臺灣博碩士論文加值系統

本研究所使用之微生物為固氮微菌叢（Nitrogen Fixing Microbiome, NMb），此菌種富含Bacillus sp.及Clostridium sp.兩種優勢菌，其所屬之變形菌門（Proteobacteria）具有脫氮功能，過去利用此微生物研究發現其具有破壞污染物結構之能力，於馴養過程中以Anammox培養基質進行培養，期盼能夠藉由此培養方法提高其污染物去除能力，並觀察NMb是否具有脫氮能力。
本研究主要分為批次及半連續式反應槽實驗，於反應槽啟動過程中，同時進行批次培養找出微生物原始特性;由批次實驗可觀察到pH值會影響微生物之脫氮能力，於中性環境培養時，總氮去除效率可達94.3%，其中氨氮與亞硝酸鹽氮之添加濃度（mg-N/L）比例以1：1最佳，加入碳源培養後，可觀察到隨著碳氮比增加（6.3、7.5、8），總氮去除效率也會提高。
半連續式反應槽馴養初期無額外添加碳源時，總氮濃度166.6 mg-N/L其去除效率即可達92.4%，隨著氮化物濃度提高，以添加醋酸鈉作為碳源及調整碳氮比（C/N ratio=7）後，總氮濃度為322 mg-N/L時，其去除效率平均可達77.8%，反應槽達穩定狀態後，總氮去除效率隨著氮負荷提高而增高，氮負荷為0.041 kg/ m3.day時，總氮去除效率可達81%。
由菌群分析結果可發現到NMb中含有Desulfovibrio硫酸還原菌，NMb反應槽中含有Desulfotomaculum硫酸還原菌，此結果可推測NMb本身具有硫酸還原菌存在。

The microorganisms used in this research is Nitrogen Fixing Microbiome（NMb）, this species rich in Bacillus sp. and Clostridium sp., its belonging Proteobacteria has dinitrogen function, Therefore, a NMb obtained from the anaerobic hydrogen fermentation was applied to answer this question. The NMb was rich in the phylum-level of Firmicutes (58.5%), Actinobacteria (25.2%), Proteobacteria (2.7%), and Bacteroidetes (2.0%), during the domestication, the growth environment of anammox was given for cultivation, to improve the pollutant removal ability and nitrogen fixiation ability to maximize.
Results from batch cultures indicated that the NMb had the characteristics of converting nitrite to nitrate. Its total nitrogen (TN) could be degraded when the cultures adding with an organic carbon. Especially, the removal efficiency of the TN increased with the increment of the ratio of carbon to nitrogen (C/N) from 6.0 to 8.0.
In the period of startup of semi-continues anaerobic de-nitrogen fermenter, the TN removal efficiency could reach 77.8% under a high TN concentration about 322 mg/L and a C/N of 7.0. As the fermenter reaching a stable condition, the removal efficiency of the TN increased with the increment of the nitrogen loading rate. A high TN removal efficiency of 81% could be obtained when its nitrogen loading rate up to 0.041kg/m3-day.
Microbial population analysis demonstrated that the NMb and the microbiome of the fermenter had sulfide reduction bacteria of Desulfovibrio sp. and Desulfotomaculum sp., respectively. Considering those bacteria with the mechanisms observed from this study could be reasoned that the NMb had the symbiotic bacteria of nitrogen reduction and sulfide oxidation (NR-SOB). This evidence was why the NMb could be acclimated to the degradation of the organic nitrogen under anaerobic condition.

目錄
摘要 I
ABSTRACT II
誌謝 IV
目錄 V
圖目錄 VII
表目錄 X
第一章 前言 1
1.1 研究緣起 1
1.2 研究目的 2
第二章 文獻回顧 3
2.1 自然界中的氮循環 3
2.1.1 氮循環 3
2.1.2 微生物氮循環機制 7
2.2 傳統氮化物處理程序 9
2.2.1 氮化物之物理處理方法 9
2.2.2 氮化物之化學處理方法 12
2.2.3 氮化物之生物處理方法 13
2.3 操作條件對於生物除氮系統所產生之差異性 16
2.3.1 環境因子對於微生物所造成之影響 16
2.3.2 碳源於生物除氮系統之表現 20
2.4 新穎氮化物處理程序 21
2.4.1 厭氧氨氧化（Anaerobic Ammonium Oxidation, ANAMMOX） 21
2.4.2厭氧氨氧化應用程序 22
2.5 NMb微生物菌群來源及特性 26
2.5.1 厭氧產氫醱酵系統（Anaerobic Hydrogen Producing Fermentation System） 26
2.5.2 優勢菌群及共生菌之特性 28
2.5.3 微生物之代謝變異與動力學之應用 30
第三章 材料與方法 33
3.1研究架構 33
3.2 實驗材料及設計 33
3.2.1 系統植種來源及特性 33
3.2.2 NMb半連續式反應槽 36
3.2.3 人工合成基質 37
3.3實驗方法 39
3.3.1 批次實驗方法及流程 41
3.3.2 NMb反應槽操作程序 46
3.4 水質分析項目及方法 47
3.5 菌群分析 58
3.6 數據分析 61
第四章 結果與討論 62
4.1 探討NMb對於脫氮之基本特性 62
4.1.1 氨氮強度對於NMb脫氮之影響 63
4.1.2 亞硝酸鹽氮對於NMb脫氮之影響 70
4.1.3 碳氮比對於脫氮能力之影響 75
4.2 厭氧脫氮的可行性 79
4.2.1 pH及ORP於脫氮過程中之變化 79
4.2.2 反應槽之氮負荷濃度變化 81
4.2.3 碳氮比對於去除效率所造成之影響 88
4.3 NMb硫化菌氧化還原變化之關聯性 92
4.4 微生物生長變化及菌群組成 94
第五章 結論與建議 97
5.1結論 97
5.2建議 97
第六章 參考文獻 99
附錄 107

石濤（2012）環境微生物學. 鼎茂圖書.
朱皓瑜（2018）HMB熟成過程中對2,4-二氯酚脫鹵之影響. 國立高雄第一科技大學, 高雄市.
吳碩安（2019）混合植種NMB與Anammox污泥啟動厭氧除氮系統之可行性研究. 國立高雄科技大學, 高雄市.
李珮芸（2011）外加碳源對生物薄膜反應器處理低碳氮比高科技業廢水之影響. 國立交通大學, 新竹市.
林后志，林舜宏（2014）以磷酸銨鎂法搭配中央合成反應曲面法實驗設計探討科學園區聯合污水廠廢水中氨氮去除之研究.
林志高（2011）新穎厭氧氨氧化技術發展及應用. 國立交通大學環境工程研究所, 新竹市.
柯勇（2016）植物生理學. 藝軒出版社.
唐存宏（2016）工業廢水氨氮處理概述. 財團法人台灣產業服務基金會.
許美芳，王俊欽，方鴻源，陳文欽，袁又罡，許昺慕，陳錫添，沈淑敏（2007）環境微生物學. 華格納出版.
許淑娟（2014）厭氧氨氧化系統為生物組成及除氮效能之探討. 國立中興大學, 台中市.
歐陽嶠輝（2011）下水道工程學. 長松文化.
鍾威振（2016）利用來自厭氧氫醱酵的生物製劑降解三氯酚的可行性. 國立高雄第一科技大學, 高雄市.
韓長龍（2015）厭氧氫醱酵生物製劑的開發與應用. 國立高雄科技大學, 高雄市.
蘇遠志，黃世佑（2002）微生物化學工程學. 華香園出版社.
Alejandra, G., Rodríguez, O., Quintero, J., Miguel, A., Morales, M., Thelma, K. Morales-Martínez., José, A. Rodríguez-De, L. G., Dávila, M. M., Aroca, G., and González, L. J. R.（2019）Clostridium strain selection for co-culture with Bacillus subtilis for butanol production from agave hydrolysates. Bioresource Technology 275: 410-415.
Alou, M. T., Ndongo, S., Frégère, L., Labas, N., Andrieu, C., Richez, M., Couderc, C., Baudoin, J. P., Abrahão, J., Brah, S., Diallo, A., Sokhna, C., Cassir, N., Scola, B. L., Cadoret, F., and Raoult, D.（2018）Taxonogenomic description of four new Clostridium species isolated from human gut: ‘Clostridium amazonitimonense’, ‘Clostridium merdae’, ‘Clostridium massilidielmoense’ and ‘Clostridium nigeriense’. New Microbe and New Infect 21: 128-139.
An de Graaf, A.A., Mulder, A., de Bruijn, P., Robertson, J. M., and Kuenen, J.G. (1995) Anaerobic Oxidation of Ammonium Is a Biologically Mediated Process. Applied and Environmental Microbiology 61: 1246-1251.
Baeza, J. A., Gabriel, D., and Lafuente, J.（2004）Effect of internal recycle on the nitrogen removal efficiency of an anaerobic/anoxic/oxic (A2/O) wastewater treatment plant (WWTP). Process Biochemistry 39: 1615-1624.
Banu, J. R., Uan, D. K., and Yeom, I. T.（2009）Nutrient removal in an A2O-MBR reactor with sludge reduction. Bioresource Technology 100: 3820-3824.
Boivin, C. M., Giraud, E., Perret, X., and Batut, J.（2009）Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes? Trends in Microbiology, Vol:17（No.10）.
Chen, X. G., Zheng, P., Qaisar, M., and Tang, C. J.（2012）Dynamic behavior and concentration distribution of granular sludge in a super-high-rate spiral anaerobic bioreactor. Bioresource Technology 111: 134-140.
Choi, C., Lee, J. K., and Kim, M.（2008） The effects on operations of sludge retention time and carbon/nitrogen ratio in an intermittently aerated membrane bioreactor (IAMBR). Bioresource Technology 99: 5397-5401.
De Melo Filho, M. E. S., Owatari, M. S., Mouriño, J. L. P., Lapa, K. R., and Soares, H. M.（2020）Application of nitrification and denitrification processes in a direct water reuse system for pacific white shrimp farmed in biofloc system. Aquacultural Engineering 88: 102-043.
Donald, E. F.（1994）Plant growth and development：A molecular approach. Trend in Cell Biology, vol:4（No.11）.
Donna, M. K., Nelson, J., Nancy, L. E., Timothy, J. T., and David, E. G.（2013）Nitrogen and sulfur requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on cellulosic substrates in minimal nutrient media. Bioresource Technology 130: 125-135.
Ersahin, M. E., Ozgun, H., Dereli, R. K., and Ozturk, I.（2011）Anaerobic Treatment of Industrial Effluents: An Overview of Applications. Waste Water-Treatment and Reutilization.
European Environment Agency.（2010）The nitrogen cycle.
Fang, F., Qiao, L. L., Cao, J. S., Li, Y., Xie, W. M., Sheng, G. P., and Yu, H. Q.（2016）Quantitative evaluation of A2O and reversed A2O processes for biological municipal wastewater treatment using a projection pursuit method. Separation and Purification Technology 166: 164-170.
Gallardo-Altamirano, M.J., Maza-Marquez, P., Montemurro, N., Rodelas, B., Osorio F., and Pozo, C.（2019） Linking microbial diversity and population dynamics to the removal efficiency of pharmaceutically active compounds (PhACs) in an anaerobic/anoxic/aerobic (A2O) system. Chemosphere 233: 828-842.
Ge, C., Liu, B., Che, J., Chen, M., Liu, G., and Wei, J.（2015）Diversity of Bacillus species inhabiting on the surface and endophyte of lichens collected from Wuyi Mountain. Microbiologica Sinica 55: 551-563.
Habicht, K.S., and Canfield, D. E.（1997）Sulfur isotope fractionation during bacterial sulfate reduction in organic-rich sediments. Marine Microbiology PII S0016-7037: 00311-6.
Hamilton, C., Calusinska, M., Baptiste, S., Masset, Julien., Beckers, L., Thonart, P., and Hiligsmann, S.（2018）Effect of the nitrogen source on the hydrogen production metabolism and hydrogenases of Clostridium butyricum CWBI1009. International Journal of Hydrogen Energy 43: 5451-5462.
Harindintwali, J. D., Zhou, J., and Yu, X. B.（2020）Lignocellulosic crop residue composting by cellulolytic nitrogen-fixing bacteria: A novel tool for environmental sustainability. Science of the Total Environment 715: 136912.
Hooper, A. B., Vannelli, T., Bergmann, D. J., and Arciero, D. M.（1997） Enzymology of the oxidation of ammonia to nitrite by bacteria. Antonie van Leeuwenhoek 71: 59-67.
Hosny, M., Abdallah, R. A., J. Khalil, B., Fontanini, A., Baptiste, E., Armstrong, N., and Scola, B. L.（2019）Clostridium pacaense: a new species within the genus Clostridium. New Microbe and New Infect 28: 6-10.
Huang, J. C., and Shang, C.（2007）Air Stripping. Water and Wastewater Treatment.
Idania, V.V., Lorena, G. C. R., Marisol, P. R., Alfonso, S. G., and Vargas, A.（2019）Enhanced hydrogen production from lignocellulosic substrates via bioaugmentation with Clostridium strains. Industrial Crops & Products 137: 105-111.
Jetten, M. S. M.（2008）The microbial nitrogen cycle. Applied Environmental Microbiology 10: 2903-2909.
John, A. F.（2017）Planctomycetes-New Models for Microbial Cells and Activities. Microbial Resources. http://dx.doi.org/10.1016/B978-0-12-804765-1.00001-1.
Jose Ramon, V. P.（2014）Autotrophic nitrogen removal in granular sequencing batch reactors. UNIVERSIDADE DE SANTIAGO DE COMPOSTELA.Santiago.
Kamarisima., Hidaka, K., Miyanaga, K., and Tanji, Y.（2018）The presence of nitrate- and sulfate-reducing bacteria contributes to ineffectiveness souring control by nitrate injection. International Biodeterioration & Biodegradation 129: 81-88.
Körner, H., and Zumft, W. G.（1989）Expression of Denitrification Enzymes in Response to the Dissolved Oxygen Level and Respiratory Substrate in Continuous. American Society for Microbiology.
KowalskI, M. S., Devlin, T. R., Oleszkiewicz, J. A.（2018） Start-up and long-term performance of anammox moving bed biofilm reactor seeded with granular biomass. Chemosphere 200: 481-486.
Lay, J. J.（2001）Biohydrogen Generation by Mesophilic Anaerobic Fermentation of Microcrystalline Cellulose. Biotechnol Bioeng 74: 280-287.
Lay, J. J., Li, Y. Y., Noike, T.（1998）The influence of pH and ammonia concentration on the methane production in high-solids digestion processes. Water Environment Research 70: 1075-1082.
Li, D., Zhang, S., Li, S., Zeng, H., and Zhang, J.（2019）The nitrogen removal of autotrophic and heterotrophic bacteria in aerobic granular reactors with different feast/famine ratio. Bioresource Technology 272: 370–378.
Li, Q., Wang, S., Zhang, P., Yu, J., Qiu, C., and Zheng, J.（2018） Influence of temperature on an Anammox sequencing batch reactor(SBR) system under lower nitrogen load. Applied Bioresource Technology 269: 50-56.
Mulder, A., Van de Graaf, A. A., Robertson, L. A., and Kuenen, J. G. （1995） Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiology Ecology 16: 177-183.
Pai, T. Y.（2007）Modeling nitrite and nitrate variations in A2O process under different return oxic mixed liquid using an extended model. Process Biochemistry 42: 978-987.
Peng, Y., Hou, Ho., Wang, S., Cui, Y., and Yuan, Z.（2007）Nitrogen and phosphorus removal in pilot-scale anaerobic-anoxic oxidation ditch system. Journal of Environmental Sciences 20: 398–403.
Pez, H. L., Puig, S., Ramon, G ., Ruscalleda, M., Balaguer, M. D., and Colprim, J.（2008）Start-up and enrichment of a granular anammox SBR to treat high nitrogen load wastewaters. Chemical Technology and Biotechnology 83: 233-241.
Purwono., Rezagama, A., Hibbaan, M., and Budihardjo, M. A.（2017）Ammonia-Nitrogen(NH3-N)and Ammonium-Nitrogen(NH4-N)Equilibrium on The Process of Removing Nitrogen By Using Tubular Plastic Media. J. Mater. Environ. Sci., Vol: 8, Issue S, Page 4915-4922.
Riegler, P., Bieringer, E., Chrusciel, T., Stärz, M., Löwe, H., and Weuster-Botz, D.（2019）Continuous conversion of CO2/H2 with Clostridium aceticum in biofilm reactors. Bioresource Technology 291: 121-760.
Romillac, N.（2019）Ammonification. Earth Systems and Environmental Sciences.
Ruiz, G., Jeison, D., and Chamy, R.（2003） Nitrification with high nitrite accumulation for the treatment of wastewater with high ammonia concentration. Water Research 37: 1371-1377.
Stein, L. Y.（2015）Microbiology: Cyanate fuels the nitrogen cycle. Microbiologhy doi: 10.1038/nature14639.
Strock, J. S.（2008）Ammonification. Ecological Processes.
Tomaszewski, M., Cems, G., and Alekandra, Z. B.（2017）Significance of pH control in anammox process performance at low temperature. Applied Chemosphere 185: 439-444.
Van de Graaf, A. A., de Bruijn, P., Robertson, L. A., Jetten, M. M., and Kuenen, J. G.（1996）Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142: 2187-2196.
Van den Berg, L., and Ashmore, M.（2008）Nitrogen. Ecotoxicology.
Van der Bruggen, B.（2015）Advances in electrodialysis for water treatment. Advances in Membrane Technologies for Water Treatment.
Watsuntorn, W., Ruangchainikom, C., Rene, E. R., Lens, P. N. L., and Chulalaksananukul, W.（2019）Comparison of sulphide and nitrate removal from synthetic wastewater by pure and mixed cultures of nitrate-reducing, sulphide-oxidizing bacteria. Bioresource Technology 272: 40–47.
Watsuntorn, W., Ruangchainikom, C., Rene, E. R., Lens, P. N. L., and Chulalaksananukul, W.（2017）Hydrogen sulfide oxidation under anoxic conditions by a nitrate- reducing, sulfide-oxidizing bacterium isolated from the Mae Um Long Luang hot spring, Thailand. International Biodeterioration & Biodegradation 124: 196-205.
Widdison, P. E., and Burt, T. P.（2008）Nitrogen Cycle. Global Ecology.
Wu, G., Li, Z., Huang, Y., Zan, F., Dai, J., Yao, J., Yang, B., Chen, G., and Lei, L.（2020）Electrochemically assisted sulfate reduction autotrophic denitrification nitrification integrated (e-SANI®) process for high-strength ammonium industrial wastewater treatment. Chemical Engineering Journal 381: 122-707.
Yang, J., Zhang, L., Fukuzaki, Y., Hira, D., and Furukawa, K.（2010）High-rate nitrogen removal by the Anammox process with a sufficient inorganic carbon source. Bioresource Technology 101: 9471–9478.
You, S.J., Hsu, C.L., Chuang, S.H., and Ouyang, C.F.（2003）Nitrification efficiency and nitrifying bacteria abundance in combined AS-RBC and A2O systems. Water Research 37: 2281-2290.
Zhang, H., Zhao, Z., Li, S., Chen, S., Huang, T., Li, N., Yang, S., Wang, Y., Kou, L., and Zhang, X.（2019）Nitrogen removal by mix-cultured aerobic denitrifying bacteria isolated by ultrasound:Performance, co-occurrence pattern and wastewater treatment. Chemical Engineering Journal 372: 26-36.
Zhang, W., Yu, C., Wang, X., Hai, L., and Hu, J.（2020）Increased abundance of nitrogen fixing bacteria by higher C/N ratio reduces the total losses of N and C in cattle manure and corn stover mix composting. Waste Management 103: 416-425.
Zhang, X., Liu, Y., Li, Z. R., Zhang, J., Chen, Y., and Wang, Q.（2020）Impact of COD/N on anammox granular sludge with different biological carriers. Science of the Total Environment 728: 138557.
Zhou, X., Zhang, Ze., and Li, Y.（2017）Four-stage biofilm anaerobic-anoxic-oxic-oxic system for strengthening the biological treatment of coking wastewater: COD removal behaviors and biokinetic modeling. The Royal Society of Chemistry 7: 23714-23726.