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研究生:邱昱誠
研究生(外文):Yu-Cheng Chiu
論文名稱:微生物發酵轉化甘油生產1,3-丙二醇之研究
論文名稱(外文):Production of 1,3-propanediol by Klebsiella sp. from glycerol
指導教授:李振綱李振綱引用關係
指導教授(外文):Cheng-Kang Lee
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
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:2008
畢業學年度:96
語文別:中文
論文頁數:115
中文關鍵詞:丙二醇微生物發酵
外文關鍵詞:propanediolKlebsiella sp.
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全球原油價格不斷攀升,能源問題備受世界各國重視,因此積極開發替代能源,其中生質柴油為主要開發項目之一。製備生質柴油過程中會生成大量之副產物甘油,因此如何提升甘油之附加價值是十分值得之研究課題。本論文主要研究運用微生物Klebsiella sp.來轉化甘油生成1,3-丙二醇(1,3-propanediol;1,3-PDO),1,3-PDO為製備人造纖維PTT(polytrimethylene polyesters)之原料,可提升甘油之附加價值。
在搖瓶中培養Klebsiella sp.,1,3-PDO之產率可達61% mol/mol glycerol,濃度為22.6 g/l,而生產力則可達0.71 gl-1h-1。在3公升發酵槽中進行批次發酵,於空氣流量為0.88 vvm時,24小時內1,3-PDO濃度可達23.15 g/l,產率可達到63% mol/mol glycerol,生產力為0.96 gl-1h-1。
當饋料批次發酵調控pH值於6.4時,26小時即可獲得53.26 g/l之1,3-PDO,生產力可達2.05 gl-1h-1,而1,3-PDO產率則下降為44.48 %,並且最終1,3-PDO濃度則可超過60 g/l,饋料發酵後期1,3-PDO濃度微幅增加,但乳酸濃度則快速增加,最終乳酸濃度可達70 g/l。
當以生質柴油副產物甘油為碳源時,在搖瓶發酵1,3-PDO之產率可達58% mol/mol glycerol,濃度為21.4 g/l,而生產力則可達0.65 gl-1h-1;當饋料批次發酵調控pH值於6.4時,32小時可獲得39.45 g/l之1,3-PDO,生產力可達1.23 gl-1h-1,而1,3-PDO產率則為31.62 %。
Biodisel is becoming an important alternative fuel in recent year. As biodiesel production scale increases, the price of its by-product glycerol is decreasing. In order to reduce the cost of biodiesel production, transformation of glycerol to a value-added product is required. Glycerol can be converted by Klebsiella sp. to 1,3-propanediol (1,3-PDO), an important precursor for manufacturing polymethylene polyesters (PTT). In the present study, batch fermentation of Klebsiella sp. was performed in flask and 3L-fermentor to produce 1,3-PDO from pure glycerol. In the flask, the molar yield, concentration, and productivity of 1,3-PDO were 61%, 22.6 g/l, and 0.71 gl-1h-1, respectively. On the other hand, the molar yield, concentration, and productivity of 1,3-PDO were improved to 63%, 23.15 g/l, and 0.96 gl-1h-1, respectively, in 3-L fermentor operation with 24 h of fermentation and 0.88 vvm of airflow. By controlling the pH at 6.4, the concentration, and productivity of 1,3-PDO were further increase to 53.26 g/l and 2.05 gl-1h-1 within 26 h, however, yield decreased to 44.48%. After 26 h of fermentation, the concentration of 1,3-PDO gradually increased and followed by a rapid increase of lactic acid concentration at the later stage of fermentation. The final concentration of 1,3-PDO and lactic acid were about 60 g/l and 70 g/l, respectively. The production of 1,3-PDO was also carried out by using crude glycerol obtained from biodiesel production. Batch fermentation of Klebsiella sp. was performed in flask. The molar yield, concentration, and productivity of 1,3-PDO were 58%, 21.4 g/l, and 0.65 gl-1h-1, respectively. For fed-batch fermentation, the concentration, and productivity of 1,3-PDO were improved to 39.45 g/l and 1.23 gl-1h-1, respectively within 32 h, however, the molar yield decrease markedly to 31.62%.
第一章 前言 1
1.1 生質能源之開發 1
1.2 生質柴油簡介 1
1.3 研究目的 4

第二章 文獻回顧 5
2.1 1,3-丙二醇 5
2.1.1 基本性質介紹 5
2.1.2 應用 5
2.2 PTT 6
2.2.1 PTT纖維特性 6
2.3 1,3-丙二醇製程 7
2.3.1 石化合成法 …. 7
2.3.2 微生物發酵法 8
2.4 微生物發酵生產1,3-丙二醇之代謝路徑 9
2.4.1 微生物之甘油代謝 9
2.4.2 Klebsiella pneumoniae發酵甘油之代謝路徑 11
2.4.2.1 Pyruvate之生成與酵素GDH 11
2.4.2.2 3-HPA之生成與酵素GDHt , PDOR 12
2.4.2.3 GDH,GDHt與PDOR三酵素之關係 14
2.5 K. pneumoniae發酵甘油生產1,3-丙二醇 15
2.5.1 厭氧與微氧條件發酵 15
2.5.2 不同通氣條件之影響 16
2.5.3 添加因子之影響 17
2.6 基因工程菌發酵醣類基質生產1,3-PDO 18
2.6.1 美國DuPunt與Genencor公司單一微生物發酵醣
類基質生產1,3-PDO之構想 18
2.6.2基因重組之大腸桿菌發酵醣類基質生產1,3-PDO
之構想 21
2.7 運用廢棄甘油發酵生產1,3-丙二醇 24

第三章 實驗材料與方法 27
3.1 實驗藥品 27
3.2 實驗儀器及設備 28
3.3 實驗菌株介紹 29
3.4 分析儀器及方法 29
3.4.1 液相代謝產物組成分析 29
3.4.2 酵素法分析甘油 31
3.4.2.1 分析原理 31
3.4.2.2 操作步驟 33
3.5 Klebsiella sp.培養基成分 34
3.5.1 儲菌培養基成分 34
3.5.2 前培養基成分 35
3.5.3 發酵生產1,3-PDO培養基成分 37
3.6 Klebsiella sp.之菌種保存 38
3.7儲菌培養基對Klebsiella sp.生長之影響 38
3.8 Klebsiella sp.發酵實驗流程 39
3.9 微氧條件下BPG plate儲菌方式之影響 40
3.10 Klebsiella sp.發酵培養基之添加因子探討 42
3.11於小型發酵槽中進行批次發酵 43
3.12氣體流量對於Klebsiella sp.發酵生產1,3-PDO之影響 43
3.13發酵過程中調控酸鹼值之影響 44
3.14 不同酸鹼值條件對於饋料批次發酵之影響 44
3.15 應用生質柴油副產物甘油發酵生產1,3-PDO 45
3.15.1 分析生質柴油副產物之組成 45
3.15.2 批次發酵crude glycerol生產1,3-PDO 45
3.15.3 饋料批次發酵crude glycerol生產1,3-PDO 45
3.16 建立Klebsiella sp.菌體濃度與細胞乾重換算檢量線
之流程 46

第四章 結果與討論 48
4.1儲菌培養基對於Klebsiella sp.生長之影響 48
4.2微氧條件下BPG plate儲菌方式之影響 50
4.3 Klebsiella sp.發酵培養基之添加因子探討 51
4.3.1 有無三種添加因子對於1,3-PDO生產之影響 51
4.3.2 未添加Acetaldehyde因子對於1,3-PDO生產之影響 56
4.3.3 未添加Fumarate與Acetaldehyde因子對於1,3-PDO生產
之影響 58
4.3.4 添加因子結果綜合比較與討論 60
4.4 空氣流量對於Klebsiella sp. 發酵甘油生產1,3-PDO之
影響 62
4.5 發酵過程中調控酸鹼值之影響 70
4.6 不同酸鹼值條件對於饋料批次發酵之影響 73
4.6.1氨水調控發酵過程之pH維持於6.0 73
4.6.2氨水調控發酵過程之pH維持於6.4 73
4.6.3氨水調控發酵過程之pH維持於6.8 76
4.6.4 氨水調控酸鹼值對於饋料批次發酵影響之結論 76
4.7應用生質柴油副產物甘油發酵生產1,3-PDO 81
4.7.1 生質柴油副產物之組成 81
4.7.2 批次發酵crude glycerol生產1,3-PDO 82
4.7.3 饋料批次發酵crude glycerol生產1,3-PDO 84

第五章 結論 88
附錄 90
參考文獻 93
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