本研究合成出一個具混營生長特性之大腸桿菌，透過2-酮戊二酸:鐵氧還蛋白氧化還原酶（2-oxoglutarate:ferredoxin oxidoreductase, OGOR）的異源表現（heterologous expression）使該菌株在以蘋果酸（malate）作為額外碳源及氫氣作為能量來源下吸收二氧化碳作為無機碳源。我們利用自行設計之生物反應器進行長時間（約180天）的二階段適應性演化實驗，使異營的大腸桿菌突變為混營生長。該生物反應器在實驗過程中展現了其優點，其可在厭氧條件下透過無線傳輸系統即時傳送生長數據，且其偵測生長數據的時間間隔短，有利於觀察大腸桿菌的生長特性，例如600奈米波長之光密度（600nm wavelength optical density, OD600）及生長速率（growth rate），一些需要較佳時間解析度的生長特性也得以觀察，例如微生物的兩階段生長（diauxic growth）。適應性演化實驗分為兩階段，第一階段進行絲氨酸（serine）的演化實驗，菌株在演化後觀察到兩階段生長的現象。第二階段進行蘋果酸（malate）的演化實驗，菌株在演化後剩下一個區間的混營生長，且二氧化碳成為該菌株生長之必須要素，使二氧化碳的吸收得到驗證。我們期望本實驗可開啟更多有關2-酮戊二酸:鐵氧還蛋白氧化還原酶（OGOR）對生物固碳可能性之研究，同時利用本生物反應器進行長期的演化實驗也顯見了其穩定性。

We report the mixotrophic growth of Escherichia coli based on recombinant 2- oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO2 by using malate as auxiliary carbon source and hydrogen as energy source. We employ a long term (~180 days) two stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli by a self-designed bioreactor. The bioreactor shows its robustness during the whole process, such as transmitting real-time growing data wirelessly under anaerobic condition. Its short time intervals of data sensing also make it easy to observe E. coli culture properties such as optical density and growth rate, properties requiring better time resolution such as diauxic growth can also be observed. The adaptive evolution is divided into two stages. In the first stage of evolution with serine, mixotrophy with CO2 assimilation is manifested as second phase in diauxic growth. In the end of the second stage of evolution with malate, the strain exhibits pure mixotrophy with CO2 as essential substrate for growth. We expect this work will open new possibilities in utilization of OGOR for microbial CO2 assimilation and future hydrogen-based electro-microbial conversion, performing adaptive evolution utilizing our bioreactor also shows the stability and robustness of our devices.

致謝..................................................... I
中文摘要.............................................. III
ABSTRACT.......................................... IV
目錄..................................................... VI
圖目錄................................................. IX
表目錄................................................. XII
一、 緒論............................................. 1
1-1研究動機......................................... 1
1-1-1全球氣候的改變........................... 1
1-1-2研究目標..................................... 4
1-2文獻回顧........................................ 5
1-2-1固碳途徑..................................... 5
1-2-1-1卡爾文循環.............................. 6
1-2-1-2 還原性三羧酸循環.................. 8
1-2-2利用微生物進行固碳反應............ 9
1-2-3 前人研究基礎............................. 13
二、 實驗材料及方法........................... 18
2-1實驗樣本........................................ 18
2-2厭氧培養基的製備......................... 18
2-3洋菜盤的製備及塗盤...................... 20
2-4菌種保存的製作............................. 21
2-5菌株預養........................................ 22
2-6演化實驗設計................................. 23
2-7硬體裝置........................................ 25
2-7-1 硬體簡介.................................... 25
2-7-2 氣體條件及實現方式.................. 28
2-7-3生物反應器之操作方式............... 30
三、 實驗結果...................................... 32
3-1實驗過程概述................................. 32
3-2演化實驗細節................................. 34
3-2-1 第一階段－以絲胺酸作為碳源.... 34
3-2-2第二階段－以蘋果酸作為碳源..... 37
3-3演化結果........................................ 40
3-3-1 第一階段演化菌株比較............... 40
3-3-2 第二階段演化菌株比較.............. 42
3-3-3驗證氫氣能量來源...................... 46
四、 結論及討論.................................. 49
五、 參考文獻...................................... 51
六、 附錄............................................. 55
A-1演化實驗全部數據.......................... 55
A-2演化實驗的重複............................. 56
A-3 ARDUINO 程式碼......................... 57
A-4 全基因定序分析........................... 66
A-5 中英對照表.................................. 69

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