本實驗設立了一組實驗室規模之缺氧/好氧 (A/O) 生物反應槽，以利模擬實廠廢水除氮程序。即時聚合酶連鎖反應及末端限制性片段長度多樣性分別作為本實驗基因定量、定性之分析方法。功能性amoA基因及nxrB基因分別用以偵測氨氧化細菌 (AOB) 及亞硝酸氧化鹽細菌 (NOB)。實驗中設計了多組情境加以探討AOB及NOB在不同環境刺激下所表現之微生物反應。A/O生物反應槽之進流氨氮濃度由100 mg-N/L調升至300 mg-N/L後，amoA及nxrB基因皆於好氧槽內氨氮、亞硝酸鹽濃度波動之前，提早反應出不同程度之相對基因表現量。在A/O生物反應槽進流氟濃度逐漸提高之時期，amoA及nxrB基因表現量皆呈現出不斷波動之趨勢，但其並未導致氨氮、亞硝酸鹽累積於好氧槽中。在為期8天25 mg/L銅之衝擊負荷時期，amoA及nxrB基因表現量立即呈下降趨勢，而A/O生物反應槽之氨氮去除率則在4天後開始迅速下滑。本實驗中藉由末端限制性片段長度多樣性分析及選殖定序法測得之優勢硝化菌為Nitrosomonas nitrosa、 Nitrospira defluvii及 Nitrospira japonica。經amoA基因於DNA及mRNA層級上之分析中指出，AOB菌群組成並未因應不同之進流刺激而產生明顯改變，然而不同群族之NOB則於不同進流時期中呈現出不同比例之nxrB基因表現量。

A lab-scale A/O bioreactor was setup to stimulate the wastewater nitrogen removal process. Quantitative polymerase chain reaction (qPCR) and terminal restriction fragment length polymorphism (T-RFLP) were used to provide quantitative and qualitive genetic information in this study. amoA and nxrB functional gene were used to target ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) respectively. Several scenarios were designed to analyze the microbial response of AOB and NOB to different environment stimulus. By increase the influent ammonia concentration of A/O bioreactor from 100 mg-N/L to 300 mg-N/L, the relative expression of both amoA and nxrB gene showed a leading trend to the concentrations of ammonia and nitrite in the aerobic tank. Fluctuation of both amoA and nxrB mRNA expression level was detected in response to the gradually increased fluoride concentration in the A/O bioreactor, though it did not lead to the accumulation of ammonia and nitrite in the aerobic tank. Shock load with 25 mg/L of copper was last for 8 days in the A/O bioreactor, causing the immediate reduction of both amoA and nxrB mRNA expression level and a sharp decline of ammonia removal rate four days later. The dominant nitrifying bacteria species in this study includes Nitrosomonas nitrosa, Nitrospira defluvii, and Nitrospira japonica, revealed by the combination of T-RFLP and cloning sequencing. Although there was no obvious change of AOB community structure in response to different influent stimulus by both amoA gene and transcriptional analysis, different levels of nxrB mRNA expression were shown in different NOB species during each influent phases.

Abell, G. C. J., Revill, A. T., Smith, C., Bissett, A. P., Volkman, J. K., &Robert, S. S. (2010). Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary. ISME Journal, 4(2), 286–300.
Ahn, J. H., Kwan, T., &Chandran, K. (2011). Comparison of partial and full nitrification processes applied for treating high-strength nitrogen wastewaters: Microbial ecology through nitrous oxide production. Environmental Science and Technology, 45(7), 2734–2740.
Amann, R. I., Ludwig, W., &Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59(1), 143–169.
Aoi, Y., Masaki, Y., Tsuneda, S., &Hirata, A. (2004). Quantitative analysis of amoA mRNA expression as a new biomarker of ammonia oxidation activities in a complex microbial community. Letters in Applied Microbiology, 39(6), 477–482.
Bae, H., Park, J. H., Jun, K. S., &Jung, J. Y. (2011). The community analysis of ammonia-oxidizing bacteria in wastewater treatment plants revealed by the combination of double labeled T-RFLP and sequencing. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 46(4), 345–354.
Baeza, J. A., &Vicent, T. (2003). Inhibition of nitrification by fluoride in high-strength ammonium wastewater in acti v ated sludge. Process Biochemistry, 39(1), 73–79.
Barbier, O., Arreola-Mendoza, L., &DelRazo, L. M. (2010). Molecular mechanisms of fluoride toxicity. Chemico-Biological Interactions, 188(2), 319–333.
Beg, S. A.Siddiqi, R. H., & Ilias, S. (1982). Inhibition of nitrification by arsenic, chromium, and fluoride. Water Pollution Control Federation, 54(5), 482–488.
Braker, G., Ayala-Del-Río, H. L., Devol, A. H., Fesefeldt, A., &Tiedje, J. M. (2001). Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes. Applied and Environmental Microbiology, 67(4), 1893–1901.
Cabezas, A., deAraujo, J. C., Callejas, C., Galès, A., Hamelin, J., Marone, A., …Etchebehere, C. (2015). How to use molecular biology tools for the study of the anaerobic digestion process? Reviews in Environmental Science and Biotechnology, 14(4), 555–593.
Church, M. J., Short, C. M., Jenkins, B. D., Karl, D. M., &Zehr, J. P. (2005). Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean. Applied and Environmental Microbiology, 71(9), 5362–5370.
Clarkson, W. W., Collins, A. G., &Sheehan, P. L. (1989). Effect of Fluoride Nitrification of a Concentrated Industrial Waste. Applied and Environmental Microbiology, 55(1), 240–245.
Collins, A. G., Clarkson, W. W., Florio, S. A., Collins, A. G., Clarkson, W. W., &Florio, S. A. (1991). Alternative fixed-film biological nitrification processes for a semiconductor waste. Research Journal of the Water Pollution Control Federation, 63(1), 60–66.
Daims, H., Nielsen, J. L., Nielsen, P. H., Schleifer, K. H., &Wagner, M. (2001). In Situ Characterization of Nitrospira-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants. Applied and Environmental Microbiology, 67(3–12), 5273–5284.
EPA, Taiwan. (2019). 水字水字第 1080028628 號令修正發布第 2、2-1 條條文.
Ge, S., Wang, S., Yang, X., Qiu, S., Li, B., &Peng, Y. (2015). Detection of nitrifiers and evaluation of partial nitrification for wastewater treatment: A review. Chemosphere, 140, 85–98.
Gruber-dorninger, C., Pester, M., Kitzinger, K., Savio, D. F., Loy, A., Rattei, T., …Daims, H. (2015). Functionally relevant diversity of closely related Nitrospira in activated sludge. The ISME Journal, 9(3), 643–655.
Harb, M., Wei, C. H., Wang, N., Amy, G., &Hong, P. Y. (2016). Organic micropollutants in aerobic and anaerobic membrane bioreactors: Changes in microbial communities and gene expression. Bioresource Technology, 218, 882–891.
Horz, H. P., Rotthauwe, J. H., Lukow, T., &Liesack, W. (2000). Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products. Journal of Microbiological Methods, 39(3), 197–204.
Hu, C. Y., Lo, S. L., Kuan, W. H., &Lee, Y. D. (2005). Removal of fluoride from semiconductor wastewater by electrocoagulation- flotation. Water Research, 39(5), 895–901.
Hu, Z., Chandran, K., Grasso, D., &Smets, B. F. (2004). Comparison of nitrification inhibition by metals in batch and continuous flow reactors. Water Research, 38(18), 3949–3959.
Huang, Z., Gedalanga, P. B., Asvapathanagul, P., &Olson, B. H. (2010). Influence of physicochemical and operational parameters on Nitrobacter and Nitrospira communities in an aerobic activated sludge bioreactor. Water Research, 44(15), 4351–4358.
Hwang, Y, H., S. & T., &Tanaka. (1994). Denitrification with isopropanol as a carbon source in a biofilm system. Water Science and Technology, 30(11), 69.
James D. Watson. (1989). Molecular Biology of the Gene. Bioelectrochemistry and Bioenergetics, 22(1), 85.
Johnston, N. R., &Strobel, S. A. (2020). Principles of fluoride toxicity and the cellular response: a review. Archives of Toxicology, 94(4), 1051–1069.
Juretschko, S., Timmermann, G., Schmid, M., Schleifer, K., &Pommerening-ro, A. (1998). Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge : Nitrosococcus mobilis and Nitrospira -Like Bacteria as Dominant Populations. Applied and Environmental Microbiology, 64(8), 3042–3051.
Kapoor, V., Elk, M., Li, X., Impellitteri, C. A., &Santo Domingo, J. W. (2016). Effects of Cr(III) and Cr(VI) on nitrification inhibition as determined by SOUR, function-specific gene expression and 16S rRNA sequence analysis of wastewater nitrifying enrichments. Chemosphere, 147, 361–367.
Kapoor, V., Li, X., Chandran, K., Impellitteri, C. A., &Domingo, J. W. S. (2016). Use of functional gene expression and respirometry to study wastewater nitrification activity after exposure to low doses of copper. Environmental Science and Pollution Research, 23(7), 6443–6450.
Kayee, P., Rongsayamanont, C., Kunapongkiti, P., &Limpiyakorn, T. (2016). Ammonia half-saturation constants of sludge with different community compositions of ammonia-oxidizing bacteria. Environmental Engineering Research, 21(2), 140–144.
Khatri, C., Field, J. A., Ochoa-herrera, V., Banihani, Q., Leo, G., &Sierra-alvarez, R. (2009). Toxicity of fluoride to microorganisms in biological wastewater treatment systems. Water Research, 43(13), 3177–3186.
Kim, K. T., Kim, I. S., Hwang, S. H., &Kim, S. D. (2006). Estimating the combined effects of copper and phenol to nitrifying bacteria in wastewater treatment plants. Water Research, 40(3), 561–568.
Kim, Y. M., Lee, D. S., Park, C., Park, D., &Park, J. M. (2011). Effects of free cyanide on microbial communities and biological carbon and nitrogen removal performance in the industrial activated sludge process. Water Research, 45(3), 1267–1279.
Kim, Y. M., Park, H., Cho, K. H., &Park, J. M. (2013). Long term assessment of factors affecting nitrifying bacteria communities and N-removal in a full-scale biological process treating high strength hazardous wastewater. Bioresource Technology, 134, 180–189.
Koops, H., &Pommerening-ro, A. (2001). Distribution and ecophysiology of the nitrifying bacteria emphasizing cultured species. FEMS Microbiology Ecology, 37(1), 1–9.
Kowalchuk, G. A., &Stephen, J. R. (2001). AMMONIA- OXIDIZING BACTERIA : A Model for Molecular Microbial Ecology. Annual Reviews in Microbiology, 55(1), 485–529.
Kuo, D. H. W., Robinson, K. G., Layton, A. C., Meyers, A. J., &Sayler, G. S. (2010). Transcription levels (amoA mRNA-based) and population dominance (amoA gene-based) of ammonia-oxidizing bacteria. Journal of Industrial Microbiology and Biotechnology, 37(7), 751–757.
Kuypers, M. M. M., Marchant, H. K., &Kartal, B. (2018). The microbial nitrogen-cycling network. Nature Reviews Microbiology, 16(5), 263–276.
Lai, C. L., &Lin, S. H. (2004). Treatment of chemical mechanical polishing wastewater by electrocoagulation: System performances and sludge settling characteristics. Chemosphere, 54(3), 235–242.
Lee, Y. W., Tian, Q., Ong, S. K., Sato, C., &Chung, J. (2009). Inhibitory Effects of Copper on Nitrifying Bacteria in Suspended and Attached Growth Reactors. Water, Air, and Soil Pollution, 203(1–4), 17–27.
Lin, S. H., &Kiang, C. D. (2003). Combined physical, chemical and biological treatments of wastewater containing organics from a semiconductor plant. Journal of Hazardous Materials, 97(1–3), 159–171.
Liu, W. T., Marsh, T. L., Cheng, H., &Forney, L. J. (1997). Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Applied and Environmental Microbiology, 63(11), 4516–4522.
Livak, K. J., Flood, S. J. A., Marmaro, J., Giusti, W., &Deetz, K. (1995). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. Genome Research, 4(6), 357–362.
Lu, H., Chandran, K., &Stensel, D. (2014). ScienceDirect Microbial ecology of denitrification in biological wastewater treatment. Water Research, 64, 237–254.
Lücker, S., Wagner, M., Maixner, F., Pelletier, E., Koch, H., &Vacherie, B. (2010). A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proceedings of the National Academy of Sciences, 107(30), 13479–13484.
M. Alawi, S. Off, M. K. and E. S. (2009). Temperature influences the population structure ofnitrite-oxidizing bacteria in activated sludge. Environmental Microbiology Reports, 1(3), 184–190.
Maixner, F., Noguera, D. R., Anneser, B., Stoecker, K., Wegl, G., Wagner, M., &Daims, H. (2006). Nitrite concentration influences the population structure of Nitrospira-like bacteria. Environmental Microbiology, 8(8), 1487–1495.
Microbe Notes. (2020). Retrieved November9, 2020, from https://microbenotes.com/real-time-pcr-principle-process-markers-advantages-applications/
Mohanakrishnan, J., Kofoed, M. V. W., Barr, J., Yuan, Z., Schramm, A., &Meyer, R. L. (2011). Dynamic microbial response of sulfidogenic wastewater biofilm to nitrate. Applied Microbiology and Biotechnology, 91(6), 1647–1657.
Nogueira, R., & Melo, L. F. (2006). Competition Between Nitrospira spp . and Nitrobacter spp . in Nitrite-Oxidizing Bioreactors. Biotechnology and Bioengineering, 95(1), 169–175.
Ochoa-Herrera, V., León, G., Banihani, Q., Field, J. A., &Sierra-Alvarez, R. (2011). Toxicity of copper(II) ions to microorganisms in biological wastewater treatment systems. Science of the Total Environment, 412–413, 380–385.
Otawa, K., Asano, R., Ohba, Y., Sasaki, T., Kawamura, E., Koyama, F., …Nakai, Y. (2006). Molecular analysis of ammonia-oxidizing bacteria community in intermittent aeration sequencing batch reactors used for animal wastewater treatment. Environmental Microbiology, 8(11), 1985–1996.
Ouyang, F., Ji, M., Zhai, H., Dong, Z., &Ye, L. (2016). Dynamics of the diversity and structure of the overall and nitrifying microbial community in activated sludge along gradient copper exposures. Applied Microbiology and Biotechnology, 100(15), 6881–6892.
Ouyang, F., Zhai, H., Ji, M., Zhang, H., &Dong, Z. (2016). Physiological and transcriptional responses of nitrifying bacteria exposed to copper in activated sludge. Journal of Hazardous Materials, 301, 172–178.
Panigrahi, S., Velraj, P., &Subba Rao, T. (2019). Chapter 21 - Functional Microbial Diversity in Contaminated Environment and Application in Bioremediation. In S.Das &H. R. B. T.-M. D. in the G. E.Dash (Eds.), Microbial Diversity in the Genomic Era (pp. 359–385).
Park, S., &Ely, R. L. (2008). Candidate stress genes of Nitrosomonas europaea for monitoring inhibition of nitrification by heavy metals. Applied and Environmental Microbiology, 74(17), 5475–5482.
Pester, M., Maixner, F., Berry, D., Rattei, T., Koch, H., Lücker, S., …Daims, H. (2014a). NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira. Environmental Microbiology, 16(10), 3055–3071.
Pester, M., Maixner, F., Berry, D., Rattei, T., Koch, H., Lücker, S., …Daims, H. (2014b). NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira. Environmental Microbiology, Vol. 16, pp. 3055–3071.
Rotthauwe, J. H., Witzel, K. P., &Liesack, W. (1997). The ammonia monooxygenase structural gene amoa as a functional marker: Molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology, 63(12), 4704–4712.
Smith, C. J., &Osborn, A. M. (2009). Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiology Ecology, 67(1), 6–20.
Soliman, M., &Eldyasti, A. (2018). Ammonia-Oxidizing Bacteria (AOB): opportunities and applications—a review. In Reviews in Environmental Science and Bio/Technology (pp. 285-321.).
Song, K., Sawayanagi, K., Numano, T., Taniichi, Y., Kikuchi, T., Takeda, T., …Terada, A. (2019). High-rate partial nitrification of semiconductor wastewater: Implications of online monitoring and microbial community structure. Biochemical Engineering Journal, 143(September 2018), 34–40.
Sun, F. L., Fan, L. L., &Xie, G. J. (2016). Effect of copper on the performance and bacterial communities of activated sludge using Illumina MiSeq platforms. Chemosphere, 156, 212–219.
Throbäck, I. N., Enwall, K., Jarvis, Å., &Hallin, S. (2004). Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiology Ecology, 49(3), 401–417.
Tokutomi, T., Shibayama, C., Soda, S., &Ike, M. (2010). A novel control method for nitritation: The domination of ammonia-oxidizing bacteria by high concentrations of inorganic carbon in an airlift-fluidized bed reactor. Water Research, 44(14), 4195–4203.
Ushiki, N., Jinno, M., Fujitani, H., Suenaga, T., Terada, A., &Tsuneda, S. (2017). Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation. Journal of Bioscience and Bioengineering, 123(5), 581–589.
Vrieze, J.De, Regueiro, L., Props, R., Vargas, R. V., Jáuregui, R., &Pieper, D. H. (2016). Presence does not imply activity : DNA and RNA patterns differ in response to salt perturbation in anaerobic digestion. Biotechnology for Biofuels, (November).
Wagner, M., Rath, G., Koops, H. P., Flood, J., &Amann, R. (1996). In situ analysis of nitrifying bacteria in sewage treatment plants. Water Science and Technology, 34(1-2–2 pt 1), 237–244.
Wang, Z., Luo, G., Li, J., Chen, S. Y., Li, Y., Li, W. T., &Li, A. M. (2016). Response of performance and ammonia oxidizing bacteria community to high salinity stress in membrane bioreactor with elevated ammonia loading. Bioresource Technology, 216, 714–721.
Whang, L. M., Wu, Y. J., Lee, Y. C., Chen, H. W., Fukushima, T., Chang, M. Y., …Chang, B. (2012). Nitrification performance and microbial ecology of nitrifying bacteria in a full-scale membrane bioreactor treating TFT-LCD wastewater. Bioresource Technology, 122, 70–77.
Wittwer, C. T., Herrmann, M. G., Moss, A. A., &Rasmussen, R. P. (1997). Continuous Fluorescence Monitoring of Rapid. BioTechniques, 22(1), 130–138.
Wu, L., Ning, D., Zhang, B., Li, Y., Zhang, P., Shan, X., …Zhou, H. (2019). Global diversity and biogeography of bacterial communities in wastewater treatment plants. Nature Microbiology, 4(7), 1183–1195.
Xiao, Y., Chen, T., Hu, Y., Wang, D., Han, Y., Lin, Y., &Wang, X. (2014). Advanced treatment of semiconductor wastewater by combined MBR-RO technology. Desalination, 336(1), 168–178.
Ye, L., &Zhang, T. (2011). Ammonia-oxidizing bacteria dominates over ammonia-oxidizing archaea in a saline nitrification reactor under low DO and high nitrogen loading. Biotechnology and Bioengineering, 108(11), 2544–2552.
Zeng, W., Zhang, Y., Li, L., Peng, Y. zhen, &Wang, S. ying. (2009). Control and optimization of nitrifying communities for nitritation from domestic wastewater at room temperatures. Enzyme and Microbial Technology, 45(3), 226–232.
Zhang, Q., Liang, G., Myrold, D. D., &Zhou, W. (2017). Variable responses of ammonia oxidizers across soil particle-size fractions affect nitrification in a long-term fertilizer experiment. Soil Biology and Biochemistry, 105, 25–36.
Zhang, T., Jin, T., &Yan, Q. (2010). Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR. Applied Microbiology and Biotechnology, 87(3), 1167–1176.
Zhang, T., Shao, M., &Ye, L. (2011). 454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. The ISME Journal, 6(6), 1137–1147.
Zhang, X., Gao, X., Li, C., Luo, X., &Wang, Y. (2019). Fluoride contributes to the shaping of microbial community in high fluoride groundwater in Qiji County, Yuncheng City, China. Scientific Reports, 9(1), 1–10.
Zhou, J., Liu, W., Deng, Y., Jiang, Y. H., Xue, K., He, Z., …Wang, A. (2013). Stochastic assembly leads to alternative communities with distinct functions in a bioreactor microbial community. MBio, 4(2), 1–8.
李雅菁，＂針對TFT-LCD製程廢水實場進行硝化效能評估與氨氧化菌群生態之研究＂國立成功大學環境工程學系碩士論文，2000