臺灣博碩士論文加值系統

本研究以狼尾草和豆渣為基質(substrate)，進行木黴菌(Trichoderma reesei ATCC 56765)及黑麴黴菌(Aspergillus niger ATCC 10864)的固態培養生產纖維素酶(cellulase)，並以狼尾草為基質測試粗酵素萃取液之水解糖化效果。為了改善木黴菌本身β-葡萄糖苷酶(β-glucosidase)不足的缺點，我們測試兩種策略：(1)混合培養，即木黴菌/黑麴黴菌的共培養; (2)粗酵素液混合，即分別培養木黴菌與黑麴黴菌，萃取其粗酵素液，再以等體積混合粗酵素液。研究結果顯示，兩種策略皆可提升β-葡萄糖苷酶活性，採混合培養時，纖維素水解酶與β-葡萄糖苷酶活性可分別達13.1 ± 1.3 FPU/g dry substrate與 13.9 ± 1.2 CBU/g dry substrate，而混合粗酵素液的纖維素水解酶與β-葡萄糖苷酶活性可分別達 12.4 ± 1.1 FPU/g dry substrate與 21.9 ± 0.5 CBU/g dry substrate。
隨後我們在10 FPU/g-glucan的酵素劑量與2 wt%的基質濃度下，測試粗酵素萃取液水解糖化狼尾草的效能，並與市售酵素液作比較。我們使用三種不同狼尾草物料，包含未經預處理的原始物料(pristine)、鹼性(5 % NaOH)和鹼性過氧化氫(alkaline hydrogen peroxide)預處理過的狼尾草，後二者有較高的葡聚醣(glucan)與較低的木質素(lignin)含量，有助於纖維素的水解。研究結果顯示，相較於市售酵素液，自產之粗酵素液有較為優越的水解糖化效果，其中又以粗酵素混合液的效果最佳，其葡萄糖產率可達市售酵素液的兩倍以上，水解基質為鹼性過氧化氫預處理過的狼尾草時，葡萄糖產率可達44 %。

Napier grass and okara (or soy pulp) were used as the substrate for the production of cellulolytic enzymes by Trichoderma reesei (ATCC 56765) and Aspergillus niger (ATCC 10864) via solid-state fermentation. To overcome the low -glucosidase activity problem of T. reesei monoculture, two strategies were investigated: (1) mix culture approach, i.e., co-cultivation of T. reesei and A. niger; (2) enzyme mixture approach, i.e., mixing crude enzyme extracts from monocultures of T. reesei and A. niger. Both approaches were able to increase the level of -glucosidase activity compared to T. reesei monoculture. The cellulosic enzyme activities achieved by the mix culture approach were 13.1 ± 1.3 FPU/g dry substrate and 13.9 ± 1.2 CBU/g dry substrate, while the enzyme mixture approach attained 12.4 ± 1.1 FPU/g dry substrate and 21.9 ± 0.5 CBU/g dry substrate.
The hydrolysis efficacy of our crude enzyme extracts was compared to commercial enzyme preparations at a cellulase loading of 10 FPU/g-glucan and 2 wt% substrate concentration. Three different substrates were used, i.e., pristine (un-pretreated), alkaline pretreated (AP), and alkaline hydrogen peroxide pretreated (AHP) napier grass. The pretreatments partially removed lignin and increased the proportion of cellulose in the substrates, which helped the conversion of glucan. Our crude enzyme extracts, whether from the mix culture or the enzyme mixture approach, show higher hydrolysis efficiency than the commercial enzyme preparations. The highest glucose yield of 44 % was obtained using AHP napier grass as the substrate and the crude extract from the enzyme mixture approach, which was more than twice the glucose yield achieved by the commercial enzyme preparations.

摘要 i
Abstract iii
誌謝 v
目錄 vi
表目錄 xi
圖目錄 xii
第一章 緒論 1
1.1 前言 1
1.2 研究動機 3
1.3 研究目的 4
第二章 文獻回顧 5
2.1 生質能源的定義 5
2.2 狼尾草 7
2.3 米糠 8
2.4 豆渣 9
2.5 木質纖維素 10
2.5.1 纖維素 12
2.5.2 半纖維素 14
2.5.3 木質素 15
2.6 木質纖維素前處理簡介 17
2.6.1 物理性前處理法 18
2.6.1.1 機械粉碎前處理法 18
2.6.1.1 熱裂解前處理法 18
2.6.2 化學性預處理法 18
2.6.2.1 酸水解預處理法 18
2.6.2.2 鹼水解預處理法 18
2.6.2.4 鹼性過氧化氫去木質素預處理法 20
2.6.2.4 臭氧解預處理法 20
2.6.3 物理/化學性預處理法 20
2.6.3.1 蒸煮爆碎法 20
2.6.3.2 濕氧爆碎法 22
2.6.3.3 鹼性蒸煮爆碎法 23
2.6.4 生物性前處理法 23
2.7 酵素水解 24
2.7.1 纖維素酵素 24
2.7.2 纖維素酵素的種類及作用方式 26
2.7.2.1 內切型纖維水解酶 26
2.7.2.2 外切型纖維水解酶 26
2.7.2.3 纖維雙醣水解酶 26
2.7.2.4 纖維水解酶的作用方式 26
2.7.3 木聚醣酵素 28
2.7.4 木聚醣酵素的種類及作用方式 28
2.7.4.1 內切型木聚醣酶 28
2.7.4.2 外切型木聚醣酶 28
2.8 影響酵素水解的關鍵因子 29
2.9 固態發酵 30
2.9.1 固態發酵菌種分類 30
2.9.2 菌種混合培養 32
第三章 材料與方法 34
3.1 實驗儀器 34
3.2 實驗藥品與材料 35
3.2.1 藥品 35
3.2.2 菌株 37
3.2.2.1 菌株保存 37
3.2.2.2 血球計數器計數 37
3.2.3 固態發酵基質 38
3.2.3.1 狼尾草 38
3.2.3.2 米糠 38
3.2.3.3 豆渣 39
3.3 實驗流程 40
3.3.1 固態發酵基質 41
3.3.2 菌株 41
3.3.3 酵素水解基質 42
3.4 菌株兼容性測試 43
3.4.1 接種方式A 43
3.4.2 接種方式B 44
3.4.3 接種方式C 44
3.5 固態發酵 45
3.5.1 固態發酵氮源改變 45
3.5.2 狼尾草和豆渣混合培養 45
3.5.3 不同種菌株混合培養 45
3.5.4 酵素萃取 46
3.6 酵素水解 47
3.6.1 酵素水解基質 47
3.6.1.1 鹼性預處理 47
3.6.1.2 過氧化氫去木質素鹼性蒸爆預處理 47
3.6.2 酵素水解步驟 48
3.7 分析方法 49
3.7.1 狼尾草成分分析 49
3.7.1.1 乾重測試 ( NREL Lap-001規範 ) 49
3.7.1.2 碳水化合物分析 ( NREL Lap-002規範 ) 50
3.7.1.3 酸不可溶木質素分析 ( NREL Lap-003規範 ) 52
3.7.1.4 酸可溶木質素分析 52
3.7.1.5 灰分分析 ( NREL Lap-005規範 ) 53
3.7.2 酵素活性測定 54
3.7.2.1 葡萄糖標準檢量線 54
3.7.2.2 p-nitrophenol標準檢量線 55
3.7.2.3 木糖標準檢量線 55
3.7.2.4 纖維素水解酶檢測 ( Cellulase ) 56
3.7.2.5 Beta-葡萄糖苷酶測定 ( β-glucosidase ) 57
3.7.2.6 木聚醣水解酶測定 ( Xylanase ) 58
3.8 高效能液相層析儀分析原理 59
第四章 結果與討論 61
4.1 固態發酵基質探討 61
4.1.1 狼尾草的組成 61
4.1.2 米糠的組成 62
4.1.3 豆渣的組成 62
4.2 以木黴菌進行固態發酵 63
4.2.1 氮源種類對酵素活性的影響 63
4.2.2 固態基質比例對酵素活性影響 64
4.2.3 培養天數對酵素活性影響 66
4.3 兼容性測試 67
4.3.1 接種方式A 67
4.3.1.1 豆渣 67
4.3.1.1 米糠 68
4.3.2 接種方式B 68
4.3.2.1 豆渣 68
4.3.2.2 米糠 69
4.3.3 接種方式C 69
4.3.3.1 豆渣 69
4.3.3.2 米糠 70
4.3.4 兼容性評估 70
4.4 以黑麴黴進行固態發酵 71
4.5 混合培養 72
4.6 粗酵素液混合 74
4.7 酵素水解 75
4.7.1 酵素水解基質探討 76
4.7.2 無處理狼尾草水解情形 77
4.7.2.1 葡萄糖 77
4.7.2.2 木糖 78
4.7.3 鹼性預處理狼尾草水解情形 79
4.7.3.1 葡萄糖 79
4.7.3.2 木糖 80
4.7.4 過氧化氫去木質素鹼性蒸爆預處理狼尾草水解情形 81
4.7.4.1 葡萄糖 81
4.7.4.1 木糖 82
4.7.5 商業酵素液水解情形 83
4.7.6 還原糖分析 85
第五章 結論 87
參考文獻 89

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