以生質柴油製程副產物粗甘油進行醱酵研究生產聚3-羥基丁酯-co-3-羥基戊酯共聚生物高分子

Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fermentation using crude glycerol derived from biodiesel manufacture process

吳佳紋(Chia-Wen Wu)

指導教授 ： 藍祺偉

元智大學/工程學院/化學工程與材料科學學系/碩士(2012年)

摘要

聚羥基烷酯(PHAs)是儲存在細胞體內的一種生物性聚酯高分子，其具有生物相容性與生物可分解性，環境中有許多細菌可以行體內合成，以做為細菌自身碳源與能量提供，分子物理性質近似於石化塑膠產品。培養微生物需要足夠的碳源，在醱酵過程中，碳源占了醱酵大部分成本。目前生質柴油廠不斷增設，轉酯化製程產生大量副產物『粗甘油』，其市場價格低廉，而本研究以粗甘油為碳源，利用菌株Ralstonia eutropha H16合成PHAs，進而提高甘油利用的經濟效益。
本研究利用化學培養基(Define medium)培養微生物Ralstonia eutropha H16菌種，先以搖瓶進行實驗，探討各個實驗因子對於細胞生長以及PHA合成之相互影響關係，其實驗因子分別為：粗甘油濃度、丙酸鈉濃度、丙酸鈉添加時間、酵母萃取粉濃度以及磷酸根濃度，最後再進入醱酵槽系統放大，探討溶氧量(Dissolved oxygen, DO)的影響。結果顯示，PHA最適化條件分別為粗甘油濃度為5 (%,w/v)；丙酸鈉濃度為7.5 g L-1；丙酸鈉添加時間為細胞培養12小時之後；酵母萃取粉由於成本考量下，最佳濃度為0.2 g L-1；磷酸根濃度為0.058 g L-1；溶氧量控制在50%。從多組實驗結果可以發現，菌類生長以及PHA合成皆會受到各個實驗因子的相互影響，因此再藉由實驗設計-反應曲面法（Response Surface Methodology）探討粗甘油濃度、丙酸鈉濃度、磷酸根濃度、溶氧量及丙酸鈉添加時間對Ralstonia eutropha H16菌種生產PHV的影響，進行二階迴歸分析得到理論方程式為Y＝7.394＋1.9288772X1＋0.091311X2－1.987625X12－0.5875X1X2－2.492625X22，最適化條件為粗甘油濃度57.77 g L-1，丙酸鈉濃度10 g L-1，磷酸根濃度0.0713 mole L-1，溶氧量50%，丙酸鈉添加時間為培養12小時之後，經過72小時培養，PHV濃度累積最大產量之理論計算值為7.86 g L-1。所合成之PHAs經由NMR鑑定分子結構為PHBV。以粗甘油為碳源合成的PHBV重量平均分子量為29000~32000Dalton；經由熱重分析儀(TGA)分析PHBV相較於PHB有較好的熱穩定性；經由示差掃描熱量分析儀(DSC)分析PHBV，其結晶度會隨著HV含量上升而下降。

關鍵字

聚羥基烷酯 ； 粗甘油 ； 醱酵 ； Ralstonia eutropha H16 ； 反應曲面法

並列摘要

Polyhydroxyalkonoates (PHAs), a type of biological polyester, are an alternative substance for conversional petrochemical plastics because of its biocompatibility and biodegradation. Many microorganisms in nature can accumulate PHAs as intracellular granules which serve as carbon and energy source under limited condition. It has been reported that cost of carbon supply is a major factor in the fermentation process of PHAs production. To reduce the cost of raw materials becomes a key to success of PHAs product in market. Crude glycerol, a derivated product from transesterfication process, is made by bio-diesel factories. The expanded capacities of biodiesel production result in the overproduction of crude glycerol with respect to low selling price. In the present research, the crude glycerol was employed as sole carbon source with addition of sodium propionate precursor for production of poly(hydroxybutyrate-co-valerate)(PHBV) from Ralstonia eutropha H16.
First of all, the microbial cell was cultured in defined medium under a variety of factors including concentration of crude glycerol and sodium propionate, the time of sodium propionate addition, and the concentration of yeast extract or phosphate. Subsequently, a 2 litres fermentation system was introduced to investigate the effect of dissolved oxygen (DO) upon cell growth and PHBV content. The results indicate that the optimum concentration of carbon source for R. eutropha H16 to accumulate PHBV are 5% (w/v) crude glycerol and 7.5 g L-1 of sodium propionate. The 69.98% of PHBV content where PHV content is 12.31% can be achieved as precursor was added after 12 hours of cultivation time. The increasing concentration of yeast extract can improve cell density but no significantly effect on PHBV accumulation was observed. The optimal concentration of phosphate in culture is 0.058 g L-1. The higher DO controlled value in fermenter displayed a significant increment in both cell density and PHBV content. In order to improve PHV production, the response surface methodology（RSM）was employed to study the optimal condition of PHV accumulation including concentration of crude glycerol or sodium propionate, the concentration of phosphate, the time of sodium propionate addition, and dissolved oxygen (DO) by R. eutropha H16. From RSM results, the theoretical equation Y＝7.394＋1.9288772X1＋0.091311X2－1.987625X12－0.5875X1X2－2.492625X22 is obtained. It showed the maximum PHV concentration 7.8658 g L-1 therotically at optimal conditions of 57.77 g L-1 of crude glycerol, 10 g L-1 of sodium propionate, 0.0713 mole L-1 of phosphate, 50% DO control and post-addition of sodium propionate at 12th hr after 72 hours incubation. The produced polymer is identified as PHBV by NMR analysis and characterized with an average molecular weight in range between 290,000 to 320,000 Daltons at PDI values in range of 1.77 to 1.99.

並列關鍵字

Polyhydroxyalkanoates ； Crude glycerol ； fermentation ； Ralstonia eutropha H16 ； response surface methodology

參考文獻

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