微生物發酵轉化甘油生產1,3-丙二醇之研究｜國立臺灣科技大學博碩士論文系統

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研究生：	邱昱誠 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 Production of 1,3-propanediol by Klebsiella sp. from glycerol
指導教授：	李振綱 Cheng-Kang Lee
口試委員:	朱義旭 Yi-Hsu Ju 張嘉修 Jo-Shu Chang
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
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：	propanediol, Klebsiella sp.
相關次數：	點閱：164 下載：4
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上一筆

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 生質能源之開發	1
2 生質柴油簡介	1
3 研究目的	4

第二章 文獻回顧	5
1 1,3-丙二醇	5
1.1 基本性質介紹	5
1.2 應用	5
2 PTT	6
2.1 PTT纖維特性	6
3 1,3-丙二醇製程	7
3.1 石化合成法 ….	7
3.2 微生物發酵法	8
4 微生物發酵生產1,3-丙二醇之代謝路徑	9
4.1 微生物之甘油代謝	9
4.2 Klebsiella pneumoniae發酵甘油之代謝路徑	11
4.2.1 Pyruvate之生成與酵素GDH	11
4.2.2 3-HPA之生成與酵素GDHt , PDOR	12
4.2.3 GDH,GDHt與PDOR三酵素之關係	14
5 K. pneumoniae發酵甘油生產1,3-丙二醇	15
5.1 厭氧與微氧條件發酵	15
5.2 不同通氣條件之影響	16
5.3 添加因子之影響	17
6 基因工程菌發酵醣類基質生產1,3-PDO 	18
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
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
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
1 實驗藥品	27
2 實驗儀器及設備 	28
3 實驗菌株介紹	29
4 分析儀器及方法	29
4.1 液相代謝產物組成分析	29
4.2 酵素法分析甘油	31
4.2.1 分析原理	31
4.2.2 操作步驟	33
5 Klebsiella sp.培養基成分	34
5.1 儲菌培養基成分	34
5.2 前培養基成分	35
5.3 發酵生產1,3-PDO培養基成分	37
6 Klebsiella sp.之菌種保存	38
7儲菌培養基對Klebsiella sp.生長之影響	38
8 Klebsiella sp.發酵實驗流程	39
9 微氧條件下BPG plate儲菌方式之影響	40
10 Klebsiella sp.發酵培養基之添加因子探討	42
11於小型發酵槽中進行批次發酵	43
12氣體流量對於Klebsiella sp.發酵生產1,3-PDO之影響	43
13發酵過程中調控酸鹼值之影響	44
14 不同酸鹼值條件對於饋料批次發酵之影響	44
15 應用生質柴油副產物甘油發酵生產1,3-PDO	45
15.1 分析生質柴油副產物之組成	45
15.2 批次發酵crude glycerol生產1,3-PDO	45
15.3 饋料批次發酵crude glycerol生產1,3-PDO	45
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
1儲菌培養基對於Klebsiella sp.生長之影響	48
2微氧條件下BPG plate儲菌方式之影響	50
3 Klebsiella sp.發酵培養基之添加因子探討	51
3.1 有無三種添加因子對於1,3-PDO生產之影響	51
3.2 未添加Acetaldehyde因子對於1,3-PDO生產之影響	56
3.3 未添加Fumarate與Acetaldehyde因子對於1,3-PDO生產
     之影響	58
3.4 添加因子結果綜合比較與討論	60
4 空氣流量對於Klebsiella sp. 發酵甘油生產1,3-PDO之
影響	62
5 發酵過程中調控酸鹼值之影響	70
6 不同酸鹼值條件對於饋料批次發酵之影響	73
6.1氨水調控發酵過程之pH維持於6.0	73
6.2氨水調控發酵過程之pH維持於6.4	73
6.3氨水調控發酵過程之pH維持於6.8	76
6.4 氨水調控酸鹼值對於饋料批次發酵影響之結論	76
7應用生質柴油副產物甘油發酵生產1,3-PDO	81
7.1 生質柴油副產物之組成	81
7.2 批次發酵crude glycerol生產1,3-PDO	82
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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