探討黃豆粉與溫度對Aspergillus terreus利用 酵素糖化稻稈生產衣康酸之影響

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黃緬雄(Mian-Shiung Huang) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

探討黃豆粉與溫度對Aspergillus terreus利用酵素糖化稻稈生產衣康酸之影響

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摘要(中)

衣康酸 (Itaconic acid) 由於其具有特殊結構特性，因此被廣泛應用於民生工業上，用來製造人造纖維、樹酯及其他合成材料，甚至具有取代石化原料的潛力。Aspergillus terreus是目前已知具有較高衣康酸產率的真菌，由於過去主要針對衣康酸代謝途徑、pH值、通氣量及反應器等進行探討，但對於中高溫度以及農業廢棄物糖化還原糖之利用的研究較少，因此為本研究之重點。

首先以具有能生產較高活性之酵素的真菌，Trichoderma reesei利用經過鹼處理之稻稈作為誘導基質，在30°C下生產纖維水解酵素，相較於豆渣，以豆粉作為氮源能得到較高活性之酵素，得到內切、外切型纖維酵素及β-glucosidase酵素活性各為0.193 IU/mL、0.152 IU/mL、0.146 IU/mL，filter paper activity為0.754 FPU/mL。接著在稻稈糖化階段，若增加稻稈添加量則會使糖化效率降低，在50°C下糖化20、40及60 g/L的稻稈，得到各別19.14、32.88及45.13 g/L的還原糖，轉化率各為84.6、73.3及66.6%。

在衣康酸發酵實驗部分，若接菌量從10%增加至30%，衣康酸產率能夠被提高至0.826 g/L day，約3.4倍。相較於 (NH4)2SO4與CSL，以豆粉作為氮源，在40°C下進行衣康酸發酵能得到較高的衣康酸轉化率，在第8天能得到0.22 g/g的單位基質衣康酸轉化率 (Yp/s)，一康酸產率為1.28 g/L day。而初始葡萄糖為50 g/L時，若將豆粉濃度從2.1g/L減半至1.05g/L，葡萄糖與豆粉比率為47.62，衣康酸產率能被提高至1.57 g/L day，約提高22.7%，Yp/s為0.3 g/g。

最後透過限制氮源，以32 g/L糖化還原糖及0.67 g/L豆粉進行衣康酸發酵，得到菌量為6.16 g/L，衣康酸產率為0.4 g/L day，雖然相較於葡萄糖控制組略低，但還原糖也能有效的被利用。

摘要(英)

Itaconic acid (IA) has been declared to be one of the most promising and flexible building blocks derived from biomass, it is a monomer for the manufacturing of synthetic fibers, synthetic resins, plastics, rubbers or other polymeric materials.

A. terreus strains can achieve high product yields of IA by utilizing glucose. For several decades, some studies focus on metabolic pathway for IA production, pH, oxygen supply and bioreactor. However, the investigation of mesophilic or thermophilic temperature and utilizing rice straw for IA fermentation is deficient.

At present, T. reesei has been reported as the excellent cellulose producer. So first, the rice straw by alkaline pretreatment and soybean powder is used for cellulase production by T. reesei Rut C-30 at 30°C, the crude enzyme with the highest activity of CMCase, Avicelase and β-glucosidase was 0.193, 0.152 and 0.146 IU/mL, and the filter paper activity was 0.754 FPU/mL. The crude enzyme is used for saccharification of 20、40 and 60 g/L rice straw at 50°C, it obtains 19.14、32.88 and 45.13 g/L reducing sugar. The saccharification efficiency is 84.6、73.3 and 66.6% respectively.

In IA fermentation, the inoculum from 10 to 30% directly increases the IA productivity 3.4 times, it were obtained 0.826 g/L day. Using soybean powder as nitrogen source is better than (NH4)2SO4 and CSL at 40°C, the higher IA productivity and Yp/s, can be obtained 0.22 g IA/g glucose and 1.28 g/L day, respectively. If using half of 2.1 g/L soybean powder, the IA productivity can be increased to 1.57 g/L day and Yp/s is 0.3 g/g. At last, 32 g/L reducing sugar and 0.67 g/L soybean powder is used for IA production by nitrogen limitation, the IA productivity is 0.4 g/L day, it slightly less than the control group of glucose.

關鍵字(中)

★ 衣康酸
★ 纖維水解酵素
★ 糖化
★ 稻稈
★ 黃豆

關鍵字(英)

★ Itaconic acid
★ Cellulase
★ Saccharification
★ Rice straw
★ soybean

論文目次

摘要 I

ABSTRACT II

目錄 III

圖目錄 VII

表目錄 X

第一章、緒論 1

1-1 研究動機 1

1-2 研究目的 2

第二章、文獻回顧 3

2-1 衣康酸 3

2-1.1 衣康酸簡介 3

2-1.2 衣康酸來源 3

2-1.3 衣康酸之應用 5

2-1.4 衣康酸之市場現況 6

2-1.5 衣康酸發酵問題 7

2-2 木質纖維素水解 10

2-2.1 木質纖維素簡介 10

2-2.2 木質纖維素前處理 13

2-2.3 纖維水解酵素簡介 16

2-2.4 纖維水解酵素之來源 18

2-2.5 纖維素糖化之環境參數 19

2-3 真菌 20

2-3.1 真菌簡介 20

2-3.2 真菌分類 20

2-3.3 土麴黴 (Aspergillus terreus) 簡介 21

2-3.4 土麴黴發酵生產衣康酸之生物合成途徑 23

2-3.5 裡氏木黴 (Trichoderma reesei) 簡介 25

2-4影響衣康酸發酵之因素 26

2-4.1 培養基組成 26

2-4.2 溫度 28

2-4.3 pH值 28

2-4.4 通氣量及攪拌速率 29

第三章、實驗材料與方法 30

3-1 實驗規劃 30

3-2 實驗材料 32

3-2.1 實驗菌株 32

3-2.2 實驗藥品 33

3-2.3 實驗儀器及設備 35

3-3 實驗方法 37

3-3.1 稻稈前處理 37

3-3.2 菌種保存 38

3-3.3 培養基組成 39

3-3.4 纖維水解酵素生產 42

3-3.5 糖化稻稈累積還原糖 43

3-3.6 衣康酸發酵實驗 44

3-3.7 糖化及衣康酸發酵最適化操作 45

3-4 分析方法 47

3-4.1 發酵液分析流程圖 47

3-4.2 菌重分析 47

3-4.3 還原糖定量分析 48

3-4.4 酵素活性分析 49

3-4.5 糖化效率計算公式 50

3-4.6 衣康酸濃度分析 50

第四章、結果與討論 53

4-1纖維水解酵素生產 53

4-1.1 種瓶碳源實驗 53

4-1.2 豆渣及豆粉在酵素生產階段之比較 55

4-1.3 A. terreus酵素活性測試 57

4-2 糖化稻稈累積還原糖 58

4-2.1 糖化時機 58

4-2.2 不同pH對酵素糖化稻稈之影響 60

4-2.3 溫度對於糖化稻稈之影響 62

4-2.4 不同稻稈添加量之糖化效率 63

4-3 衣康酸發酵實驗 64

4-3.1 接種量對衣康酸發酵之影響 64

4-3.2 不同氮源對衣康酸發酵之影響 69

4-3.3 不同溫度對衣康酸發酵之影響 71

4-3.4 不同豆粉濃度對衣康酸發酵之影響 75

4-4 衣康酸發酵測試 78

4-4.1 還原糖利用之衣康酸發酵測試 78

4-4.2 還原糖利用及限制氮源之衣康酸發酵測試 80

第五章、結論與建議 83

5-1 結論 83

5-2 建議 85

參考文獻 86

附錄一 91

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指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2015-8-27

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