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研究生:邱雅蓮
研究生(外文):Ya-Lian Ciou
論文名稱:利用Klebsiella pneumoniae醱酵生產泛用纖維單體-1,3-丙二醇之研究
論文名稱(外文):Study on the development of bioprocess for the monomer of widly used fiber-1,3-propanediol by Klebsiella pneumoniae
指導教授:魏毓宏
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:88
中文關鍵詞:13-propanediolKlebsiella pneumonia回應曲面法醱酵策略
外文關鍵詞:13-propanediolKlebsiella pneumoniaResponse surface methodologyfermentation strategy
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1,3-丙二醇為合成泛用纖維-對苯二甲酸丙二醇(polytrimethyleneterephthalate, PTT)之單體,其產業應用性高。本研究主要目的,係探討菌株Klebsiella pneumoniae醱酵合成1,3-丙二醇之相關生產製程,以加速其日後工業化之應用。Klebsiella pneumoniae為革蘭氏陰性兼性厭氧菌。因此,首先考量不同供氧程度,對Klebsiella pneumoniae合成目標產物1,3-丙二醇之影響。結果顯示,菌株在有氧(200 rpm)條件下,有利菌株合成1,3-丙二醇,其產量可達7.6 g/L。最佳前培養條件之確立方面,研究顯示:以10 % LB前培養基菌液,接種於主培養,在培養條件為轉速200 rpm、30 ℃、初始pH 9,可得最佳之1,3-丙二醇產能,達9.5 g/L。其次,藉由回應曲面實驗設計法,進行培養基最適化。研究結果顯示:Glycerol、(NH4)2SO4、CoCl2•2H2O為影響Klebsiella pneumoniae合成1,3-丙二醇之三個主要參數,當使用Glycerol (34 %)、(NH4)2SO4 (3.4 mM)、CoCl2•2H2O(14μM)為主培養基,可獲得1,3-丙二醇最終產量為16.5 g/L,其產率為0.4 g/L/h。在醱酵策略方面,於24小時饋入1 %甘油,最終1,3-丙二醇產量、產率分為21.53 g/L及0.29 g/L/h。進一步利用批次饋料方式提升產量,分別於24、36、48小時,饋入1 %甘油及36、48小時饋入3.4 mM硫酸銨,1,3-丙二醇可達最大產量為27.85 g/L。其次,藉由 1H-NMR、13C-NMR 與EI-MS 鑑定其產物化學結構及分子量,研究證實:本研究K. pneumoniae醱酵所產生1,3-丙二醇,與標準品比對確實相符合。
1,3-Propanediol has numerous industrial applications in synthesize the monomer of widely used fiber-polytrimethyleneterephthalate (PTT). The purpose of this thesis is to study the related fermentation process for the production of 1,3-propanediol (1,3-PD) by Klebsiella pneumonia. Klebsiella pneumoniae is a Gram-negative, facultative anaerobic bacterium. Therefore, effect of different oxygen supply levels on 1,3-propanediol production was the first consideration condition. This work shows 1,3-propanediol can be produced by Klebsiella pneumoniae under aerobic condition (200 rpm), the production was 7.6 g/L. Also, 1,3-PD production was optimal in batch cultures when the temperature, pH, agitation rate were controlled at 30 oC, pH 9 and 200 rpm, respectively. This study shows that the concentration of 1,3-PD was 9.5 g/L under the optimal conditions. To further enhance 1,3-PD production, statistical experimental design methodology was applied to optimize the culture medium composition favoring 1,3-PD synthesis. Three key parameters (glycerol, (NH4)2SO4, CoCl2•2H2O) were selected by two-level factorial design. Response surface methodology was then used to identify the optimal composition of the three key parameters, giving an optimal concentration of 34 %, 3.40 mM, and 14 μM for glycerol, (NH4)2SO4,and CoCl2•2H2O, respectively. With this optimal medium, the 1,3-PD production could be markedly elevated to a yield of 16.5 g/L and a volumetric productivity of 0.40 g/L/h. For fermentation strategies, feeding 1 % glycerol at 24 hours could produce 21.53 g/L of 1,3-PD. With feed-batch strategy, the maximum concentration of 1,3-PD could be improved to 27.85 g/L,while the with feeding concentration of glycerol maintained at a value of 1%. Mass spectrometry (EI-MS) and nuclear magnetic resonance (13C-NMR) analysis indicate that the purified product from the broth of Klebsiella pneumonia is in consistent with sample purchased from Fluka Co.
中文摘要 i
英文摘要 iii
致謝 v
目錄 vi
表目錄 x
圖目錄 xi
第一章 前言 1
1-1 菌株Klebsiella pneumoniae 1
1-2 泛用纖維-聚對苯二甲酸丙二醇 3
1-3 1,3-丙二醇(1,3-propanediol, 1,3-PD) 4
1-3-1 物理及化學性質 4
1-3-2 生產1,3-丙二醇之方法 4
1-3-3 生產1,3-丙二醇未來之潛力 5
1-4 微生物利用甘油之代謝路徑 6
1-5 回應曲面法 ( Response surface methodology, RSM ) 9
1-5-1 二水準因子設計( Two-level factorial design ) 11
1-5-2 陡升路徑法 ( Method of path steepest ascent ) 11
1-5-3 中心混程設計 ( Central composite design, CCD ) 12
1-6 研究動機 13
第二章 材料與方法 14
2-1 實驗藥品及分析儀器 14
2-1-1 實驗藥品 14
2-1-2分析儀器 16
2-2菌株及培養基 17
2-2-1菌株 17
2-2-2 培養基 17
2-3 粗萃物1,3-丙二醇之分離純化 19
2-4 實驗與方法 20
2-4-1 前培養條件測試之實驗流程 20
2-4-2 供氧需求之培養方法 21
2-4-3 1,3-丙二醇之定性分析 22
2-4-4 定量分析 22
2-4-5 微生物生長之分析 23
第三章 結果與討論 26
3-1 論文架構 26
3-2 探討培養條件 27
3-2-1 探討培養K. pneumoniae之不同供氧需求 27
3-2-2 探討K. pneumoniae代謝之副產物 36
3-2-3 篩選最適前培養條件 41
3-3 開發最適化培養基之前置步驟--篩選因子 47
3-3-1 篩選碳源 47
3-3-2 篩選無機氮源 47
3-3-3 篩選有機氮源 47
3-3-4 篩選主培養其他成分 48
3-3-5 篩選微量元素各成分 48
3-4 依實驗設計法探討最適化培養基之組成 52
3-4-1 二水準因子設計 52
3-4-2 陡升路徑法 55
3-4-3 回應曲面法 56
3-5 探討醱酵過程有無控pH對1,3-丙二醇之影響 61
3-6 化學結構確認和分子量分析 64
3-7 醱酵策略之探討 68
3-7-1搖瓶批次饋料醱酵 (Fed-batch fermentation) 68
3-7-1-1第一次饋料時機及甘油濃度 68
3-7-1-2 批次饋料 73
3-7-1-3 探討饋料成份對1,3-丙二醇產量之影響 73
3-7-2 醱酵槽培養條件測試 78
第四章 結論與未來展望 82
4-1 結論 82
4-2未來展望 83
參考文獻 84
附錄 88
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蒙哥馬利(Montgomery, Douglas C.) :實驗設計與分析(2003)
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