利用Bacillus sp. 分解纖維素進行兩階段式發酵生產特用化學品

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林文仕(Wen-shi Lin) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用Bacillus sp. 分解纖維素進行兩階段式發酵生產特用化學品
(Study of converting cellulose to Specialty Chemical on the two-step fermentation by Bacillus sp.)

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摘要(中)

纖維素為地球上龐大的可利用的再生能源，如果能將農業廢棄物中的纖維素藉由纖維水解酵素轉換為葡萄糖並且供應後續菌種發酵利用來生產產物，如此不僅有效率的利用農業廢棄物並且能降低操作成本，提高產能。
本論文所使用的菌種Bacillus sp.經由實驗尋找得到纖維水解酵素活性最高的pH值為pH5；最高的酵素活性反應溫度為40℃。而在pH4.8時能停止菌體的生長，並且酵素保有活性，發酵液中的還原醣濃度也會開始累積，而有進行pH值調控的還原醣累積量為1.501g/L比沒有調控的控制組的還原醣累積量0.231g/L高了6.5倍。而經過還原糖累積後的發酵液用來培養Aspergillus terreus則可以提高菌體的重量，菌重是控制組的1.68倍但是在產物衣康酸的生產方面只有控制組的47.7%，所以要再尋找適合衣康酸生產的條件才會有更好的纖維素轉化為衣康酸的效果。
累積完的還原醣經由HPLC及GPC分析後認定為纖維寡醣，所以可能必須要再尋找能生產β-葡萄糖苷酵素的菌種將反應後的纖維寡醣再進一步轉換成葡萄糖，才能更有效的被大部分菌種利用來生產所想要的產物。

摘要(英)

Cellulose, an abundant and renewable energy resource, the cellulose in agricultural waste can be conversion by cellulase for glucose，and glucose can supply the second-step fermentation use to produce products. It will not only efficient use of agricultural waste and can reduce operating costs.
The strain used in this study was Bacillus sp. and Aspergillus terreus. The cellulase activity of Bacillus sp. was found optimum at pH 5 and temperature 40 ℃. When pH is 4.8, the cell growth was stopped, and the cellulase retained activity, the reducing sugar in the broth was accumulated. The reducing sugar in the broth with pH controlled could be accumulated to 1.501g/L but without pH controlled could only be accumulated to 0.231g/L. Then we use the broth of reducing sugar was accumulated to culture Aspergillus terreus. And the biomass was 1.68 times to the control group but the Itaconic acid was only 47.7% to the control group. We will have to find the condition which is favorable to Produce Itaconic acid.
The reducing sugar was analysis with GPC and HPLC. We found that the reducing sugar was cellooligosaccharide. We will have to find the microorganism with the ability of producing β-1,4-glucosidase for conversion cellooligosaccharide to glucose. The glucose will simple to be used by the most microorganism.

關鍵字(中)

★ 芽孢桿菌
★ 土麴黴
★ 衣康酸
★ 纖維水解酵素
★ 纖維素

關鍵字(英)

★ Aspergillus terreus
★ Bacillus sp.
★ cellulose
★ cellulase
★ Itaconic acid

論文目次

摘要 i
Abstractii
誌謝 iii
目錄 iv
圖目錄 viii
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 纖維素之簡介3
2-2 纖維素水解酵素簡介5
2-2-1 內切型纖維水解酵素(Endo-1-4-β-D-glucanase)7
2-2-2 外切型纖維水解酵素(Exo-1-4-β-D-glucanase)8
2-2-3 β-葡萄糖苷酵素(β-glucanase)8
2-2-4 纖維素分解酵素的協同作用機制9
2-2-5 纖維素水解酵素之誘導9
2-3 纖維水解酵素來源10
2-4芽孢桿菌(Bacillus)簡介14
2-5 影響芽孢桿菌(Bacillus sp.) 所生產之纖維素水解酵素活性之發酵條件 14
2-5-1 碳源14
2-5-2 氮源15
2-5-3 溫度16
2-5-4 pH值17
2-5-5溶氧值(DO 值)18
2-6 特用化學品-衣康酸19
2-6-1 衣康酸簡介19
2-6-2 衣康酸來源20
2-6-3 衣康酸之應用與市場22
2-7 土麴黴 (Aspergillus terreus) 之簡介24
第三章實驗規劃、材料與方法26
3-1 實驗材料26
3-1-1 實驗菌株26
3-1-2 實驗藥品27
3-2 實驗儀器與設備28
3-3 實驗方法30
3-3-1實驗規劃30
3-3-2 菌種保存31
3-3-3 培養基組成31
3-3-4 Bacillus sp.搖瓶實驗與pH值調控35
3-3-5 Bacillus sp.發酵槽實驗與pH值及溫度調控36
3-3-6 利用Bacillus sp.發酵液培養Aspergillus terreus36
3-4 分析方法38
3-4-1 分析流程38
3-4-2 菌重定量39
3-4-3還原醣定量40
3-4-4剛果紅測試42
3-4-5酵素活性測試43
3-4-6以HPLC分析組成44
3-4-7分子量測定45
3-4-8 衣康酸含量分析47
第四章實驗結果與討論48
4-1 剛果紅測試48
4-2 pH值對纖維水解酵素活性的影響49
4-3 溫度對酵素活性的影響50
4-4 酵素活性對溫度的穩定性測試51
4-5 發酵槽培養時間對酵素活性的影響56
4-6 pH值調控之搖瓶實驗57
4-7 調控pH值與溫度對發酵槽還原醣累積的影響59
4-8以HPLC分析發酵液組成63
4-9 以GPC分析發酵液組成64
4-10利用Bacillus sp.發酵液培養Aspergillus terreus65
第五章結論與建議67
5-1 結論67
5-2 建議68
第六章參考文獻69

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指導教授

徐敬衡(Chin-hang Shu)

審核日期

2012-8-15

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