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

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黃柏傑(Po-Chieh Huang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

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

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摘要(中)

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 ％。

摘要(英)

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%.

關鍵字(中)

★ 稻稈
★ 生質酒精
★ 纖維水解酵素
★ 黑麴黴
★ 酵母菌

關鍵字(英)

★ Aspergillus niger
★ Saccharomyces cerevisiae
★ bioethanol
★ cellulase
★ rice straw

論文目次

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

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指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2013-7-29

推文