利用Klebsiella pneumoniae醱酵生產泛用纖維單體-1,3-丙二醇之研究

Study on the development of bioprocess for the monomer of widly used fiber-1,3-propanediol by Klebsiella pneumoniae

邱雅蓮(Ya-Lian Ciou)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2009年)

https://doi.org/10.6838/YZU.2009.00248

摘要

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。

關鍵字

1,3-propanediol ； Klebsiella pneumonia ； 回應曲面法 ； 醱酵策略

並列摘要

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.

並列關鍵字

1,3-propanediol ； Klebsiella pneumonia ； Response surface methodology ； fermentation strategy

參考文獻

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