探討以Aspergillus niger分解稻桿及Saccharomyces cerevisiae生產生質酒精之研究

簡易檢索 / 詳目顯示

回結果列表

研究生：	黃柏傑 Po-Chieh Huang
論文名稱：	探討以Aspergillus niger分解稻桿及Saccharomyces cerevisiae生產生質酒精之研究
指導教授：	徐敬衡 Chin-Hang Shu
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	91
中文關鍵詞：	稻稈、生質酒精、纖維水解酵素、黑麴黴、酵母菌
外文關鍵詞：	Aspergillus niger, Saccharomyces cerevisiae, bioethanol, cellulase, rice straw
相關次數：	點閱：12 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

近年來由於能源危機和石油價格高漲，以農業廢棄物做為基質生產生質酒精為研究議題漸受重視，也有許多文獻及研究成果，足以說明生質能源目前占有的地位。
本研究透過固態發酵培養Aspergillus niger，大量生產纖維水解酵素，並於醣化步驟中提出有別於傳統的醣化方法。傳統的醣化方法為添加純化的纖維水解酵素，近來亦有自行生產酵素的操作，但仍會經過粗製的純化或是分離菌體的步驟。在本研究中提出控制環境參數以省略上述步驟，抑制菌體生長及使還原糖累積，達到節省成本之效果。在醣化步驟中發現纖維水解酵素為醣化的主要因素，進而提出延長酵素活性之方法。最後用醣化後發酵液利用Saccharomyces cerevisiae進行酒精發酵。
由實驗結果顯示，固態發酵能生產較高單位的纖維水解酵素，利用黃豆及稻稈培養最高可得到23.85 U/gdm，單位基質對酵素活性轉化率最高可到12.88 U/gds。第二階段醣化步驟中可藉由提升溫度使酵素活性增高並抑制Aspergillus niger生長；及藉由厭氧在第三階段中抑制Aspergillus niger利用還原糖。在醣化步驟中加入20g/L經鹼處理過的稻稈可得到最高11.58 g/L的還原糖，轉化效率為58.42 ％。實驗亦說明酵素活性是影響還原糖累積的主要因素。透過添加PMSF或持續添加自行生產的酵素可延長酵素活性。而應用於酒精發酵，可得最大酒精濃度2.00 g/L，轉化效率為86.71 ％。

The strain used in this study was Aspergillus niger which could product lots of cellulase by solid state fermentation. Traditional saccharification method used commercial purified cellulase. In the recent year, there were some operating which producing celluase from microorganisms, but it still need to extract crudely or separate the microorganism. A novel method in saccharification step is proposed to easily operate in this study. By controlling environmental parameters like temperature or oxygen to inhibit the growth of Aspergillus niger, the culture resulted in the accumulation of reducing sugar. The operation not only remove the complex steps but also achieve the target of cost down. Cellulase activity is the main controlling factor in the saccharification step, thus we proposed to extend the method of cellulase activity. Finally, ethanol was obtained by Saccharomyces cerevisiae after saccharification fermentation broth.
The experimental results show that solid-state fermentation can produce higher unit cellulase activity, and the highest cellulase activity obtained as 23.85 U/gdm by soybean and rice straw. Saccharification step could be utilized to enhance the enzyme activity and to inhibit the growth of Aspergillus niger by increasing temperature. Anaerobic operating can inhibit Aspergillus niger as a result that the reducing sugar was produced at the second step. In the saccharification step, adding 20 g/L of alkali-treated rice straw can obtain the highest 11.58 g / L of reducing sugar, the conversion efficiency was 58.42%. Adding PMSF as a protease inhibitor or adding more cellulase was can extend the cellulase activity. Applied to the fermentation of ethanol, the maximum ethanol concentration can be obtained as 2.00 g / L and the conversion efficiency as 86.71%.

摘要    i
Abstract    ii
誌謝    iii
目錄    iv
圖目錄    viii
表目錄    x
第一章 緒論    1
1-1 研究動機    2
1-2 研究目的    2
第二章 文獻回顧    3
2-1 木質纖維素    3
2-1-1 木質纖維素簡介    3
2-1-2 木質纖維素之前處理    4
2-2 纖維水解酵素    8
2-2-1 纖維水解酵素簡介    8
2-2-2 纖維水解酵素來源    11
2-2-3 纖維水解酵素適合之環境參數    13
2-3 黑麴黴(Aspergillus niger)    13
2-4 固態發酵    14
2-5 再生能源    16
2-5-1 再生能源簡介    16
2-5-2 生質能源    16
2-5-3 生質乙醇    17
2-6 釀酒酵母(Saccharomyces cerevisiae)    19
第三章  實驗規劃、材料與方法    23
3-1 實驗流程設計    23
3-2 實驗材料    24
3-2-1 實驗菌株    24
3-2-2 實驗藥品    26
3-2-3 實驗設備    28
3-3 實驗方法    29
3-3-1 菌種保存    29
3-3-2 培養基組成    29
3-3-3 種菌操作條件    32
3-3-4 稻稈之前處理    33
3-3-5 纖維水解酵素生產    33
3-3-6 醣化生產還原糖    34
3-3-7 Saccharomyces cerevisiae發酵生產酒精    37
3-4 分析方法    38
3-4-1 還原糖定量    38
3-4-2 酵素活性測試    39
3-4-3 葡萄糖濃度分析    41
3-4-4 乙醇濃度分析    42
3-4-5 菌重定量    42
第四章 實驗結果與討論    45
4-1 纖維水解酵素生產    45
4-1-1 液態發酵槽實驗    45
4-1-1 固態發酵槽實驗    46
4-2 醣化生產還原糖    53
4-2-1 不同溫度下酵素活性測試    53
4-2-2 環境參數對Aspergillus niger之影響    54
4-2-3 基質處理與否對醣化之影響    57
4-2-4 不同濃度之稻稈醣化結果    59
4-2-5 還原糖無法持續上升之原因    60
4-2-6 探討延長酵素活性之方法    62
4-3 Saccharomyces cerevisiae發酵生產酒精    66
4-3-1 以不同基質醣化應用於酒精生產    66
4-3-2以不同濃度稻稈醣化應用於酒精生產    67
第五章 結論與建議    70
5-1 結論    70
5-2 建議    71
第六章 參考文獻    72

                                

1. Bansal, N., Tewari, R., Soni, R., and Soni, S.K., Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues. Waste Management, 2012. 32(7): p. 1341-6.

2. McMillan, J.D., Enyzmatic Conversion of Biomass for Fuels Production. Vol. 566. 1994, Washington: American Chemical Society.

3. Tanaka, M., Matsuno, R., and Converse, A.O., N-Butylamine and acid-steam explosion pretreatments of rice straw and hardwood: Effects on substrate structure and enzymatic hydrolysis. Enzyme and Microbial Technology, 1990. 12(3): p. 190-195.

4. Sun, Y. and Cheng, J., Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 2002. 83(1): p. 1-11.

5. Sánchez, C., Lignocellulosic residues: Biodegradation and bioconversion by fungi. Biotechnology Advances, 2009. 27(2): p. 185-194.

6. Taiz, L. and Zeiger, E., Cell walls: structure, biogenesis, and expansion. Plant Physiology. Sinauer Associates, Sunderland, MA, 1998: p. 409-443.

7. Kim, J.Y., Hur, S.H., and Hong, J.H., Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810. Biotechnol Lett, 2005. 27(5): p. 313-6.

8. Lee, B.H., Kim, B.K., Lee, Y.J., Chung, C.H., and Lee, J.W., Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53. Enzyme and Microbial Technology, 2010. 46(1): p. 38-42.

9. Bisaria, V.S., Mishra, S., and Eveleigh, D.E., Regulatory Aspects of Cellulase Biosynthesis and Secretion. Critical Reviews in Biotechnology, 1989. 9(2): p. 61-103.
10. Sohail, M., Siddiqi, R., Ahmad, A., and Khan, S.A., Cellulase production from Aspergillus niger MS82: effect of temperature and pH. N Biotechnol, 2009. 25(6): p. 437-41.

11. Sukumaran, R.K., Singhania, R.R., Mathew, G.M., and Pandey, A., Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production. Renewable Energy, 2009. 34(2): p. 421-424.

12. Béguin, P., Gilkes, N.R., Kilburn, D.G., Miller, R.C., O'neill, G.P., and Warren, R.A.J., Cloning of Cellulase Genes. Critical Reviews in Biotechnology, 1987. 6(2): p. 129-162.

13. Uusitalo, J.M., Helena Nevalainen, K.M., Harkki, A.M., Knowles, J.K.C., and Penttilä, M.E., Enzyme production by recombinant Trichoderma reesei strains. Journal of Biotechnology, 1991. 17(1): p. 35-49.

14. Bollók, M. and Réczey, K., Cellulase enzyme production by various fungal strains on different carbon sources. Acta Alimentaria, 2000. 29(2): p. 154-168.

15. Tamás Juhász*, K.K., Zsolt Szengyel and Kati Réczey, Production of β-Glucosidase in Mixed Culture of Aspergillus niger BKMF 1305 and Trichoderma reesei RUT C30. Food Technol. Biotechnol., 2003. 41(1): p. 49-53.

16. 林文仕, 利用Bacillus sp. 分解纖維素進行兩階段式發酵生產特用化學品, 化學工程與材料工程研究所, 國立中央大學, 碩士論文, 2012

17. Schlochtermeier, A., Walter, S., Schröder, J., Moorman, M., and Schrempf, H., The gene encoding the cellulase (Avicelase) Cell from Streptomyces reticuli and analysis of protein domains. Molecular Microbiology, 1992. 6(23): p. 3611-3621.

18. Alexander, M., Introduction to Soil Microbiology, 1977, John Wiley & Sons: New York.

19. Schinner, F. and Mersi, W.v., Xylanase-,CM-cellulase- and Invertase Actvity in Soil: an Improved Method. Soil Biol. Biochem, 1990. 22(4): p. 511-515.

20. Sattler, K., Schimmelpilze – Lebensweise Nutzen Schaden Bekämpfung. Berlin: Springer-Verlag, 1986, 230S., 69 Abb., 55 Tab., 58 DM. Acta Biotechnologica, 1987. 7(5): p. 446-446.

21. Schuster, E., Dunn-Coleman, N., Frisvad, J.C., and Van Dijck, P.W., On the safety of Aspergillus niger--a review. Appl Microbiol Biotechnol, 2002. 59(4-5): p. 426-35.

22. Bhargav, S., Panda, B.P., Ali, M. and Javed S., Solid-state Fermentation: An Overview. Chem. Biochem. Eng. Q., 2008. 22(1): p. 49-70.

23. Sato, K. and Sudo, S., Small-scale solid-state fermentations. Manual of industrial microbiology and biotechnology, 1999. 2: p. 61-63.

24. Mussatto, S.I., Dragone, G., Guimarães, P.M.R., Silva, J.P.A., Carneiro, L.M., Roberto, I.C., Vicente, A., Domingues, L., and Teixeira, J.A., Technological trends, global market, and challenges of bio-ethanol production. Biotechnology Advances, 2010. 28(6): p. 817-830.

25. 黃俊凱, 探討光照對Saccharomyces cerevisiae生產乙醇之影響, 化學工程與材料工程研究所, 國立中央大學, 碩士論文, 2008

26. 黃浩宸, 探討可控式包埋Saccharomyces cerevisiae對於乙醇醱酵之影響化學工程與材料工程研究所, 國立中央大學, 碩士論文, 2011

27. Ingledew, W.M., Alcohol production by Saccharomyces cerevisiae: a yeast primer, L. T.P., J. K.A., and K. D.R, Editors. 1999, Nottingham University Press: United Kingdom.

28. Romero, M.D., Aguado, J., Gonza'lez, L., and Ladero, M., Cellulase production by Neurospora crassa on wheat straw. Enzyme and Microbial Technology, 1999. 25: p. 244-250.

29. Miller, G.L., Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 1959. 31(3): p. 426-428.

30. Ghose, T.K., Measurement of Cellulase Activities. Pure & Appl. Chem., 1987. 59(2): p. 257-268.

31. Tsuji, A., Kinoshita, T., and Hoshino, M., Analytical chemical studies on amino sugars. II. Determination of hexosamines using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Chem Pharm Bull (Tokyo), 1969. 17(7): p. 1505-10.

32. Cunha, F.M., Esperanca, M.N., Zangirolami, T.C., Badino, A.C., and Farinas, C.S., Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulase. Bioresour Technol, 2012. 112: p. 270-4.

33. Suto, M. and Tomita, F., Induction and catabolite repression mechanisms of cellulase in fungi. J Biosci Bioeng, 2001. 92(4): p. 305-11.

34. Liu, Y.K., Yang, C.A., Chen, W.C., and Wei, Y.H., Producing bioethanol from cellulosic hydrolyzate via co-immobilized cultivation strategy. Journal of Bioscience and Bioengineering, 2012. 114(2): p. 198-203.

35. 施智雄, 利用鹼和Aspergillus niger處理稻稈以提升甲烷生產於厭氧共發酵系統, 化學工程與材料工程研究所, 國立中央大學, 碩士論文, 2013
36. Li, Y., Park, J., Shiroma, R., and Tokuyasu, K., Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation. Journal of Bioscience and Bioengineering, 2011. 111(6): p. 682-686.

簡易檢索 / 詳目顯示

相關論文