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標題: 以基因重組大腸桿菌建立二氧化碳固定平台
Development of the bio-mitigation platform for carbon dioxide fixation by recombinant Escherichia coli
作者: 莊宗諭
Zhuang, Zong-Yu
關鍵字: 基因重組大腸桿菌;recombinant E. coli;二氧化碳固定;發酵條件;carbon dioxide fixation;fermentation condition
出版社: 化學工程學系所
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由於近百年來大量使用各式石化燃料造成大氣中二氧化碳濃度不斷攀升,若放任大氣中二氧化碳濃度不斷增加,必會對未來造成不可挽救後果。大氣中二氧化碳可藉卡爾文循環中ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO)蛋白而被固定再利用,因此本實驗在大腸桿菌內建立部分卡爾文循環,藉此發展一生物緩解平台基礎,使此平台之基因重組大腸桿菌具備固定自身排放二氧化碳之能力,並經由理論計算,此平台最大可減少40%之二氧化碳排放。在好氧批次培養中,以LB培養基(Luria-Bertani medium)添加20g/L L-arabinose為碳源後,基因重組大腸桿菌之菌體濃度即提升1.26~1.66倍,可達到較高生長水平。而以發酵槽控制pH值為7.0,並以含20g/L L-arabinose之LB培養基進行半厭氧發酵下,再藉由計算L-arabinose消耗量、乙酸乙醇代謝物產量與二氧化碳生產量相互間之分率,如(ethanol + acetic acid)/sugar consumption (mol/mol)、CO2/sugar consumption (mol/mol)分率變化,來比較量基因重組大腸桿菌固定二氧化碳能力。基因重組大腸桿菌與原生大腸桿菌之(ethanol + acetic acid)/sugar consumption (mol/mol)皆接近為0.72,而基因重組大腸桿菌之CO2/sugar consumption (mol/mol)較原生大腸桿菌下降16.2%,而理論最大下降值為40%,經目前實驗基因重組大腸桿菌固定二氧化碳能力以達成理論之40.5%,顯示此生物緩解平台具有以基因重組大腸桿菌固定二氧化碳之潛力。而以NaHCO3、MgSO4、K2HPO4等鹽類作為因子探討在半厭氧批次培養之固碳效率時,可知NaHCO3與MgSO4無法改善固碳效率,但K2HPO4與ATP生成及合成固碳作用之前驅物皆有所相關,故在此可藉K2HPO4改善醣消耗量與固碳效率。

Since all kinds of fossil fuels are currently used on a large-scale, the carbon dioxide concentration in the atmosphere has continued to rise. If this condition cannot be controlled, it will cause irreversible consequences in the future. The carbon dioxide can be fixed and re-used by the 1,5-bisphosphate carboxylase/oxygenase(RuBisCO) protein, which is the key protein in the Calvin cycle. This study utilizes this mechanism to establish part of the Calvin Cycle in E. coli for development of the bio-mitigation platform for carbon dioxide fixation. In theory, this platform can reduce carbon dioxide emissions by 40%. The experimental results indicated that the LB (Luria-Bertani) medium contained 20g/L L-arabionse as a carbon source which in aerobic batch culture, the O.D.600 value of this recombinant E. coli will be 1.26~1.66-flO.D. of the wild type E. coli. This recombinant E. coli can achieve a higher growth level. In the fermentation experiment, the LB medium contained 20g/L L-arabionse and it was used as a carbon source and it had controlled pH value at 7.0 in the semi-anaerobic fermentation. The data of L-arabinose consumption, ethanol accumulation, acetic acid accumulation, and carbon dioxide evolution can be used to calculate for the yield of (ethanol + acetic acid)/sugar consumption (mol/mol) and CO2/sugar consumption (mol/mol). With this information, the carbon dioxide fixation rate of recombinant E. coli can be compared with the wild type. The yield of (ethanol + acetic acid)/sugar consumption (mol/mol) on recombinant and wild type E. coli are both close to 0.72, while the yield of CO2/sugar consumption (mol/mol) on recombinant E. coli is decreased by 16.2% compared to wild type. The theoretical maximum reduced value is 40%. This study achieved 40.5% of theoretical, which indicated the bio-mitigation platform has the potential of carbon dioxide fixation. In order to improve the carbon dioxide fixation efficiency, this study used NaHCO3, MgSO4, K2HPO4 as the factor. The semi-anaerobic batch culture indicated that NaHCO3 and MgSO4 cannot improve the carbon dioxide fixation efficiency. In addition, K2HPO4 related to ATP generation and synthetic precursor of carbon dioxide fixation can enhance the sugar consumption and carbon dioxide fixation efficiency.
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