to the physiology and biochemistry of the Genus Clostridium, pp: 27-53. In Minton Nigel P. and Clarke David J. (ed.), Biotechnology handbooks ．3, Plenum Press．New York and London.

Asada,Yasuo and Miyake Jun. 1999. Photobiological hydrogen production. Journal of Bioscience and Bioengineering 88(1):1-6.

Bockris, J. O’ M.1999. Hydrogen economy in the future. International Journal of Hydrogen Energy 24:1-15.

Carolyn, C. E. and Catherine, E. G. P. 2001. International energy agency agreement on the production and utilization of hydrogen. Proceddings of the 2001 DOE Hydrogen Program Review. Nrel/CP-570-30535.

Cato, E. P., George, W. Lance and Finegold Sydney M. 1986. Clostridium, pp:1141-1200. In Butler John P. (ed.), Bergey’s Manual of Systematic Bacteriology. Williams & Wilkins.

Chen, C.K. and Blaschek, H. P. 1999. a. Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101. Appl. Microbiol. Biotechnol 52(1):170-173.

Chen, C.K.and Blaschek, H. P. 1999. b. Effect of acetate on molecular and physiological aspects of Clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration. Appl. Microbiol. Biotechnol 65(2) : 499-505.

Das, D. and Veziroglu, N. 2001. Hydrogen production by biological processes: a survey of literature. International Journal of Hydrogen Energy 26:13-28.

Ginkel, S. V., Shih ,Wu Sung and Li, Ling. 2001. Role of initial sucrose and pH levels on natural, hydrogen-producing anaerobe germination. Proceddings of the 2001 DOE Hydrogen Program Review. Nrel/CP-570-30535.

Girbal, L., Vasconcelos, I. and Soucaille, P. 1994. Transmembrane pH of Clostridium acetobutyricum is inverted( more acidic inside ) when the in vivo activity of hydrogenase is decreased. Journal of Bacteriology 176:6146-6147.

Guedon, E., Payot, S., Desvaux, M. and Petitdemange, H. 1999. Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthesis medium. Journal of Bacteriology 181(10):3262-3269

Gottschalk, G. and Peinemann, S. 1992. The anaerobic way of life, pp:300-311. In Balows, A., Turper, H. G., Dworkin,
M., Harder W. and Schleifer K. H. (ed.), Prokaryote, Spring-Verlag, New York.

Holt, R. A., Stephens, G. M. and Morris, J. G. 1984. Production of solvents by Clostridium acetobutyricum cultures maintained at neutral pH. Applied and Environmental Microbiology 48:1166-1170.

Kashket, E. R. and Cao, Zhi-Yi. 1995. Clostridial strain degeneration. FEMS Microbiology Review 17:307-315.

Kroll, R. G. and Booth, I. R. 1983. The relationship between intracellular pH, the pH gradient and potassium transport in Escherichia coli. Biochem. J. 216:709-716.

Lamed, R. J., Lobos, J. H. and Su, T. M. 1988. Effects of stirring and hydrogen on fermentation products of Clostridium thermocellum. . Applied and Environmental Microbiology 54(5):1216-1221

Ljungdahl, A. G., Hugenholtz, J. and Wiegel, J. 1989. Acetogenic and acid-producing Clostridia, pp: 145-180. In Minton Nigel P. and Clarke David J. (ed.), Biotechnology handbooks ．3, Plenum Press．New York and London.

Miller, D. N., Bryant, J. E., Madsen, E. I. and Ghiorse, W. C. 1999. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Applied and Environmental Microbiology 65(11):4715-4724.

Momirlan, M and Veziroglu, T.N..2002. Current status of hydrogen energy. Renewable and Sustainable Energy Reviews. 6:141-179.

Nandi, R. and Sengupta, S. 1998. Microbial Production of Hydrogen:An Overview. Critical Reviews in Microbiology 24(1):61-84.

Rogner H. H. 1998. Hydrogen technologies and the technology learning curve. International Journal of Hydrogen Energy 23(9):833-840.

Hefner III, Robert A. 2002. The age of energy gases. International Journal of Hydrogen Energy 27:1-29.

Seth Dunn. 2002. Hydrogen futures: toward a sustainable
energy system. International Journal of Hydrogen Energy 27:235-264.

Stackebrandt E. and Rainey F. A. 1997. Phylogenetic Relationships, pp: 3-19. In Rood, J. I., McClane, B. A., Songer, J. G. and Titball, R.W. (ed.), The Clostridia : Molecular Biology and Pathogenesis, Academic Press.

Stim-Herndon, K. P., Nair, R., Papoutsakis, E. T., and Bennett, G. B. 1996. Analysis of degeneration variants of Clostridium acetobutyricum ATCC824. Anaerobe 2:11-18.

Terracciano, J. S., Schreurs, W. J. A. and Kashket, E. R. 1987. Membrane H+ conductance of Clostridiun thermoaceticum and Clostridiun acetobutyricum: Evidence for electrogenic Na+/H+ antiport in Clostridiun thermoaceticum. Applied and Environmental Microbiology 53(4):782-786.

Vasconcelos, I., Girbal, L. and Soucaille, P. 1994. Regulation of carbon and electron flow in Clostridium acetobutyricum growth in chemostat culture at natural pH on mixtures of glucose and glycerol. Journal of Bacteriology 176(3):1443-1450.

Woods, D. R. 1993. History and Future Potential of the Clostridia in Biotechnology, pp: 1-23. In Minton Nigel P. and Clarke David J. (ed.), Biotechnology handbooks ．3, Plenum Press．New York and London.

Zajic, J. E., Kosaric, N. and Brosseau, J. D. 1978. Microbial Production of Hydrogen. Adv. Biochem.Eng 7:57-109.