厭氧氨氧化程序( Anaerobic Ammonium oxidation)簡稱為Anammox程序，此程序藉由Anammox菌將水中的氨氮與亞硝酸鹽氮轉換為硝酸鹽氮及氮氣，這項技術擁有幾個特點如Anammox菌因其於厭氧環境生長的特性，所以能夠減少曝氣量，產生較少的污泥，透過使用厭氧氨氧化技術，可以將運營成本和溫室氣體排放量分別減少60%和90%，進而減少能源的消耗，使Anammox程序較傳統的處理方法更具備競爭優勢，是未來處理含氮廢水的主要發展方向之一。
　　本研究實驗分為兩個部分，(一)第一部分分為三個階段，分別為第一階段馴養期、第二階段氮負荷增加期及第三階段連續流操作期，在這三階段中調整反應槽進流污水中的氮濃度及水力停留時間(HRT)方式改變氮負荷量(0.025 g N/L/D到0.172 g N/L/D)，前兩個階段操作為批式活性污泥法( Sequencing Batch Reactor, SBR)，第三階段改變為連續流操作的方式，實驗中觀察反應槽內因氮負荷量的改變而造成的出流水水質狀況及反應槽處理效率；(二) 第二部分，藉由次世代定序方法分析反應槽中菌群結構的變化。
    第一階段馴養期，分別提高了一次的氮負荷量，從0.0125 g N/L/D提高到0.025 g N/L/D，氨氮消耗量與亞硝酸鹽氮消耗量的比為1：1.53，硝酸鹽氮產生量與氨氮的消耗量比為1 : 1.31，因此反應槽內可能有亞硝酸鹽氧化菌參與反應。第二階段氮負荷增加期，總共調整了五次的氮負荷量，從0.025 g N/L/D提高到0.172 g N/L/D，第一次到第三次調整氮負荷中，氨氮消耗量與亞硝酸鹽氮消耗量的比分別為1：1.13、1：1.22、1：1.5，而硝酸鹽氮產生量與氨氮的消耗量比分別為1：0.851：0.76、1：0.88，推測這些時期反應槽內可能有亞硝酸鹽氧化菌參與反應，第四次調整氮負荷中，氨氮消耗量與亞硝酸鹽氮消耗量的比為1：1.37，硝酸鹽氮產生量與氨氮的消耗量比為1：0.76，同時發現有氣體產生，推測反應槽內可能有亞硝酸鹽氧化菌參與反應，第五次調整氮負荷中，氨氮消耗量與亞硝酸鹽氮消耗量的比為1：2.13，硝酸鹽氮產生量與氨氮的消耗量比為1：2.13，推測反應槽內可能有亞硝酸鹽氧化菌參與反應。而第三階段連續流操作期，總共調整了兩次的氮負荷量，從0.172 g N/L/D提高到0.2 g N/L/D，第一次調整氮負荷，氨氮消耗量與亞硝酸鹽氮消耗量的比為1：2.45，硝酸鹽氮產生量與氨氮的消耗量比為1：1.43，推測反應槽內可能有氨氧化菌及亞硝酸鹽氧化菌的存在，第二次調整氮負荷中，氨氮消耗量與亞硝酸鹽氮消耗量的比為1：2.86，硝酸鹽氮產生量與氨氮的消耗量比為1：0.97，推測反應槽內可能有氨氧化菌及亞硝酸鹽氧化菌的存在。
　　本研究在第一部分，根據水質分析結果，藉由厭氧氨氧化反應式，觀察反應槽，藉由氨氮去除率與亞硝酸鹽氮去除率的比及硝酸鹽氮產生量與氨氮的去除率比，第一階段馴養期，推測含有亞硝酸鹽化菌的存在，第二階段氮負荷增加期，推測含有氨氧化菌、亞硝酸鹽化菌及厭氧氨氧化菌的存在。第三階段連續流操作期，推測含有亞硝酸鹽化菌的存在。
    在SBR反應槽操作模式下，低氮負荷(0.025 g N/L/D到0.050 g N/L/D)的氨氮去除效率(62%)優於高氮負荷(0.1 g N/L/D到0.172 g N/L/D)的氨氮去除效率(57%)，可能是產生基質毒害的效應，因此後續改採連續流操作的反應槽，在連續流操作模式下，因更換反應槽而有氨氮與亞硝酸鹽氮下降的趨勢，分別為7%和29%，但回穩之後氨氮及亞硝酸鹽氮的去除率皆可達到97%和90%。
    藉由次世代定序方法分析的方式進行菌群分析，顯示反應槽內有厭氧氨氧化菌(Ca. Brocadia和Ca. Kuenenia)，氨氧化菌(Nitrosomonas)、亞硝酸鹽化菌(Nitrospira )及硝化菌(Denitratisoma)的存在。未來若配合定量分析的方法，針對槽中可能參與氮循環的關鍵菌種、菌屬進行分析，將可提供更精確的功能性微生物族群之變化資料。

The Anammox process is one of the technologies for the treatment of ammonia nitrogen wastewater in the world. In the anammox reaction, nitrite and ammonium ions are converted into diatomic nitrogen and nitrate. It can reduce operating costs and greenhouse gas emissions by 60% and 90% respectively, thereby reducing energy consumption, making the Anammox process more competitive than traditional treatment methods.
This research experiment is divided into two parts. (1) The first part is divided into three stages, namely the first acclimatization stage, the second nitrogen loading increasing stage and the third continuous-flow operation stage. In these three stages, change of the nitrogen loading (0.025 g N/L/D to 0.172 g N/L/D) by adjusting nitrogen concentrations in the influent and hydraulic retention time (HRT) of the reactor. The first two stages was operation in sequencing batch reactor (SBR) format while the third stage was conducted in a continuous flow operation mode. In the experiment, the quality of effluent and the treatment efficiency of the reactor corresponding the change of the nitrogen loading were monitored; (2) The second part is to analyze the dynamics of prokaryotic community structure in the reactor by the next-generation sequencing method.
In the first period of acclimatization stage, the nitrogen load was increased from 0.0125 g N/L/D to 0.025 g N/L/D, and the ratio of ammonia nitrogen consumption to nitrite nitrogen consumption was 1:1.53. The ratio of nitrate nitrogen production to ammonia nitrogen consumption is 1:1.31, so nitrite oxidizing bacteria may participate in the reaction in the reaction tank. In the second phase of nitrogen load increase period, the nitrogen load was adjusted five times in total, from 0.025 g N/L/D to 0.172 g N/L/D. During the first to third adjustment of nitrogen load, ammonia nitrogen consumption The ratios of nitrate nitrogen to nitrite nitrogen consumption are 1:1.13, 1:1.22, 1:1.5, and the ratios of nitrate nitrogen production to ammonia nitrogen consumption are 1:0.851:0.76, 1:0.88, respectively. It is inferred that these During the period, there may be nitrite oxidizing bacteria participating in the reaction in the reaction tank. In the fourth adjustment of nitrogen load, the ratio of ammonia nitrogen consumption to nitrite nitrogen consumption is 1:1.37, and the ratio of nitrate nitrogen production to ammonia nitrogen consumption It is 1:0.76, and gas is found to be generated. It is speculated that there may be nitrite oxidizing bacteria participating in the reaction in the reaction tank. In the fifth adjustment of nitrogen load, the ratio of ammonia nitrogen consumption to nitrite nitrogen consumption is 1:2.13, The ratio of nitrate nitrogen production to ammonia nitrogen consumption is 1:2.13. It is speculated that nitrite oxidizing bacteria may participate in the reaction in the reaction tank. In the third stage continuous flow operation period, the nitrogen load was adjusted twice, from 0.172 g N/L/D to 0.2 g N/L/D. The nitrogen load was adjusted for the first time, and the ammonia nitrogen consumption and nitrous acid were adjusted. The ratio of salt nitrogen consumption is 1:2.45, and the ratio of nitrate nitrogen production to ammonia nitrogen consumption is 1:1.43. It is speculated that there may be ammonia oxidizing bacteria and nitrite oxidizing bacteria in the reaction tank. Adjust the nitrogen for the second time. In the load, the ratio of ammonia nitrogen consumption to nitrite nitrogen consumption is 1:2.86, and the ratio of nitrate nitrogen production to ammonia nitrogen consumption is 1:0.97. It is speculated that ammonia oxidizing bacteria and nitrite oxidizing bacteria may exist in the reaction tank.
In the SBR operation mode, the ammonia removal efficiency (62%) at low nitrogen loading (0.025 g N/L/D to 0.050 g N/L/D) is better than that of high nitrogen loading (0.1 g N/L/D to 0.050 g N/L/D). The ammonia nitrogen removal efficiency of 0.172 g N/L/D) (57%) may be caused by the effect of substrate inhibition. Therefore, reactor was then operated in the continuous flow mode. The removal rate of ammonia nitrogen and nitrite nitrogen can reach 97% and 90%, respectively after stabilization.
Through the analysis of the prokaryotic community, anammox bacteria (Ca.Brocadia and Ca. Kuenenia), and ammonia oxidizing bacteria (Nitrosomonas), nitrite bacteria (Nitrospira) and nitrifying bacteria (Denitratisoma) were detected in the reactor. In the future, if quantitative analysis methods are used to analyze the core species and genera of bacteria that may participate in the nitrogen cycle, it will provide more detail data on the changes of functional microbial populations.

第一章	研究緣起	1
第二章	文獻回顧	3
2.1	氮於環境中造成的危害	3
2.2	除氮處理程序	4
2.2.1	MLE (Modified Ludzack-Ettinger process)	5
2.2.2	Bardenpho	5
2.2.3	MBR (Membrane Bioreactor)程序	6
2.2.4	氧化渠程序	6
2.3	厭氧氨氧化程序	7
2.3.1	厭氧氨氧化微生物	7
2.3.2	厭氧氨氧化程序的原理與特性	8
2.4	微生物參與的氮循環路徑	9
2.5	硝化作用	10
2.5.1	硝化作用原理	10
2.5.2	硝化菌	11
2.6	脫硝作用	13
2.6.1	脫硝作用原理	13
2.7	NGS 次世代定序分析	14
第三章	實驗方法	16
3.1	實驗流程圖	16
3.2	厭氧氨氧化生物反應槽	17
3.3	反應槽營養基	22
3.4	含氮化合物分析	23
3.4.1	氨氮	23
3.4.2	亞硝酸鹽氮	25
3.4.3	硝酸鹽氮	27
3.5	微生物菌群分析方法	29
3.5.1	反應槽染色體核酸(genomic DNA)萃取步驟	29
3.5.2	染色體核酸定量及純度分析	30
3.6	微生物菌群分析方法	32
第四章	實驗結果與討論	33
4.1	厭氧氨氧化反應槽水質與處理效能	33
4.1.1	第一階段：馴養期(第0天~第64天)	34
4.1.2	第二階段：氮負荷增加期(第64天~第308天)	38
4.1.3	第三階段：連續流系統操作期(第309天~第431天)	47
4.1.4	結論	51
4.2	微生物菌群分析	52
4.2.1	菌群分析	52
4.2.2	相關性分析	57
4.2.3	結論	62
第五章	結論與建議	63
5.1	結論	63
5.2	建議	64
參考文獻	65

 
圖片目錄
圖 2-1 部分事業現行氨氮管制標準	3
圖 2-2 公共衛生下水道氨氮及總氮現行排放標準	4
圖 2-3 MLE程序流程圖(中華技術, 2012)	5
圖 2-4  Bardenpho流程圖(中華技術, 2012)	5
圖 2-5 MBR程序流程圖 (中華技術, 2012)	6
圖 2-6 氧化渠程序(中華技術, 2012)	6
圖 2-7本實驗所可能發生之氮循環圖 (Marcel M. M. Kuypers*, 2018)	9
圖 2-8 Illumin系統的簡易流程圖 (Mardis 2008)	15
圖 3-1實驗流程圖	16
圖 3-2馴養期反應槽	18
圖 3-3馴養期系統槽示意圖	18
圖 3-4氮負荷增加期反應槽	19
圖 3-5氮負荷增加期系統槽示意圖	20
圖 3-6連續流系統操作期反應槽	21
圖 3-7連續流系統操作期示意圖	21
圖 3-8微量分光光度計面板	31
圖 4-1馴養期-氨氮進、出流及去除率變化圖	35
圖 4-2馴養期-亞硝酸鹽氮進、出流及去除率變化圖	36
圖 4-3馴養期-硝酸鹽氮出流變化圖	36
圖 4-4馴養期-總氮進、出流及去除率變化圖	37
圖 4-5氮負荷增加期樣品採樣	39
圖 4-6氮負荷增加期-氨氮進、出流及去除率變化圖	41
圖 4-7氮負荷增加期-亞硝酸鹽氮進、出流及去除率變化圖	41
圖 4-8氮負荷增加期-硝酸鹽氮出流變化圖	42
圖 4-9氮負荷增加期-總氮進、出流及去除率變化圖	43
圖 4-10連續流系統操作期-氨氮進、出流及去除率變化圖	48
圖 4-11連續流系統操作期-亞硝酸鹽氮進、出流及去除率變化圖	48
圖 4-12連續流系統操作期-硝酸鹽氮出流變化圖	49
圖 4-13連續流系統操作期-總氮進、出流及去除率變化圖	49
圖 4-14反應槽菌群分布圖-以門來分類	53
圖 4-15物種稀釋曲線	57
圖 4-16排序階梯圖(Rank abundance curve)	58
圖 4-17 Weighted Unifrac PCoA 分析圖	59
圖 4-18物種豐度聚類熱圖……………………………………….……...60 
 
表目錄
表 2-1 厭氧氨氧化菌分類 (王氏.2015)	7
表 2-2厭氧氨氧化菌生長條件	8
表 2-3氨氧化古菌	11
表 2-4 氨氧化菌	11
表 2-5亞硝酸鹽氧化菌	12
表 2-6脫硝菌 (Jeter and Ingraham 1981)	13
表 3-1營養鹽配置	22
表 4-1厭氧氨氧反應槽參數	34
表 4-2馴養期階段之氮化物反應計量表	37
表 4-3氮負荷增加期階段之氮化物反應計量表	43
表 4-4連續流系統操作期之氮化物反應計量表	50
表 4-5反應槽菌群分布比例	53
表 4-6反應槽內各採樣階段氨氧化菌、亞硝酸鹽化菌以及脫硝菌分布比例 55	
表 4-7反應槽內各採樣階段Anammox分布比例	56

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