本研究從台中市工業區污水處理廠以及台北市民生污水處理廠之污泥中分離出數株純種之脫硝菌株，並從中擇一脫硝效率最佳者 (暫訂名為THU菌株) 對其進行生化特性之研究，寄望能對脫硝菌株的特性與脫硝反應能有更深的了解，並進一步將菌株以固定化處理方式來提高其脫硝效率。
最後選出之脫硝菌株THU為一革蘭氏陰性桿菌，具有鞭毛，在絕對厭氧的環境中無法生長，為一絕對好氧性脫硝菌。THU菌株在溶氧濃度約為 5.3 mg/L 的情況下依然能進行脫硝作用，生長過程中能同時消耗氧氣及硝酸氮，並將硝酸氮轉換成氮氣而排放至環境中，而經由 pH 及ORP的即時監控亦可以觀察到脫硝反應進行的過程。而THU菌株進行脫硝反應時的最佳溫度為37oC，最佳 pH 為8.05，最適利用碳源為醋酸鈉。當碳氮比控制為7時，培養基中的溶解性有機碳與硝酸氮均可同時去除完畢，而經由生長及脫硝動力模式的推求，可以得到THU菌株之最大比生長速率為0.58 hr-1，最大比脫硝速率為0.7 mg-NO3/mg-VSS·hr。
當培養基中氮源置換成氯化氨後，發現THU菌株亦可以利用氨氮進行生長，並可進一步將 NH4+ 經由 NO3? 轉換至 N2O，顯示THU菌株是一株可進行同時硝化-脫硝 (SND) 反應之菌株，且當NH4+起始濃度為80 mg/L 時，總氮去除率幾乎可達100%。
利用 Alginate 進行固定化生物擔體用以去除硝酸氮時，發現菌株經固定化後之硝酸氮去除效率比懸浮系統中之效率有顯著的提升，而擔體強度不足的問題藉由聚乙二醇及聚乙烯亞胺的添加後可以得到大幅的改善。

Several strains of denitrifying bacteria were isolated from wastewater treatment plant of Taichung industrial park and Men-Shen sewage treatment plant in this study. In order for further investigation of the characteristics of denitrifying bacteria and denitrification process, an isolate, tentatively named strain THU was chosen and its biochemical characteristics were investigated in this study. Immobilization of strain THU for nitrate removal was done to improve the denitrification efficiency.
Strain THU was a gram-negative, strictly aerobic bacterium with monotrichous flagellum. It could not grow in strictly anaerobic conditions. Denitrification process still occurred even when the concentration of dissolved oxygen was 5.3 mg/L. Nitrate was reduced to nitrogen gas by strain THU. Oxygen and nitrate-nitrogen were consumed simultaneously during denitrification process. The optimal temperature of strain THU to reduce nitrate was 37oC, the optimal pH was 8.05, and the optimal carbon source was sodium acetate. Both of nitrate and dissolved organic carbon in the medium could be removed completely when the C/N ratio was 7. The specific growth rate and the specific denitrification rate of strain THU were 0.58 hr-1 and 0.70 mg-NO3﷓-N/mg-VSS·hr, respectively.
It was found that strain THU could still grow by using ammonia when NH4Cl was used as the N source in the medium. When the initial concentration of ammonia was 80 mg/L, total nitrogen removal could be achieved by almost 100%. By the transfer of NH3 to N2O, it showed that strain THU had the capacity of nitrification and denitrification simultaneously.
After strain THU was entrapped by alginate, the nitrate removal rate by immobilization system was much better than that of suspended-growth system. The strength of the immobilized beads can be enhanced by mixing alginate with polyethyleneimine and polyethylene glycol.

第一章前 言………………………………………………1
第二章文獻回顧………………………………………………3
2.1生物除氮反應…………………………………………3
2.1.1生物脫硝反應…………………………………………5
2.1.2生物硝化反應…………………………………………9
2.1.3同硝化-脫硝反應之原理與機制……………………10
2.2脫硝作用之環境影響因子 …………………………14
2.2.1溶氧 …………………………………………………14
2.1.1.1厭氧脫硝 ……………………………………………14
2.1.1.2好氧脫硝 ……………………………………………16
2.2.2溫度 …………………………………………………22
2.2.3pH ……………………………………………………23
2.2.4碳源與碳/氮比對脫硝效率之影響…………………23
2.2.5氧化還原電位 ………………………………………25
2.3純種菌之添加對於脫硝效率之影響 ………………26
2.4固定化細胞生長系統 ………………………………27
2.4.1固定化技術之發展 …………………………………27
2.4.2固定化微生物技術之優點及限制 …………………31
2.4.3固定化處理技術之應用 ……………………………32
2.4.4以褐藻膠鈣包覆菌體之固定化處理技術 …………35
2.5研究目的 ……………………………………………36
第三章實驗方法與設計 ……………………………………37
3.1實驗流程設計 ………………………………………37
3.2菌種來源採樣 ………………………………………37
3.3菌種之分離、培養與選取 …………………………39
3.3.1菌種之分離與培養 …………………………………39
3.3.2菌種之選取 …………………………………………41
3.4菌種生化特性之探討 ………………………………41
3.4.1菌相觀察 ……………………………………………42
3.4.2生長速率 ……………………………………………42
3.4.3溫度 …………………………………………………44
3.4.4pH ……………………………………………………44
3.4.5碳源 …………………………………………………45
3.4.6碳氮比 ………………………………………………45
3.4.7基質抑制效應 ………………………………………46
3.4.8休眠細菌試驗 ………………………………………46
3.4.9絕對厭氧環境培養 …………………………………46
3.4.10Lab VIEW電腦即時監測 ……………………………47
3.4.11BNP (Biochemical Nitrogen Potential) 反應系
統 ……………………………………………………49
3.5固定化THU菌株脫硝反應……………………………50
3.5.1細菌固定化程序 ……………………………………50
3.5.2固定化生物擔體反應槽裝置 ………………………52
3.5.3固定化擔體於不同CaCl2 溶液浸泡時間下之強度
探討 …………………………………………………53
3.6菌種保存 ……………………………………………55
3.7菌種鑑定 ……………………………………………55
3.7.1鑑別染色 ……………………………………………55
3.7.2Biolog microstation system菌種鑑定 …………57
3.7.3API 20E菌種鑑定……………………………………57
3.8實驗分析項目與方法 ………………………………58
3.8.1含氮化合物之分析 …………………………………58
3.8.2溶解性有機碳 (DOC) 之測定………………………62
3.9電子顯微鏡照相 ……………………………………63
3.9.1掃瞄式電子顯微鏡照相 ……………………………63
3.9.2穿透式電子顯微鏡照相 ……………………………64
第四章結果與討論 …………………………………………66
4.1研究菌株之選取 ……………………………………66
4.2菌相觀察 ……………………………………………69
4.3菌種鑑定 ……………………………………………71
4.3.1革蘭氏染色 …………………………………………71
4.3.2Biolog 菌種鑑定……………………………………72
4.3.3API 20E 菌種鑑定 …………………………………73
4.4生化特性之探討 ……………………………………76
4.4.1休眠細菌脫硝反應 …………………………………76
4.4.2溶氧對脫硝菌株之影響 ……………………………77
4.4.3最佳化溫度 …………………………………………80
4.4.4最佳pH ………………………………………………83
4.4.5最佳碳源 ……………………………………………87
4.4.6最佳碳氮比 …………………………………………91
4.4.7細胞生長及脫硝反應動力學 ………………………95
4.4.8硝酸氮濃度抑制效應 ……………………………101
4.4.9進行脫硝反應之氧化還原電位 (Oxidation-Reduction
Potential, ORP) 及 pH 變化即時監測…………104
4.4.10脫硝產氣反應之探討………………………………107
4.4.11THU菌株與其他脫硝菌株脫硝效率之比較 ………111
4.5脫硝菌株THU進行同硝化-脫硝 (Simultaneous
Nitrification and Denitrification, SND)反應
之探討………………………………………………112
4.5.1THU菌株之硝化能力測試 …………………………112
4.5.2休眠細菌硝化反應…………………………………114
4.5.3最佳pH………………………………………………116
4.5.4進行SND反應之氧化還原電位 (Oxidation-Reduction
Potential, ORP) 及 pH 變化即時監測…………120
4.5.5SND 產氣……………………………………………124
4.6THU菌株與鑑定菌株之比較 ………………………126
4.7固定化菌株THU ……………………………………128
4.7.1褐藻膠鈣包覆THU菌株脫硝效率之探討 …………129
4.7.2不同固定化浸泡時間對擔體強度之影響…………131
4.7.3改良式固定化生物擔體……………………………134
第五章結論與建議…………………………………………138
5.1結論…………………………………………………138
5.2建議…………………………………………………140
第六章參考文獻……………………………………………141
附 錄………………………………………………………155
附錄一Biolog 鑑定系統之鑑定結果 ……………………155
附錄二THU菌株於Biolog 鑑定微盤中之培養結果………156
附錄三API 20E 鑑定系統之鑑定結果……………………157
附錄四THU菌株於API 20E鑑定帶中之培養結果…………158

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