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研究生:王馨儀
研究生(外文):Hsin-Yi Wang
論文名稱:以牛糞肥中嗜熱厭氧菌群分解纖維素產乙醇之研究
論文名稱(外文):Ethanol production from thermophilic anaerobic digestion of cellulose by a microbial community in dairy cattle manure
指導教授:黃啟裕黃啟裕引用關係
指導教授(外文):Chi-Yu Huang
學位類別:碩士
校院名稱:東海大學
系所名稱:環境科學系
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:99
中文關鍵詞:纖維素嗜熱厭氧菌乙醇DGGE
外文關鍵詞:cellulosethermophilic anaerobesethanolDGGE.
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纖維素可提供大量生質能的來源。本研究由東海大學牧場飼養的乳牛所排放出之糞便中,培養出能降解纖維素產生乙醇的嗜熱厭氧穩定菌群。研究菌群以濾紙(Whatman NO.1)為纖維素基質,由GC-FID分析乙醇產量,其最佳乙醇生成溫度及pH值分別為55-60℃左右及pH 7.7;增加氮源(yeast extract, peptone)濃度至培養基內的最佳乙醇產率之添加濃度均為1g/l;最佳產乙醇率之濾紙濃度為8g/l;降解不同紙類產生乙醇能力為濾紙>影印紙>卡紙>報紙(無法被降解)。培養基內濾紙(10g/l)可於九天內被研究菌群完全降解,其最終乙醇的產率約為0.29g ethanol/g cellulose。
本研究藉由分子生物技術,以巢式聚合酶鏈反應-變性梯度電泳凝膠法(Nested PCR-DGGE)分析此菌群16S rDNA中V3-V5的變異區段,利用亮帶擷取,再由PCR擴增萃取核酸片段後,進行定序分析菌群結構及親緣關係。在本研究中發現馴養之後的牛糞菌群之16S rDNA核酸片段與梭菌目(Clostridiales)及嗜厭氧桿菌目(Thermoanaerobacteriales)有接近之親緣關係。
Cellulose can be a major source of biomass energy. Thermophilic anaerobic biodegradation of cellulose by mixed microbial community from dairy cattle manure in Tunghai University range is demonstrated in this study. The pure cellulose (Whatman NO.1 filter paper) is used as the carbon source for producing ethanol by the mixed microbial community. GC-FID is used to analyze ethanol production from cellulose degradation. The optimal ethanol production temperature of the mixed thermophilic anaerobes is 55-60℃. The optimal ethanol production pH of the mixed thermophilic anaerobes is 7.7. Ethanol production can be increased in the mixed culture by adding 1g/l of yeast extract and peptone respectively as nitrogen sources. The optimal substrate concentration for ethanol production is 8g/l filter paper. Various used papers (filter paper, office paper, newspaper and cardboard) were used in this study to test their potential for ethanol production. The result showed the potential of tested used paper for ethanol production by this mixed culture is filter paper > office paper > cardboard > newspaper by the mixed microbial community in this study. The mixed microbial community of this study can completely degrade 10g/l of filter paper in 9 days, during which the ethanol yield is 0.29g ethanol/g cellulose.
Phylogenetic and sequence similarity of the variable region (V3-V5) of 16S rDNA from the mixed thermophilic anaerobes is analyzed for their revolutionary relationship by using Nested PCR-denaturing gradient gel electrophoresis (Nested PCR-DGGE) technology. Clostridiales and Thermoanaerobacteriales are the two closest predominant groups in this thermophilic, anaerobic community.
第一章 前言……………………………………………………………1
第二章 文獻回顧………………………………………………………3
2.1 再生能源(Renewable energy)………………………………3
2.1.1 能源危機……………………………………………………3
2.1.2 生質能………………………………………………………4
2.1.3 酒精能源……………………………………………………6
2.2纖維素(Cellulose) ……………………………………………8
2.2.1 纖維素的構造………………………………………………8
2.2.2 纖維素水解…………………………………………………10
2.3生物轉化(Bioconversion) ……………………………………12
2.4 分解纖維素之微生物(Cellulolytic microorganism)……15
2.5 纖維素分解酵素系統(Cellulase systems)…………………16
2.5.1 纖維素分解酵素 ……………………………………………16
2.5.2 細菌纖維素分解酵素系統 …………………………………18
2.5.3 嗜熱性厭氧纖維素分解細菌 ………………………………24
2.6 研究目的 …………………………………………………………26
第三章 實驗材料與方法…………………………………………………27
3.1 實驗流程設計………………………………………………………27
3.2 菌株來源採樣………………………………………………………27
3.3 菌種之培養…………………………………………………………29
3.3.1 除氧操作系統………………………………………………29
3.3.2 嗜熱厭氧菌培養基及還原劑配置…………………………31
3.3.3 培養基接菌…………………………………………………31
3.4 嗜熱厭氧菌最佳生長條件…………………………………………35
3.4.1 溫度……………………………………………………………35
3.4.2 pH值……………………………………………………………35
3.4.3 最佳yeast extract、peptone添加量……………………36
3.4.4 濾紙濃度………………………………………………………36
3.4.5 不同紙張作為纖維素來源之利用測試………………………37
3.5 分析方法……………………………………………………………38
3.5.1 蛋白質分析…………………………………………………………38
3.5.2 還原糖含量測試……………………………………………………39
3.5.3 纖維素降解含量測試………………………………………………40
3.5.4 纖維素醱酵產乙醇測試……………………………………………41
3.6 牛糞菌群之分析……………………………………………………42
3.6.1 DNA萃取………………………………………………………42
3.6.2 聚合酶連鎖反應(Polymerase Chain Reaction, PCR)…43
3.6.3 變性梯度凝膠電泳法(Denaturing Gradient Gel
Electrophoresis,DGGE)……………………………………46
3.6.4 變性梯度凝膠膠片萃取(QIAGEN gel extraction kit)…48
3.6.5 核酸序列之分析…………………………………………………50
第四章 結果與討論…………………………………………………………51
4.1 研究菌群之選取………………………………………………………51
4.2 纖維素降解含量與還原糖濃度測試…………………………………52
4.3乙醇產生測試…………………………………………………………55
4.4 最佳生長條件…………………………………………………………59
4.4.1 溫度………………………………………………………………59
4.4.2 pH值………………………………………………………………62
4.5 最佳Yeast Extract及Peptone添加量……………………………63
4.6 不同紙張作為纖維素來源之利用測試………………………………67
4.7 濾紙濃度對乙醇生產之影響探討……………………………………70
4.8 以變性梯度電泳凝膠(DGGE)進行菌群結構分析……………………74
4.8.1 聚合酶連鎖反應(PCR)擴增16S rDNA…………………………74
4.8.2 以Nested PCR擴增V3-V5變異區段……………………………75
4.8.3 變性梯度凝膠電泳 (DGGE)分析…………………………………77
4.8.4 菌群結構分析………………………………………………………79
第五章 結論與建議……………………………………………………………86
5.1 結論………………………………………………………………………86
5.2 建議………………………………………………………………………88
第六章 參考文獻…………………………………………………………………89
Altaras, N. E., M. R. Etzel and D. C. Cameron. 2001. Conversion of sugars to 1,2-propanediol by Thermoanaerobacterium
thermosaccharolyticum HG-8. Biotechnology Progress 17: 52-56.

Askarieh, M.M., A.V. Chambers, F.B.D. Daniel, P.L. FitzGerald, G.J. Holtom, N.J. Pilkington and J.H. Rees. 2000. The chemical and microbial degradation of cellulose in the near field of a repository for radioactive wastes. Waste Management 20: 93-106.

Bayer, E. A., H. Chanzyi, R. Lamed and Y. Shoham. 1998. Cellulose, cellulases and cellulosomes. Current Opinion in Structural Biology 8:548-557

Bertoldo, C. and G. Antranikian. 2002. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Current Opinion in Chemical Biology 6:151–160.

Bhat, M.K. and S. Bhat. 1997. Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances 15: 583-620.

Chen, J. and P. J. Weimer. 2001. Competition among three predominant ruminal cellulolytic bacteria in the absence or presence of non-cellulolytic bacteria. Microbiology 147: 21-30.

Desvaux, M., E.Guedon and H. Petitdemange. 2000. Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium. Applied and Environmental Microbiology 66: 2461-2470.

Feng, P., S. Berensmeier, K. Buchholz and P. J. Reilly. 2002. Production, purification, and characterization of Thermoanaerobacterium thermosaccharolyticum glucoamylase. Starch. Stärke 54: 328-337.

Haki,G.D. and S.K. Rakshit. 2003. Developments in industrially important thermostable enzymes: a review. Bioresource Technology 89: 17-34.

Hamelinck, C. N., G. Hooijdonk and A. P. Faaij. 2005. Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle- and long-term. Biomass and Bioenergy 28: 384-410.

Haruta, S., Z. Cui, Z. Huang, M. Li, M. Ishii and Y. Igarashi. 2002. Construction of a stable microbial community with high cellulose-degradation ability. Applied Microbiology and Biotechnology 59:529-534.

Hu, Z.H., G. Wang and H.Q. Yu. 2004. Anaerobic degradation of cellulose by rumen microorganisms at various pH values. Biochemical Engineering Journal 21:59-62.

Hu, Z.H., H.Q. Yu and R.F. Zhu. 2005. Influence of particle size and pH on anaerobic degradation of cellulose by ruminal microbes. International Biodeterioration and Biodegradation 55: 233-238.

Huang, C. Y., B. K. Patel, R. A. Mah and L. Baresi. 1998. Caldicellulosiruptor owensensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacterium. International Journal of Systematic Bacteriology 48: 91-97.

Hungate, R. E. 1969. A roll tube method for cultivation of strict anaerobes. In: J. R. Norris and D. W. Ribbons (ed.), Methods in Microbiology vol. 3b, Academic Press, Inc., New York. pp.117-132.

Ingram, O. Lonnie, A. Barbosa and D. F. Maria. 1999. Ethanol production in gram-positive microbes. US patent No. 5,916,787.

Karita, S., K. Nakayama, M. Goto, K. Sakka, W. Kim and S. Ogawa.
2003. A novel cellulolytic, anaerobic, and thermophilic bacterium, Moorella sp. strain F21. Bioscience, Biotechnology, and Biochemistry. 67:183-185.

Kato, S., S. Haruta, Z. J. Cui, M. Ishii, A. Yokota and Y. Igarashi. 2004a. Clostridium straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. International Journal of Systematic and Evolutionary Microbiology 54: 2043-2047.
Kato, S., S. Haruta, Z. J. Cui, M. Ishii and Y. Igarashi. 2004b. Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria. FEMS Microbiology Ecology 51: 133-142.

Klapatch, T. R., A. L. Demain and L. R. Lynd. 1996. Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum. Applied Microbiology and Biotechnology 45:127-131.

Kristjansson, J. K. 1992. Thermophilic bacteria. CRC Press, Inc. Boca Raton, Florida, USA. pp.120-129.

Leschine, S. B. 1995. Cellulose degradation in anaerobic environments. Annual Review of Microbiology 49: 399-426.

Leung, K and E. Topp. 2001. Bacterial community dynamics in liquid swine manure during storage: molecular analysis using DGGE/PCR of 16S rDNA. FEMS Microbiology Ecology 38: 169-177.

Liu, W. T., T. L. Marsh, H. Cheng and L. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Applied and Environmental Microbiology 63: 4516-4522.


Lovitt, R. W., G Shen and J. G. Zeikus. 1988. Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum. Journal of Bacteriology 170: 2809-2815.

Lyautey, E., B. Lacoste, L. Ten-Hage, J. Rols and F. Garabetian. 2005. Analysis of bacterial diversity in river biofilms using 16S rDNA PCR-DGGE: methodological settings and fingerprints interpretation. Water Research 39: 380-388.

Lynd, L. R., P. J. Weimer, W. H. Zyl and I. S. Pretorius. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews 66: 506-577.

Macy, J. M., J. E. Snellen and R. E. Hungate. 1972. Use of syringe methods for anaerobiosis. American Journal of Clinical Nutrition 25: 1318-1323.

Mansfield, S. D. and R. Meder. 2003. Cellulose hydrolysis – the role of monocomponent cellulases in crystalline cellulose degradation. Cellulose 10: 159-169.

McBee, R. H. 1954. The characteristics of Clostridium thermocellum. Journal of Bacteriology 67: 505-506.

Mielenz, J. R. 2001. Ethanol production from biomass: technology and commercialization status. Current Opinion in Microbiology 4: 324-329.

Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31: 426-428.

Murphy J.D. and K. McCarthy. 2005. Ethanol production from energy crops and wastes for use as a transport fuel in Ireland. Applied Energy 82: 148-166.

National Renewable Energy Laboratory. Technology Brief: Cellulose conversion key to fuel of the future, NREL improving key step in producing ethanol from biomass.

Ozkan, M., S. G. Desai1, Y. Zhang, D.M. Stevenson, J. Beane, E. A. White, M. L. Guerinot and L. R. Lynd. 2001. Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria. Journal of Industrial Microbiology and Biotechnology 27: 275-280.

Pavlostatvis, S. G., T. L. Miller and M. J. Wolin. 1988. Fermentation of insoluble cellulose by continuous cultures. Applied and Environmental Microbiology 54: 2655-2659

Prescott, L. M., J. P. Harley and D. A. Klein. 1999. Microbiology. Chapter 6. p.p 125-129.
Rani, K. S., M.V. Swamy and G. Seenayya. 1997. Increased ethanol production by metabolic modulation of cellulose fermentation in Clostridium thermocellum. Biotechnology Letters 19: 819-823.

Rani, K. S., M. V. Swamy and G. Seenayya. 1998. Production of ethanol from various pure and natural cellulosic biomass by Clostridium thermocellum strains SS21 and SS22. Process Biochemistry 33: 435-440.

Rani, D. S., S. Thirumale and K. Nand. 2004. Production of cellulase by Clostridium papyrosolvens CFR-703. World Journal of Microbiology and Biotechnology 20: 629-632.

Sato, K., S. Goto, S. Yonemura, K. Sekine, E. Okuma, Y. Takagi, K. Hon-Nami and T. Saiki. 1992. Effect of yeast extract and Vitamin B12 on ethanol production from cellulose by Clostridium thermocellum I-1-B. Applied and Environmental Microbiology 58: 734-736.

Schwarz, W. H. 2001. The cellulosome and cellulose degradation by anaerobic bacteria. Applied Microbiology and Biotechnology 56: 634-649.

Schwarz, W. H. 2004. A list of cellulolytic bacteria. http://www.wzw.tum.de/mbiotec/cellmo.htm


Stevenson, D. M. and P. J. Weimer. 2002. Isolation and characterization of a Trichoderma strain capable of fermenting cellulose to ethanol. Applied Microbiology and Biotechnology 59: 721-726.

Sun, Y and J. Cheng. 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresoure Technology 83: 1-11.

Tailliez, P., H. Girard, J. Millet and P. Beguin. 1989. Enhanced cellulose fermentation by an asporogenous and ethanol-tolerant mutant of Clostridium thermocellum. Applied and Environmental Microbiology 55: 207-211.

Tsai, W. T., Y. H. Chou and Y. M. Chang. 2004. Progress in energy utilization from agrowastes in Taiwan. Renewable and Sustainable Energy Reviews 8: 461-481.

Updegraff, D. M. 1969. Semimicro determination of cellulose in biological materials. Analytical Biochemistry 32: 420-424.

Van Wyk, J. P. H. 2002. Biodevelopment of wastepaper as a resource of renewable energy: Influence of enzyme concentration and paper amount on the bioconversion process. Energy and Fuels 16: 1277-1279.

Van Wyk, J. P. H. and M. Mohulatsi. 2003. Biodegradation of waste cellulose. Journal of Polymers and the Environment 11: 23-28.

Viles, F. J., Jr. and L. Silverman. 1949. Determination of starch and cellulose with anthrone. Analytical Chemistry 21: 950-953.

Wang, D. I. C., G. C. Avgerinos, I. Biocic, S. D. Wang and H. Y. Fang. 1983. Ethanol from cellulosic biomass. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 300: 323-333.

Weimer, P. J. 1996. Why don’t ruminal bacteria digest cellulose faster? Journal of Dairy Science 79: 1496-1502.

Yu, Z. and H. Zhang. 2003. Pretreatments of cellulose pyrolysate for ethanol production by Saccharomyces cerevisiae, Pichia sp. YZ-1 and Zymomonas mobilis. Biomass and Bioenergy 24: 257-262.

行政院環保署。一般事業廢棄物處理後續推動方案。民國九十一年一月。

吳敬謙。2003。懸掛式固定化纖為酵素反應器之廢紙纖維素水解與醣類產物分析。中原大學化學系研究所碩士論文。

吳耿東,李宏台。2004。生質能源化腐朽為能源。科學發展,383期,pp.20-27。


吳晟。2004a。國際能源新寵-甘蔗「酒精汽油」。能源報導,2004年1月,pp.25-28。

吳晟。2004b。明日綠色能源之星-氫能源。能源報導,2004年2月,pp.33-36。

李堅明。2005。京都議定書生效後台灣二氧化碳減量整體因應策略。能源報導,2005年7月,pp.8-10。

曹祖寧。1998。再生資源的利用及生物工程。化工技術,11期,pp.122-128。

黃其聰。2004。醇類燃料對汽油引擎排氣分析研究。中原大學機械工程學系研究所碩士論文。

郭博堯。2002。全球化石能源危機時代與我國所面臨挑戰。財團法人國家政策研究基金會國政報告。

經濟部能源局。再生能源發展方案。民國九十一年一月。

路明。2004。開發生物質能發展能源農業。http://new.cast.org.cn/saomiao/neirong/110100537112.shtml

廖經裕。2004。以分子生物方法與培養方式分析台灣兩處溫泉之嗜熱菌族群。東海大學環境科學系研究所碩士論文。

蔡信行。2003。替代燃料與再生能源。科學發展,365期,pp.62-67。

賴嫈苓。2005。京都風向球全球生物能源的市場趨勢。能源報導,2005年5月,pp.28-30。

顧洋。2005。國際的環境議題。科學發展,387期,pp.32-37。
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