臺灣博碩士論文加值系統

溫室氣體所造成的全球暖化現象日益嚴重，其中又以二氧化碳之影響最甚，因此近年來二氧化碳減量之議題備受全世界關注。先前本實驗室已透過轉殖綠硫菌之還原性三羧酸循環之關鍵酵素至大腸桿菌中，開發微生物固碳平台，於先前研究中藉固碳效率計算證實該菌株確可額外固定二氧化碳為其生物質。本研究將探討具有還原性三羧酸循環之大腸桿菌，其對於厭氧無糖環境之化學自營的可行性以及分子特性。首先將本研究之固碳菌株培養在厭氧無糖的 M9 基礎培養基下，透過額外添加電子提供者、接受者 (DMSO) 與氮源，觀察其生長狀況發現，固碳菌株的生菌落數以及 O.D600值都比質體對照組高出許多。並且在在無二氧化碳添加的條件下可發現固碳菌株幾乎不生長，這表示固碳菌株會利用二氧化碳來進行生長。在分析固碳菌株與對照組的轉錄體差異中，發現固碳菌株的 GABA shunt 相關酵素基因具有顯著的提升表現，特別是 gad 基因所構築之各個酵素，因此菌株的固碳作用可能與 GABA shunt 具有關連性。接著在 Aminoacyl-tRNA biosynthesis 的相關基因表現量也觀察到了固碳菌株表現量較高的趨勢，這可能說明了固碳菌株生長優勢的原因。在分析胞內代謝物的結果中，本研究觀察到了固碳菌株與對照菌株的 Energy Charge 趨勢相同，並且都有增加的現象，這可說明菌株會使用培養基中的電子提供者與接受者來產生能量，而固碳菌株的 Total Adenylate 總量明顯較高，可能是造成生長優勢的原因。最後本研究將電子接受者更改為中點電位差更大的硝酸根，此環境使得固碳菌株生長狀況更佳。由上述結果可推論出氧化磷酸化驅動此菌株的電子傳遞使其產生能量，而 GABA shunt 中的 GAD 酵素會消除胞內氫離子並促進質子動力勢的形成，以持續產生能量用於固碳作用生長。

The increase of atmospheric carbon dioxide, which is considered as a major greenhouse gas, plays an important role in global warming. Therefore, finding a method to decrease CO2 is an attractive global issue. A set of reductive tricarboxylic acid (rTCA) cycle genes have been introduced from Chlorobium tepidum TLS to Escherichia coli. It was confirmed that genetic engineering strain could fix CO2 in anaerobic culture using 0.2% glucose as carbon sources. In this study, the chemoautotrophic potential and characteristics of E. coli with rTCA cycle was determined under anaerobic and sugar-free condition. The rTCA strain was cultured in an anaerobic, sugar-free M9 medium, adding electron donors, receptors and nitrogen sources. The growth curve of rTCA strain was much higher than control strain. However, the rTCA strain did not grow without CO2 in anaerobic conditions. According to transcriptome data, significant up-regulation of GABA shunt related genes were in rTCA strains, such as gad gene. Therefore, the carbon fixation of rTCA strain may be related to GABA shunt. In the results of intracellular metabolite analysis, the phenomenon of increased energy charges in the two groups of strains was the same.. The total adenylate of the rTCA strain was significantly higher, possibly cause its growth advantages. And then the electron acceptor DMSO was changed to nitrate, which increased the rTCA strain growth. This study proposed a hypothesis for rTCA strain that GAD enzyme from GABA shunt decrease intracellular hydrogen ions from oxidative phosphorylation and promotes the proton-motive force of the cell. It makes energy be continuously produced for carbon fixation and growth.

第一章、前言 1
一、 二氧化碳減量處理法 2
(一) 物理處理 2
(二) 化學處理 3
(三) 生物固定 4
二、 自然界中固定二氧化碳的六種途徑 4
三、 化學自營性 ( Chemoautotroph)、混營性 ( Mixotroph ) 及電子傳遞 9
四、 前人研究 10
(一) rTCA基因來源 10
(二) 三羧酸循環 (TCA cycle) 與還原性三羧酸循環 (rTCA cycle) 12
(三) 大腸桿菌中建構還原性三羧酸循環之特性 13
五、 研究動機與目的 18
第二章、實驗材料與方法 19
一、實驗菌株 19
二、培養基 19
三、萃取質體DNA 20
四、聚合酶連鎖反應 (polymerase chain reaction, PCR) 22
五、洋菜膠體電泳分析 (agarose gel electrophoresis) 23
六、生長曲線測定 24
七、RNA定序 25
八、差異性基因表現之分析 (Analysis of Differential Expressed Gene) 26
九、胞內代謝物與碳 13 標定分析 28
第三章、結果 30
一、化學自營模式之生長探討 30
(一) 批次厭氧無糖生長曲線 30
(二) 厭氧無糖培養之碳源確立 33
二、厭氧無糖培養條件下之轉錄體分析 36
(一) 固碳菌株與對照菌株基因表現差異 39
(二) 固碳菌株經時變化之基因表現差異 46
三、胞內代謝物產量及標定碳 13CO2 碳流分析 52
四、更改電子接受者測試生長能力 60
第四章、檢討與討論 62
一、碳 13 標定分析偵測量較少 62
二、厭氧無糖的分子特性推測 62
第五章、結論與未來研究方向 64
第六章、參考文獻 65
附錄 69

行政院環境保護署溫減管理室 (2014). 溫室氣體排放統計. Retrieved from http://www.epa.gov.tw/ct.asp?xItem=10052&ctNode=31352&mp=epa
談駿嵩，鄭旭翔 (2006). 台灣在二氧化碳回收及再利用上之研究現況.
林佳璋，劉文宗 (2006). 二氧化碳回收技術
李中光，劉新校, et al. (2012). 淺談利用微藻固定CO2實現碳減排.
沈怡蓉 (2010). 微藻減碳-打造綠色世代新紀元.
王冠翡 (2011). 以藻類去除半導體業廢氣中二氧化碳之可行性研究.
劉仲康 (2012). 無氧下的生命現象：談無氧呼吸與發酵作用.
黃琡涵 (2014). 應用合成生物學技術於大腸桿菌中建立一固碳途徑-還原性三羧酸循環.
余佳樺 (2016). 以合成生物學建構還原性三羧酸循環於大腸桿菌之特性研究.
Berg, I. A. (2011). Ecological aspects of the distribution of different autotrophic CO2 fixation pathways. Applied and environmental microbiology, 77(6), 1925-1936.
Dixon, R. K., Solomon, A., Brown, S., Houghton, R., Trexier, M., & Wisniewski, J. (1994). Carbon pools and flux of global forest ecosystems. Science, 263(5144), 185-190.
Downing, T. E. (2013). Climate change and world food security (Vol. 37): Springer Science & Business Media.
Duckett, M., & Ruhemann, M. (1985). Cryogenic gas separation. Chemical engineer(420), 14-17.
Eisen, J. A., Nelson, K. E., Paulsen, I. T., Heidelberg, J. F., Wu, M., Dodson, R. J., . . . Haft, D. H. (2002). The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proceedings of the National Academy of Sciences, 99(14), 9509-9514.
Falkow, S., Rosenberg, E., Schleifer, K-H., Stackebrandt, E. (2006) The Prokaryotes: Vol. 2: Ecophysiology and Biochemistry
Gong, F., Liu, G., Zhai, X., Zhou, J., Cai, Z., Li, Y. (2015). Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Biotechnology for Biofuels (2015) 8:86
Hassol, S. J., Arctic Climate Impact Assessment., et al. (2004). Impacts of a warming Arctic : Arctic Climate Impact Assessment. Cambridge, U.K. ; New York, N.Y., Cambridge University Press.
Harvell, C. D., Mitchell, C. E., Ward, J. R., Altizer, S., Dobson, A. P., Ostfeld, R. S., & Samuel, M. D. (2002). Climate warming and disease risks for terrestrial and marine biota. Science, 296(5576), 2158-2162.
Huber, H., Gallenberger, M., Jahn, U., Eylert, E., Berg, I. A., Kockelkorn, D., . . . Fuchs, G. (2008). A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis. Proceedings of the National Academy of Sciences, 105(22), 7851-7856.
Itaya, M., & Kaneko, S. (2010). Integration of stable extracellular DNA released from Escherichia coli into the Bacillus subtilis genome vector by culture mix method. Nucleic acids research, 38(8), 2551-2557.
Jin H. F, Lim B. R, Lee K. Influence of nitrate feeding on carbon dioxide fixation by microalgae. Journal of environmental science and health. Journal of Environmental Science and Health, Part A 2006;41:2813–24.
Kaster, A.-K., Goenrich, M., Seedorf, H., Liesegang, H., Wollherr, A., Gottschalk, G., & Thauer, R. K. (2011). More than 200 genes required for methane formation from H2 and CO2 and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus. Archaea, 2011.
Kockelkorn, D., & Fuchs, G. (2009). Malonic semialdehyde reductase, succinic semialdehyde reductase, and succinyl-coenzyme A reductase from Metallosphaera sedula: enzymes of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in Sulfolobales. Journal of bacteriology, 191(20), 6352-6362.
Lashof, D. A., & Ahuja, D. R. (1990). Relative contributions of greenhouse gas emissions to global warming.
Mitchell, J. F. (1989). The “greenhouse” effect and climate change. Reviews of Geophysics, 27(1), 115-139.
Foster, B. (2001). IPCC’s Third Assessment Report.
Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008). Lehninger principles of biochemistry: Macmillan.
Stocker, T., Qin, D., Plattner, G., Tignor, M., Allen, S., Boschung, J., . . . Midgley, B. (2013). IPCC, 2013: climate change 2013: the physical science basis. Contribution of working group I to the fifth assessment report of the intergovernmental panel on climate change.
Sydney, E.B., Sturm W., Carvalho J.C., Thomaz-Soccol V., Larroche C., Pandey A., et al. Potential carbon dioxide fixation by industrially important microalgae. Bioresource Technology 2010;101:5892–6.
Tang, K.-H., & Blankenship, R. E. (2010). Both forward and reverse TCA cycles operate in green sulfur bacteria. Journal of Biological Chemistry, 285(46), 35848-35854.
Thakker, C., Martínez, I., San, K. Y., & Bennett, G. N. (2012). Succinate production in Escherichia coli. Biotechnology journal, 7(2), 213-224.
Tsuge, K., Matsui, K., & Itaya, M. (2003). One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic acids research, 31(21), e133-e133.
Zhuang, Z. Y., Li, S. Y. (2013). Rubisco-based engineered Escherichia coli for in situ carbon dioxide recycling. Bioresource Technology 150 (2013) 79–88
Lam MKLK. (2013) Effect of carbon source towards the growth of Chlorella vulgaris for CO2 bio-mitigation and biodiesel production. International Journal of Greenhouse Gas Control 2013;14:169–76.
Ramskold, D., Wang, E. T., Burge, C. B., & Sandberg, R. (2009). An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data. PLoS Comput Biol, 5(12), e1000598.
Ingledew W. J., Poole R. K. (1984). The respiratory chains of Escherichia coli. Microbiol. Rev. 48 (3): 222–71.
Jinshui W., Yinjie S., Feng Jia and Huali J. (2013). Characterization of casein hydrolysates derived from enzymatic hydrolysis. Chemistry Central Journal. 7:62.
Yiling Y., Abiola M. P., Carolin H., Gernot P., Markus W., Rüdiger H. and Victor S. (2015). Relation between chemotaxis and consumption of amino acids in bacteria. Molecular Microbiology. 96(6), 1272–1282.
Yike N., Xuejiao W., Chenglin Z., Qingyang X., Ning C., Xixian X. (2016). Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli. Metabolic Engineering 36, 10–18.