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研究生:葉宗葦
研究生(外文):Yeh Tsung Wei
論文名稱:利用微生物菌體生產生質能源乙醇之最適化共培養條件探討
論文名稱(外文):Studies on optimal co-cultural conditions for microorganism culture producing biomass generating ethanol
指導教授:劉裕國
學位類別:碩士
校院名稱:長庚大學
系所名稱:化工與材料工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:88
中文關鍵詞:纖維素生質乙醇微生物共培養系統農業廢棄物
外文關鍵詞:biomass energyethanol productionco-culture systemcellulose
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本研究的目的是利用農業廢棄物來生產可做為替代能源之生質乙醇。由於農業廢棄物中包括大量的纖維素、半纖維素與木質素等,故本研究之實驗設計以纖維素作為原料,經由系統之開發將纖維素糖化水解與乙醇發酵反應同時進行於微生物共培養系統中,以生成乙醇。本研究已進行由富含纖維素的林地或山區,篩選193株菌株,並藉由實驗得知Trichoderma reesei ATCC 26921優於所篩選之菌種。然而本研究亦證明T. reesei在30℃、200rpm與pH6.0~7.0的培養條件下,進行96小時培養,其僅產生還原糖0.4070g/l,轉化率7.33%,且將其移至Zymomonas mobilis ATCC 29191之培養系統,其乙醇產量不足0.1%,此產糖能力極差。故本研究利用Aspergillus niger ATCC 16888以彌補T. reesei在作用中產糖能力之不足。其初步結果顯示,在共懸浮培養T. reesei-A. niger中,其相同培養條件下,其還原糖產量為2.5708g/l,轉化率為46.27%,若同時將糖化發酵液置入Z. mobilis培養系統中,可得約4.24g/l的乙醇產量,可知其共同生產是可行的,但需增加原料以產生還原糖。另外,在固定化培養方面亦發現菌體可均勻地附著於實驗之載體上,故推測利用此生產體系來生產乙醇是可以期待的。
The purpose of this study is to produce alternative energy - ethanol from the agricultural waste including cellulose, hemicellulose and lignin, etc. We will develop a new co-culture system of the simultaneous saccharification and fermentation to deal with cellulose costly and effectively. Cellulose-degrading microbes were isolated from samples obtained from different environments and selected. 193 strains of cellulose-degrading microbes isolated from different environments were screened in this study. Trichoderma reesei ATCC26921 was found to be the most efficient cellulose-degrading microbe. By the use of T. reesei, a reducing sugars concentration (0.4070g/l) was obtained with a hydrolysis yield of 7.33% in culture condition of 30℃, 200rpm, initial pH6.0~7.0, and 96h. Unfortunately, the ethanol concentration was 0.1% only, when combine with Zymomonas mobilis ATCC29191 fermentation system. However, in the same culture condition, when supplementing the T. reesei with Aspergillus niger ATCC16888, we could increase the reducing sugars concentration up to 2.5708g/l and hydrolysis yield of 46.27%. The ethanol concentration was also increasing up to 4.24g/l when Z. mobilis fermentation system was added thereafter. Further development of the immobilized-cell system including the feasibility of using carrier and the optimum immobilization method were also investigated in this study. The preliminary results showed the microbe can be immobilized in the carrier completely with uniformly distribution. The total results showed high potential of ethanol production from agricultural waste in new co-culture system.
目錄
指導教授推薦書……………………………………………………………i
論文口試委員會審定書……………………………………………………ii
授權書………………………………………………………………………iii
致謝…………………………………………………………………………iv
中文摘要……………………………………………………………………v
英文摘要……………………………………………………………………vi
目錄…………………………………………………………………………vii
圖目錄………………………………………………………………………xiii
表目錄………………………………………………………………………xv
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 農業廢棄物…………………………………………………………5
2.2 纖維素………………………………………………………………6
2.3 半纖維素……………………………………………………………7
2.4 木質素………………………………………………………………8
2.5 纖維素生物能源的開發……………………………………………9
2.6 纖維素分解酵素……………………………………………………10
2.6.1 纖維素分解酵素的種類…………………………………………10
2.6.1.1 內切型纖維素分解酵素………………………………………10
2.6.1.2 外切型纖維素分解酵素………………………………………11
2.6.1.3 纖維二醣酵素…………………………………………………11
2.6.2 纖維素分解酵素的作用機制……………………………………12
2.6.2.1 C1-Cx假說……………………………………………………12
2.6.2.2 修正假說………………………………………………………13
2.6.3 纖維素分解酵素之產物反饋抑制作用…………………………13
2.6.4 纖維素水解酵素的誘導…………………………………………41
2.6.5 纖維素水解酵素的熱穩定性……………………………………14
2.7 分解纖維素的微生物種類…………………………………………15
2.7.1 細菌………………………………………………………………16
2.7.2 真菌………………………………………………………………16
2.7.3 放線菌……………………………………………………………17
2.8 Zymomonas mobilis之介紹……………………………………17
2.9 影響微生物的環境因子……………………………………………19
2.9.1 微生物的生長溫度………………………………………………19
2.9.2 微生物的氧需求性………………………………………………19
2.9.3 酸鹼度對微生物的影響…………………………………………20
2.9.4 震盪與攪拌………………………………………………………20
2.9.5 微生物之培養基成分……………………………………………21
2.9.5.1 碳源……………………………………………………………21
2.9.5.2 氮源……………………………………………………………21
2.9.5.3 無機鹽類………………………………………………………22
2.10 纖維素物質的預處理法……………………………………………22
2.11 固定化細胞培養技術………………………………………………24
2.12 微生物之共固定化…………………………………………………26
2.13 系統開發與流程規劃………………………………………………28
第三章 材料與方法…………………………………………………………30
3.1 菌株來源與採樣………………………………………………………30
3.2 設備與藥品……………………………………………………………31
3.2.1 設備儀器……………………………………………………………31
3.2.2 實驗藥品……………………………………………………………32
3.2.3 培養基之配製………………………………………………………33
3.2.3.1 Bushnell-Haas selection medium(BHS)……………33
3.2.3.2 Zymomonas mobilis medium培養基之配製………………34
3.2.4 試劑…………………………………………………………………35
3.2.4.1 磷酸鹽緩衝溶液…………………………………………………35
3.2.4.2 DNS試劑…………………………………………………………36
3.3 實驗方法………………………………………………………………37
3.3.1 優秀菌種的篩選與純化……………………………………………37
3.3.1.1 菌種的篩選………………………………………………………37
3.3.1.2 菌種的分離………………………………………………………37
3.3.1.3 纖維素分解酵素活性的初步測定………………………………38
3.3.1.4 還原糖測定法……………………………………………………38
3.3.1.5 優秀纖維素分解菌種之選取……………………………………38
3.3.1.6 菌種的保存………………………………………………………39
3.3.1.7 熱穩定性測定……………………………………………………39
3.3.2 懸浮培養體系的建立………………………………………………39
3.3.2.1 濾紙分解活性測定………………………………………………40
3.3.2.2 細胞乾重測定……………………………………………………40
3.3.2.3 乙醇濃度測定……………………………………………………41
3.3.2.4 最佳的液態培養酸鹼度…………………………………………41
3.3.2.5 最佳的液態培養轉速……………………………………………42
3.3.2.6 最佳的液態培養培養溫度………………………………………43
3.3.2.7 最佳的液態培養接種量…………………………………………44
3.3.3 纖維素水解菌之共培養體系………………………………………44
3.3.3.1 培養溫度對液態共培養之影響…………………………………44
3.3.3.2 最佳的液態共培養接種比例……………………………………45
3.3.3.3 最佳的液態共培養菌體總接種量………………………………45
3.3.3.4 三菌共培養之糖化水解與乙醇發酵之探討……………………46
3.3.4 固定化培養體系的探討……………………………………………46
3.3.4.1 凝膠包埋法………………………………………………………47
3.3.4.2 多孔載體固定化法………………………………………………48
第四章 結果與討論…………………………………………………………51
4.1 菌種的篩選……………………………………………………………51
4.2 纖維素水解步驟最適化溫度之探討…………………………………54
4.3 纖維素水解步驟最適化轉速之探討…………………………………56
4.4 纖維素水解步驟最適化酸鹼值之探討…………………………………57
4.5 纖維素水解步驟最適化接種量之探討…………………………………59
4.6 乙醇生產步驟最適化酸鹼值之探討……………………………………60
4.7 乙醇生產步驟最適化轉速之探討………………………………………61
4.8 乙醇生產步驟最適化溫度之探討………………………………………62
4.9 T. reesei與A. niger之共培養效應…………………………………63
4.10 溫度對T. reesei與A. niger的共懸浮培養之影響………………65
4.11 不同接種比例之T. reesei與A. niger的共懸浮培養………………66
4.12 T. reesei與A. niger共懸浮培養之最佳接種量……………………68
4.13 共懸浮培養之乙醇發酵能力之探討………………………………………70
4.14 相異固定化載體於T. reesei與A. niger之固定化效率……………72
4.15 預培養與共固定方法之比較………………………………………………73
4.16 天然載體之固定化效率的探討……………………………………………75
第五章 結論與建議………………………………………………………………78
5.1 結論…………………………………………………………………………78
5.2 建議…………………………………………………………………………80
參考文獻……………………………………………………………………………82
附錄 標準曲線……………………………………………………………………88
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