Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5884
標題: 突變 NifA 蛋白質結構提升 Rhodopseudomonas palustris WP3-5氨氮容忍能力之產氫研究
Evaluating the feasibility of H2 production in ammonium-rich medium with ammonium-tolerant Rhodospeudomonas palustris WP3-5 improved by NifA mutation
作者: 王思穎
Wang, Szu-Ying
關鍵字: Rhodopseudomonas palustris WP3-5
Rhodopseudomonas palustris WP3-5
NifA
氨氮
氫氣
NifA
ammonium
hydrogen
出版社: 環境工程學系所
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摘要: 氫氣是乾淨的替代性能源,燃燒後只產生水及熱能,對環境不會造成傷害,且熱值含量高達122 kJ/g。產氫方式分為物理、化學及生物產氫。紫色不含硫光合細菌是微生物產氫之主要菌群,其產氫機制是藉由其固氮酵素在固氮的過程中產生氫氣為副產物。固氮酵素會受到其活化子NifA蛋白質的調控,然而,NifA是否有活性會受到環境當中氨氮濃度所影響。當含有高濃度氨氮時會使得NifA活性受到抑制,導致無法調控下游固氮酵素相關基因的轉錄,因此固氮酵素不能表現,也造成沒有氫氣的產生。本研究藉由分子生物技術製造對氨氮容忍能力較高的Rhodopseudomonas palustris WP3-5之突變株,以便應用在利用含氨氮廢水的光醱酵產氫上面。利用PCR將nifA的Q區段進行基因序列刪除法,再以接合作用使得含有nifAnQ基因之質體進入Rhodopseudomonas palustris WP3-5,與其染色體之nifA進行二次基因重組,得到Rhodopseudomonas palustris NifAnQ突變株。以glutamate為氮源之下,野生株WP3-5及突變株NifAnQ 24產氫能力並無差異,證實突變株的固氮酵素表現沒有受到影響。另一方面,WP3-5在氮源為氯化銨時,濃度需低於0.9 mM才有氫氣產生,NifAnQ 24在濃度為7.5、18.7、及28.0 mM皆可產氫,且無論其碳源為醋酸或是乳酸。當NifAnQ 24以乳酸為碳源且基質含有高濃度氨氮時,其比產氫速率可達到97.36 mL-H2/g-biomass/day,基質轉換效率為27.8%。由此可知突變株NifAnQ 24在富含氨氮的基質中可以成功地利用光合醱酵產氫。嘗試以高初始菌體濃度進行產氫試驗,預期可達到將較多碳源分佈於產氫而非生長,然而,結果顯示NifAnQ 24並沒有因為提高菌體初始濃度而使得氫氣有較好的表現。
Hydrogen is a clean alternative energy. The advantage of hydrogen is only produce energy and water after burning, and it has high energy content (122kJ/g). Hydrogen can be produced by physical, chemical or biological process. Purple non-sulfur bacteria (PNSB) is the major microbial group to produce hydrogen. The mechanism of PNSB to produce hydrogen is mainly mediated through nitrogenase, evolved to catalyze nitrogen fixation. Nitrogenase expression is regulated by NifA, which is a nitrogenase activator. However, NifA activity is affected by ammonium concentration. The high ammonium concentration can inhibit NifA activity, therefore it can’t regulate the gene, which related nitrogenase, to transcription, and nitrogenase can not be expressed. In this study, a R. palustris NifAnQ 24 mutant with an in-frame deletion of the Q region of chromosomal nifA gene was constructed from R. palustris WP3-5 to overcome the inhibition of nitrogenase activity by ammonium. A series of ammonium-rich mediums with acetate or lactate as carbon sources and ammonium chloride or glutamate as nitrogen sources were used to compare the hydrogen production between the wild-type strain WP3-5 and the mutant strain NifAnQ 24. The result showed that, hydrogen production of NifAnQ 24 was not significantly different from WP3-5 in glutamate-contained medium, indicated that nitrogenase expression were not affected in NifAnQ 24. On the other hand, WP3-5 could produce hydrogen only when the ammonium chloride in medium was below 0.9 mM, while NifAnQ 24 could produce hydrogen in the medium when ammonium chloride concentration was 7.5, 18.7, and 28.0 mM with acetate or lactate as carbon source. In the medium with lactate as carbon source and rich in ammonium, the NifAnQ 24 SHPR was 97.36 mL-H2/g-biomass/day, and SCE is 27.8%. The result showed that NifAnQ 24 could successfully produce hydrogen by photo fermetation in ammonium rich medium. The possibility of increasing more carbon source distribution to hydrogen production than to growth by increasing initial cell concentration was also evaluated. The results indicated that, the hydrogen production of NifAnQ 24 couldn’t be improved by high initial cell concentration.
URI: http://hdl.handle.net/11455/5884
其他識別: U0005-1807201300044500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1807201300044500
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