臺灣博碩士論文加值系統

Ochrobactrum strain THU是一株在高溶氧下具有同時硝化脫硝能力的菌株。Ochrobactrum strain THU在溶氧4.80 mg/L的環境下，其NH4+的去除率可以達到99.3%，而當溶氧高於5.40 mg/L以上時，NH4+的去除率則只在60-70%之間，當溶氧低於3.84 mg/L以下時，strain THU的去除率則降到1-60%。因此，Ochrobactrum strain THU在溶氧4.80 mg/L的環境下有最佳的硝化能力。
本研究也針對在不同的NH2OH濃度對strain THU的影響，發現隨著NH2OH的濃度上升，會造成strain THU的攝氧率下降，且NH4+的去除率及生長速率也隨著NH2OH的濃度增加而下降，當NH2OH濃度達到6 mM時，strain THU的生長速率及NH4+的去除率完全停止。因此Ochrobactrum strain THU會受到NH2OH的影響。
將Ochrobactrum strain THU及Paracoccus denitrificans在溶氧4.80 mg/L的環境下進行同時硝化脫硝能力的比較。發現在溶氧4.80 mg/L的環境下，Ochrobactrum strain THU及Paracoccus denitrificans都能將水中的NH4+及亞硝酸鹽完全去除。
在酵素AMO及HAO的測試方面，發現到Ochrobactrum strain THU的粗細胞萃取物中，具有類似Paracoccus denitrificans酵素AMO及HAO的酵素存在。且在酵素AMO的測試中，Ochrobactrum strain THU及Paracoccus denitrificans的粗細胞萃取物能將反應液中的NH4+全部移除，說明strain THU去除氨氮的反應是經由酵素氧化反應而非同化作用。

Ochrobactrum strain THU is a bacterium capable of simultaneous nitrification and denitrification (SND) under high aerobic conditions. The NH4+ removal efficiency of Ochrobactrum strain THU can achieve 99.3% at 4.80 mg/L of dissolve oxygen (DO). The NH4+ removal efficiency by Ochrobactrume strain THU will decrease to 60-70% when DO is higher than 5.40 mg/L. When dissolved oxygen is lower than 3.84 mg/L, the NH4+ removal efficiency by Ochrobactrum strain THU will decrease to 1-60%. Ochrobactrum strain THU has the highest ability of nitrification when DO is 4.80 mg/L.
The research also investigates the concentration effect of NH2OH on growth and SND of Ochrobactrum strain THU. When concentration of NH2OH rises, the oxygen uptake rate (OUR) of Ochrobactrum strain THU decreases. The growth rate and NH4+ removal rate by Ochrobactrum strain THU will cease at 6mM of NH2OH. Therefore, the growth rate and nitrogen removal of Ochrobactrum strain THU will be inhibited by presence of NH2OH.
Simultaneous nitrification and denitrification capability of Ochrobactrum strain THU and Paracoccus denitrificans is compared at 4.80 mg/L of DO in this study. Ochrobactrum strain THU and Paracoccus denitrificans can both remove NH4+ and NO2- completely under the same condition (DO = 4.80 mg/L).
Cell extract of Ochrobactrum strain THU shows similar enzyme activities to the enzymes AMO and HAO of Paracoccus denitrificans. Both cell extract of Ochrobactrum strain THU and Paracoccus denitrificans can remove NH4+ completely in the reaction mixture, indicating that NH4+ removal by Ochrobactrum strain THU is an enzyme-catalyzed oxidation instead of assimilation process.

第一章 前 言…………………………………………….……. 1
第二章 文獻回顧…………………………………………………. 3
2.1 自然界中的氮循環……………………………………….. 3
2.2 生物除氮反應…………………………………………….. 4
2.2.1 硝化反應………………………………………………….. 4
2.2.2 脫硝反應………………………………………………….. 6
2.3 同時硝化脫硝反應……………………………………….. 7
2.4 影響同時硝化脫硝反應因素…………………………….. 12
2.4.1 溶氧……………………………………………………….. 12
2.4.2 pH…………………………………………………………. 13
2.4.3 溫度……………………………………………………….. 13
2.4.4 有機碳與碳氮比………………………………………….. 14
2.4.5 基質濃度………………………………………………….. 15
2.4.6 NH2OH的影響……………………………………………. 15
2.5 amo基因的研究介紹及AMO和HAO酵素介紹……….. 17
2.6 研究目的………………………………………………….. 18
第三章 實驗設備與方法………………………………………….. 19
3.1 菌種來源………………………………………………….. 19
3.2 菌種之培養……………………………………………….. 19
3.3 strain THU在不同溶氧之下的SND測試………………. 20
3.3.1 反應槽之架設…………………………………………….. 21
3.3.2 反應槽之操作……………………………………….……. 22
3.4 水中羥氨(hydroxylamine; NH2OH)對THU菌種的影響... 22
3.5 酵素AMO (ammonia monooxygenase)及HAO (hydroxylamine oxidoreductase)之測試………………….. 23
3.5.1 AMO酵素活性之測試…………………………………… 24
3.5.2 HAO酵素活性之測試……………………………………. 25
3.6 Strain THU amo gene PCR擴增………………………….. 26
3.6.1 菌種染色體DNA之抽取………………………………… 26
3.6.2 聚合酶連鎖反應………………………………………….. 27
3.6.3 洋菜膠體水平電泳(Agarose Gel Electrophoresis)………. 27
3.7 實驗分析項目與方法…………………………………….. 28
3.7.1 氨氮分析方法…………………………………………….. 28
3.7.2 硝酸鹽及亞硝酸鹽分析方法…………………………….. 29
3.7.3 水中羥氨分析方法……………………………………….. 31
3.7.4 蛋白質分析方法………………………………………….. 32
3.7.5 生長曲線方法…………………………………………….. 32
第四章 結果與討論……………………………………………….. 33
4.1 不同溶氧對strain THU的影響………………………….. 33
4.2 NH2OH對strain THU的影響…………………………… 35
4.2.1 不同濃度的NH2OH對strain THU的影響……………… 35
4.2.2 NH2OH對strain THU硝化作用的影響…………………. 37
4.2.3 NH2OH對strain THU脫硝作用的影響………………… 38
4.3 Strain THU SND反應酵素研究………………………….. 39
4.3.1 酵素AMO的測試………………………………………… 40
4.3.2 酵素HAO活性測試……………………………………… 41
4.4 Strain THU與Paracoccus denitrificans的比較…………. 42
4.5 PCR擴增酵素amoA基因片段………………………….. 44
第五章 結論與建議……………………………………………….. 46
5.1 結論……………………………………………………….. 46
5.2 建議……………………………………………………….. 48
參考文獻……………………………………………………………… 49

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