Azotobacter vinelandii ATCC 13705（A. vinelandii）是一株革蘭氏陰性好氧固氮菌，在本研究探討A. vinelandii對四氰化鎳（Tetracyanonickelate，簡稱TCN）行降解代謝之可能途徑。當此菌在生長時，可降解TCN（1、10及20 mM）為終產物氨（NH3）和甲烷（CH4）；此菌之細胞萃取液（cell-free extracts）並無法降解TCN，推測固氮酵素（nitrogenase）可能是唯一有參與TCN分解代謝過程之酵素。菌株分解1 mM TCN時，在第19小時降解速率最快速，結果顯示此時固氮酵素活性和氧呼吸率最明顯，而推測在此菌胞內溶氧濃度大量被利用，以排除胞內氧氣對nitrogenase去活化並產生大量ATP供細菌生長利用。另外，本研究亦發現當含有其他氮源如ammonia和nitrite時，會抑制菌株降解TCN。在靜止細胞（resting cells）中降解1 mM TCN時，加入8%葡萄糖時則可加速TCN之降解。此研究可瞭解A. vinelandii分解TCN之機制，以作為應用於含氰化物廢水的工廠處理之參考。

In this study, Azotobacter vinelandii ATCC13705 (A. vinelandii), which is a free-living, nitrogen-fixing, gram-negative, and aerobic rod bacterium, was need to evaluate its ability to biodegrade tetracyanonickelate (TCN) under different conditions. Results show that A. vinelandii was able to biodegrade various concentrations of TCN (1, 10, and 20 mM) under aerobic conditions. Oxygen consumption and nitrogenase activity were investigated at 1 mM of TCN. Results indicate that the production of ammonia and methane was observed when TCN was consumed. Results suggest that nitrogenase was possibly involved in the enzymatic degradation of TCN. Moreover, higher degradation rate of TCN, higher nitrogenase activity, higher oxygen consumption, and higher specific growth rates were also observed at log growth period. Results suggest that the hypothesis of respiratory protection of nitrogenase is supported. Moreover, the addition of ammonia (1, 5, and 10 mM) would cause the decrease of TCN degradation rate (28%) during a 24-hr incubation period. Inhibition of TCN degradation (degradation rate：16% for 24 hrs) was observed when nitrite (5 and 10 mM) was added into the growth medium. Furthermore, the addition of 8% of glucose would significantly enhance the TCN degradation by the resting cells (degradation rate：43% for 8 hrs) . Results from this study provide us insight into the characteristics and mechanisms of TCN conversion by A. vinelandii.

謝誌 I
摘要 II
英文摘要 III
目錄 IV
圖目錄 VII
表目錄 IX
第一章 前言 1
1-1 固氮菌的發現與Azotobacter vinelandii的簡介 1
1-2 氰化物的來源與特性 3
1-3 氰化物的處理機制 5
1-3-1 化學處理法 5
1-3-2 生物處理法 7
1-4 氰化物分解酵素及其代謝途徑 10
1-4-1 Rhodanese 11
1-4-2 Mercaptopyruvate sulfurtranase 11
1-4-3 Cyanide hydratase 12
1-4-4 Cyanidase 13
1-4-5 Nitrilase 13
1-4-6 Cyanide oxygenase 14
1-4-7 Nitrogenase 15
1-5 固氮酵素的保護策略及其在氰分解過程中所擔任的角色 16
第二章 研究目的 20
第三章 實驗設備與方法 21
3-1 實驗材料 21
3-1-1 實驗菌種 21
3-1-2 化學藥品 21
3-1-3 培養基 21
3-2 實驗方法與步驟 22
3-2-1 菌種之製備 22
3-2-2 細胞質量之測量 23
3-2-3 四氰化鎳濃度標準曲線之製備 23
3-2-4 TCN降解能力之實驗 24
3-2-5 細胞萃取液之製備 24
3-2-6 靜止細胞之製備 25
3-2-7 銨離子濃度之測定 25
3-2-8 甲醯胺濃度之測定 26
3-2-9 甲酸濃度之測定 26
3-2-10 固氮酵素及氰化物分解活性之分析 27
3-2-11 呼吸率之分析 28
第四章 結果與討論 29
4-1 A. vinelandii分解四氰化鎳之生長情況 29
4-2 A. vinelandii降解四氰化鎳之產物檢測 31
4-3 添加其它氮源對四氰化鎳降解能力之影響 35
4-3-1 銨離子 36
4-3-2 硝酸根離子 37
4-3-3 亞硝酸根離子 38
第五章 結論 39
第六章 圖表 41
第七章 參考文獻 58

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