探討以混合菌固態發酵生產酵素並用於分解稻桿以生產衣康酸之研究

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研究生：	林家煒 Chia-wei Lin
論文名稱：	探討以混合菌固態發酵生產酵素並用於分解稻桿以生產衣康酸之研究
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：	2014
畢業學年度：	102
語文別：	中文
論文頁數：	119
中文關鍵詞：	衣康酸、稻桿、纖維酵素、水解、固態發酵
外文關鍵詞：	itaconic acid, rice straw, cellulase, hydrolysis, solid-state fermentation
相關次數：	點閱：12 下載：0
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衣康酸(Itaconic acid)為一種用途廣泛的化學物，常被作為單體應用於製造塑膠、樹脂及橡膠等製品，也常被作為添加劑用於除草劑、乳化劑及高分子螯合劑等，是一種應用價值高並且具有發展潛力的一種化學物質。但目前的生產流程陷入瓶頸，原因是作為原料的葡萄糖成本過高，限制了衣康酸的經濟價值發展，因此若能找尋到較為便宜的原料以生產衣康酸，勢必可以解決這個問題，使衣康酸的經濟價值提高，能夠被更進一步的廣泛利用。
台灣早期為一個農業為主的國家，水道為台灣的第一大宗作物，每年約產生300萬噸的廢棄稻稈，若能利用這些屬於木質纖維材料的廢棄物—稻稈，並使用菌種生產脂纖維酵素做分解，將之做為原料生產衣康酸，則可以大幅降低衣康酸的生產成本，達到節省成本的目的。
本研究即是，利用黃豆加鹼處理過後之稻稈為原料，分別培養接種Aspergillus niger及Trichoderma ressei進行固態發酵，結束後將發酵殘餘物以適當比例做混合，添加檸檬酸緩衝溶液進行萃取，可得到三種纖維水解酵素之最高酵素濃度為3.88、3.95及4.11 U/ml，並發現使用稻稈作為誘導劑，可提升月46%的酵素產量。
接下來，將所得固液混合物，不經過繁瑣的過濾、濃縮等步驟，直接於攝氏50度下進行水解反應，可得最高的還原醣濃度為25.16 g/L。並發現酵素濃度高於1.3 U/ml時，可大幅提升水解效果。最後，將所得醣類進行衣康酸發酵，可得最高之衣康酸濃度為2.89g/L，轉化率為 23.1%。

Biotechnologically produced itaconic acid (IA) is a promising organic acid with a wide range of applications. But the cost problem of the raw material limited its capability of newer and wider applications.
In this study, agricultural waste—straws were hydrolyzed and used to replace the relatively expensive glucose or molasses in IA production. Solid-state fermentation was carried out with Trichoderma ressei Rut C-30 and Aspergillus niger BCRC 31130 separated-cultured at 28°C. 7.5ml/g sub of 50mM citric acid buffer solution were used to extract enzyme from SSF residual. The crude enzyme without any purification and concentration with the highest enzyme activity of exoglucanase, endoglucanase and β-glucosidase was 3.88, 3.95 and 4.11 U/ml, respectively. Using straws as precursor could increase 46% of the enzyme yield. Hydrolysis was carried out at 50°C for 72h to get the highest cumulative sugar concentration of 25.16 g/L and the decomposition ratio of 88.1%. Additionally, we found enzyme activity of 1.3 U/ml for three kinds of cellulases was needed to promote the decomposition ratio dramatically. IA fermentation was carried out in a 2-L airlift fermenter with the sugar gain from hydrolysis and obtained the highest IA concentration of 2.89g/L.
The results showed the feasibility of using straws as a replacement of molasses or glucose to decrease the cost of IA production, and it’s a valuable and potential subject worth of putting effort in.

第一章 緒論    1
1-1    研究動機    1
1-2    研究目的    3
第二章、文獻回顧    5
2-1 衣康酸    5
2-1.1 衣康酸簡介    5
2-1.2 衣康酸之應用    6
2-1.3 衣康酸來源    7
2-1.4 土麴黴 (Aspergillus terreus) 之簡介    8
2-1.5 衣康酸的近況發展    8
2-1.5 衣康酸成本的改進方向[8]    10
2-2 木質纖維素    14
2-2-1稻稈    14
2-2-2 木質纖維素簡介    14
2-2-3 木質纖維素之前處理    16
2-3 纖維水解酵素    20
2-3-1 纖維水解酵素簡介    20
2-3-2 纖維水解酵素來源    23
2-3-3 纖維水解酵素適合之環境參數    26
2-4裡氏木黴 (Trichoderma ressei)    26
2-5 黑麴黴 (Aspergillus niger)    27
2-6 固態發酵    28
第三章、實驗規劃、材料與方法    30
3-1 實驗流程規劃    30
3-2 實驗材料    31
3-2-1 實驗菌株    31
3-2-2 實驗藥品    33
3-2-3 實驗設備    34
3-3 實驗方法    35
3-3-1 菌種保存    35
3-3-2 培養基組成    35
3-3-3 種菌操作條件    39
3-3-4 稻稈之前處理    40
3-3-5 標準衣康酸發酵試驗    41
3-3-6確認稻稈是否能生產衣康酸    42
3-3-7 固態發酵生產纖維水解酵素    43
3-3-8 醣化階段的調控    46
3-3-9大型發酵槽實驗—葡萄糖    47
3-3-10 衣康酸發酵確認最佳化條件    48
3-3-11 大型發酵槽實驗    49
3-4 分析方法    50
3-4-1 還原糖定量[40]    50
3-4-2 酵素活性測試[23, 41]    52
3-4-4 衣康酸含量分析    54
第四章、實驗結果與討論    56
4-1 衣康酸發酵    56
4-2 確認稻稈是否能生產衣康酸    57
4-3 固態發酵生產纖維水解酵素    59
4-3-1 共培養與分開培養再混合之探討    60
4-3-2 稻稈添加時機的探討    64
4-3-3 萃取酵素方法與添加比例之探討    67
4-4 醣化階段的調控    76
4-4-1 商業酵素之醣化動力曲線    77
4-4-2 自行生產水解酵素之醣化動力曲線    83
4-5 大型發酵槽實驗—葡萄糖    90
4-6 衣康酸發酵確認最適化條件    92
4-7 大型發酵槽實驗    96
第五章 結論與建議    99
5-1 結論    99
5-2 建議    101
第六章 參考文獻    102

                                

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