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研究生:黃郁菁
研究生(外文):Yuching-Huang
論文名稱:同步纖維素糖化與酒精發酵之新型反應器與技術之開發
論文名稱(外文):Bioreactor development and fermentative strategies for bioethanol production using the process of simultaneous saccharification and fermentation
指導教授:魏毓宏
指導教授(外文):Yu-Hong Wei
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:78
中文關鍵詞:新型反應器同步纖維素糖化與酒精發酵生質酒精
外文關鍵詞:Novel BioreactorSimultaneous Saccharification and Bioethanol FermentationBioethanol production
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本研究主要目的,係開發具同步纖維素糖化與酒精發酵之新型反應器與相關技術。本研究結合好氧纖維素水解菌株A. niger、T. reesei及厭氧酒精發酵菌株Z. mobilis之特性,成功設計出同時水解纖維素及酒精發酵之生物反應器。首先,以羧甲基纖維素(Carboxymeth-ylcellulose, CMC)為基質,且利用聚氨基甲酸酯發泡體為載體,固定纖維素水解菌株A. niger、T. reesei,同時以海藻酸鈣凝膠為載體,固定酒精生產菌Z. mobilis,並將上述三菌同時培養於該新型生物反應器,水解並代謝酒精,以完成同步纖維素糖化與酒精發酵。研究結果顯示:當以10 g/L之CMC為基質,在三菌共固定情況下,基質羧甲基纖維素,在21小時可順利水解為纖維二糖,隨後降解為葡萄糖,並生成酒精濃度達553.75 mg/L。研究近一步指出:提高基質CMC濃度達15 g/L,且利用該新型生物反應器及三菌共培養方式,纖維素轉化生成酒精濃度在45小時可達777.80 mg/L,酒精濃度增加了1.40倍。研究最後以農業廢棄物—狼尾草取代CMC,研究亦證實開發之新型生物反應器,亦具有同時將狼尾草水解糖化及生成酒精之能力。研究顯示:10 g/L狼尾草在三菌共培養及該新型生物反應器中,得以同時水解糖化並發酵在13小時生成酒精達223.67 mg/L。提升基質濃度顯示:狼尾草濃度提升至15 g/L,於此新型反應器中培養,在14小時狼尾草糖化與生成酒精濃度更可達到507.44 mg/L,酒精濃度增加了2.27倍。再次證實:本研究成功開發新型生物反應器與技術,確實具有同步使狼尾草糖化及生成酒精之能力。未來將針對該新型生物反應器之製程放大,進行探討,以落實生質酒精之工業化生產。
In this study, we developed a novel bioreactor that simultaneously enhanced the saccharification and fermentation process for bioethanol production. Initially, A. niger and T. reesei are co-immobilized on PU (polyurethane) carrier, and co-culture in our bioreactor by using Carboxymethylcellulose (CMC) as substrate, then Z. mobilis alginate beads were added into this bioreactor to overcome the problem of simultaneous saccharification and bioethanol fermentation. We show that the yield of the bioethanol could obtain 553.75 mg/L at 21 hours, when we added 10 g/L CMC in this co-culture system. It has recently been reported that the maximum ethanol concentration (777.8 mg/L) increased nearly 1.40-fold when the CMC concentration was increased from 10 to 15 g/L. In order to predict the possibility of agriculture wastes (such as napier grass and rice straw) replace CMC to produce bioethanol by the novel bioreactor. We found that the concentration of ethanol could obtain 223.67 mg/L at 13 hour under the napier grass (10 g/L) was used as the carbon source. Furthermore, the ethanol concentration (507.44 mg/L) increased nearly 2.27-fold when the napier concentration was increased from 10 to 15 g/L. In future, this modified bioreactor will be used to scale-up to insure the bioethanol industry.
中文摘要 ii
Abstract iv
誌謝 vi
目錄 viii
表目錄 xiii
圖目錄 xiv
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 1
第二章 文獻回顧 3
2.1 生質酒精 3
2.2 纖維素 3
2.3 纖維素分解酵素 4
2.3.1 外切型纖維素分解酵素 5
2.3.2 內切型纖維素分解酵素 6
2.3.3 葡萄糖苷酵素 6
2.4 具植物細胞壁分解酵素之微生物 7
2.4.1 纖維素分解菌:Aspergillus niger 8
2.4.2纖維素分解菌:Trichoderma reesei 9
2.5 酒精生產菌:Zymomonas mobilis 9
2.6 發酵製程 12
2.6.1 SHCF 12
2.6.2 SHF 13
2.6.3 SSF 13
2.6.4 SSCF 13
2.6.5 CBP 13
2.7 固定化 14
2.7.1 聚氨基甲酸酯發泡體 16
2.7.2 海藻酸鈉固定法 17
2.8 生物反應器 19
2.8.1 攪拌式反應器 19
2.8.2 填充式反應器 20
2.8.3 膜型反應器 20
第三章 材料與方法 21
3.1菌株 21
3.2 設備儀器 21
3.3 實驗藥品 25
3.3.1 培養基 26
3.3.1.1 PDA培養基 26
3.3.1.2 BH培養基 26
3.3.1.3 Z. mobilis培養基 27
3.3.2 試劑 28
3.3.2.1 磷酸鹽緩衝溶液 28
3.3.2.2 DNS試劑 28
3.4 實驗方法 29
3.4.1 菌種培養 29
3.4.2 收集細胞懸浮液 29
3.4.3 菌種保存 30
3.4.4 細胞計數 30
3.4.5 聚氨基甲酸酯發泡體載體製備 31
3.4.6 纖維分解菌之接菌量 31
3.4.7 A. niger及T. reesei共同預培養 32
3.4.8 Z. mobilis之預培養 32
3.4.9 同時糖化與發酵,三菌共培養 34
3.4.10 狼尾草處理方法 34
3.4.10.1 狼尾草簡易處理 34
3.4.10.2 狼尾草鹼處理 34
3.4.10.3 狼尾草酸處理 35
3.5 分析方法 36
3.5.1 pH分析法 36
3.5.2 酵素活性分析法 36
3.5.3 酵素活性測試 36
3.5.3.1 內切型纖維素分解酵素活性測試 36
3.5.3.2 外切型纖維素分解酵素活性測試 37
3.5.3.3 β-葡萄糖甘酵素活性測試 37
3.5.4 纖維二糖、葡萄糖、酒精濃度分析方法 38
第四章 結果與討論 39
4.1 論文架構 39
4.2 反應器上層好氧區之探討 40
4.2.1 載體改良 40
4.2.2 雙菌預培養酸鹼值之探討 42
4.2.3 酵素協同作用 42
4.2.4 酵素活性測定 43
4.3 反應器上層好氧區與下層厭氧區之探討 47
4.3.1 雙菌預培養對酒精發酵的影響 47
4.3.2 不同預培養方式對主發酵之纖維二糖及酒精的影響 47
4.3.2.1一段式降低液面之預培養 48
4.3.2.2三段式降低液面之預培養 48
4.3.2.3 縮短預培養時間 49
4.3.3 預培養液體之顏色及菌絲變化 50
4.4 CMC基質與反應器之間的探討 55
4.4.1 最適化條件 55
4.4.2 提高CMC基質濃度 55
4.2.3 重複進料與反應器 56
4.2.3.1 重複進料之100 %置換 56
4.2.3.2 重複進料之50 %置換 59
4.2.3.3 置換酒精生產菌 60
4.5 狼尾草與反應器之間的探討 63
4.5.1 簡易處理狼尾草 63
4.5.2 鹼處理狼尾草 63
4.5.3 酸處理狼尾草 64
4.5.4 提高狼尾草基質 65
第五章 結論 70
附錄 標準曲線 72
參考文獻 74
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