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研究生：	黃裕仁 Huang, Yu-Jen
論文名稱：	特定硝化抑制化合物對高與低鹽環境馴養富含氨氧化古細菌族群影響之研究 Inhibitory effects of specific nitrification inhibitors on ammonia oxidizing archaeal community enriched under high and low salinity environments
指導教授：	黃良銘 Whang, Liang-Ming
學位類別：	碩士 Master
系所名稱：	工學院 - 環境工程學系 Department of Environmental Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	139
中文關鍵詞：	硝化作用、氨氧化古細菌、硝化抑制作用、硝化抑制化合物、鹽度
外文關鍵詞：	Nitrification, Ammonia oxidizing archaea, Nitrification inhibition, Nitrification inhibitor, Salinity
相關次數：	點閱：110 下載：4
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　　硝化作用在生物除氮程序當中為不可或缺的機制，其主要分成兩階段(氨氧化作用及亞硝酸鹽氧化作用)，其中在氨氧化階段被認為是整體硝化作用的速率限制步驟，因此氨氧化作用的活性將會影響到整體硝化活性的進行。一直以來氨氧化階段被認為主要由氨氧化細菌族群所負責，然而近年來分生及純化技術的進步成熟，新發現了另一氨氧化古細菌族群也同時參與了氨氧化作用。在許多學者研究進一步發現了在某些特定環境當中氨氧化古細菌族群相對於氨氧化細菌族群佔主導地位，也有許多指出氨氧化古細族群在條件嚴苛的環境具有存活能力，這些氨氧化古細菌族群的研究發現也使學者對整體硝化作用產生了相當的改觀。

　　在活性污泥系統當中許多因子會影響到硝化作用，如pH、溫度、溶氧、硝化抑制化合物…等。其中硝化抑制化合物廣泛存在廢水當中，進入活性污泥系統後即會造成硝化系統有明顯的抑制行為，許多學者已針對氨氧化細菌族群研究確立了不同種類的化合物於氨氧化細菌族群不同程度的影響研究。然而這些硝化抑制化合物是否對於氨氧化古細菌族群有影響仍尚未有一定了解。因此，本研究利用實驗室已馴化多年存在於高和低鹽環境中富含豐富的氨氧化古細菌族群作為研究對象，針對已知會造成氨氧化細菌族群有明顯抑制行為的化合物，做系統性的批次實驗來探討這些化合物是否會造成氨氧化古細菌族群的影響。

　　本研究主要針對的特定硝化抑制化合物分為三類，分別為芳香族碳氫化合物、有機硫化合物以及有機氮化合物。在批次實驗中發現這三類的化合物皆會對氨氧化古細菌族群造成不同程度上的影響。在芳香族碳氫化合物批次實驗中發現，當氨氧化古細菌族群暴露不同濃度的苯和甲苯會造成60%以上的抑制行為，而存在於低鹽度環境中的氨氧化古細菌族群暴露於苯酚時，相較於文獻中指出對於氨氧化細菌的抑制性來說有較高的耐受能力。有機氮化合物的批次實驗中，無論存在於高鹽度或低鹽度環境的氨氧化古細菌族群長時間暴露於DMS以及ATU會造成比氨氧化速率的下降及氨氧化作用的遲滯。在有機氮化合物(EDA和Pyridine)的測試當中發現相較於文獻中對於不同硝化系統的抑制行為，整體有機氮化合物造成氨氧化古細菌族群的抑制影響是相當明顯的。

　　Nitrification is a key step in biological nitrogen removal process, and ammonia oxidation had been considered as rate limit step in nitrification process. It was believed that ammonia oxidizing bacteria (AOB) were the main group responsible for ammonia oxidation however, several new ammonia-oxidizing organisms belonged to the archaeal domain were found also involving in ammonia oxidation had changed this view. Archaea was thought to have advantages over bacteria in extreme environments, such as harsh temperature, pH, and the existence of toxic chemicals. Therefore, it was considered that ammonia oxidizing archaea (AOA) may play more important role than ammonia oxidizing bacteria on ammonia oxidation in specific environment. Many chemicals, existing in wastewater treatment process, reported to inhibit ammonia oxidizing bacteria on ammonia oxidation activity. However, there is much less information about those chemicals inhibition effects on ammonia oxidizing archaea. Different responses of ammonia oxidizing archaea and bacteria to inhibitive chemicals would provide alternative choices for wastewater treatment process. Therefore, it is very important to establish the inhibition information of AOA. In this study, two laboratory-scale reactors were operated under high (34‰) and low salinity (2.5‰) respectively, which both contain high level of AOA enrichments were used to evaluate the resistance of AOA to specific nitrification inhibitors by batch tests. Three different types of specific nitrification inhibitors were chosen in this study, including aromatic hydrocarbon, organic sulfur compound and organic nitrogen compound, in order to systematically investigate the impact of ammonia oxidation on AOA. In the batch tests, all of selected specific nitrification inhibitors had different levels of inhibitory effect on AOA enrichments in high and low salinity condition. Benzene and Toluene decreased over 60% of ammonia oxidation activity on AOA during batch tests. The batch tests with phenol were found that AOA in low salinity condition had high resistance to phenol than ammonia oxidizing bacteria. AOA in high and low salinity condition had decreased ammonia oxidation activity under long-time exposure to DMS and ATU. In the batch tests with organic nitrogen compound, the ammonia oxidation activity of AOA significantly decreased with increasing concentrations of EDA and pyridine. The inhibitory effect on AOA in high and low salinity sludge might have more tolerance to specific nitrification inhibitors than nitrifying community.

摘要	I
Abstract	III
Acknowledgements	V
Table of Content	VIII
List of Tables	XII
List of figures	XIV
Chapter 1	Introduction	1
Chapter 2	Literature Review	3
2-1	Nitrogen cycle	3
2-2	Effect of nitrogen to the Aquatic system	5
2-3	Nitrification	7
2-4	Kinetic of nitrification	9
2-5	Ammonia oxidizing bacteria (AOB)	10
2-5-1	The metabolism of AOB	10
2-5-2	The phylogeny of AOB	10
2-6	Ammonia oxidizing archaea (AOA)	13
2-6-1	The history of discovering AOA	13
2-6-2	Distribution of AOA habitat	14
2-6-3	Physiological property of AOA	15
2-7	Occurrence of AOB in wastewater treatment plant	16
2-8	Occurrence of AOA in wastewater treatment plant	17
2-9	Effect of environmental factors on nitrification 19
2-9-1	Effect of pH on nitrification	19
2-9-2	Effect of dissolved oxygen on nitrification	20
2-9-3	Effect of temperature on nitrification	21
2-10	Nitrification inhibitor	22
2-10-1	Inhibitory tests in nitrification	22
2-10-2	Mechanism of nitrification inhibitors	24
2-11	Molecular methods for studying nitrify communities 25
2-11-1	Polymerase chain reaction (PCR)	25
2-11-2	Terminal restriction fragment length polymorphism
(T-RFLP)	 26
2-11-3	Real time PCR	28
Chapter 3	Materials and Methods	31
3-1	Research overview	31
3-2	CSTR Bioreactor	32
3-3	Analytical methods	35
3-3-1	Water quality analysis	35
3-3-2	Chemical analysis of inhibitors	35
3-4	Molecular methods	37
3-4-1	Protein extraction	37
3-4-2	DNA extraction	38
3-4-3	PCR Amplification	40
3-4-4	T-RFLP	42
3-4-5	Real-time PCR	42
3-5	Batch tests to evaluate the inhibition effect of specific nitrification inhibitor	45
3-5-1	Selected specific nitrification inhibitor	45
3-5-2	Specific nitrification inhibition on enriched AOA	45
Chapter 4	Results and Discussion	49
4-1	Performance of continuous-flow stirred tank reactors (CSTR)	49
4-2	AOA community analysis by T-RFLP in high and low salinity reactors	53
4-3	AOA and AOB abundance in high and low salinity reactors	55
4-4	Batch test- Effect of ammonia concentration on high and low salinity AOA enrichments	57
4-5	Batch test- Effect of streptomycin on high and low salinity sludge	60
4-6	Batch test-Effect of aromatic hydrocarbon compound on high and low salinity sludge	63
4-6-1	Effect of phenol on high and low salinity sludge	63
4-6-1-1	Effect of phenol on high salinity sludge	63
4-6-1-2	Effect of phenol on low salinity sludge	66
4-6-2	Effect of benzene on high and low salinity sludge	69
4-6-2-1	The ratio of benzene concentration in headspace and liquid phase during batch test	69
4-6-2-2	Effect of benzene on high salinity sludge	70
4-6-2-3	Effect of benzene on low salinity sludge	73
4-6-3	Effect of Toluene on high and low salinity sludge	76
4-6-3-1	The ratio of Toluene concentration in headspace and liquid phase during batch test	76
4-6-3-2	Effect of Toluene on high salinity sludge	77
4-6-3-3	Effect of Toluene on low salinity sludge	80
4-6-4	Summary	83
4-7	Batch test-Effect of organic sulfur compound on high and low salinity sludge	84
4-7-1	Effect of DMS on high and low salinity sludge	84
4-7-1-1	The ratio of DMS concentration in headspace and liquid phase during batch test	84
4-7-1-2	Effect of DMS on high salinity sludge	85
4-7-1-3	Effect of DMS on low salinity sludge	91
4-7-2	Effect of ATU on high and low salinity sludge	96
4-7-2-1	Effect of ATU on high salinity sludge	96
4-7-2-2	Effect of ATU on low salinity sludge	99
4-7-3	Summary	101
4-8	Effect of organic nitrogen compound on high and low salinity sludge	102
4-8-1	Effect of EDA on high and low salinity sludge	102
4-8-1-1	Effect of EDA on high salinity sludge	102
4-8-1-2	Effect of EDA on low salinity sludge	105
4-8-2	Effect of pyridine on high and low salinity sludge	108
4-8-2-1	Effect of pyridine on high salinity sludge	108
4-8-2-2	Effect of pyridine on low salinity sludge	111
4-8-2-3	Summary	113
4-9	Summary of effect of nitrification inhibitors on AOA enrichments in high and low salinity condition	114
Chapter 5	Conclusions and Suggestions	116
Reference	119

                                    

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