Influence of FA and FNA to Microbial Community in Sequencing Batch Ammonium Partial Nitrification System

암모니아 부분산화 공정의 제어와 미생물 군집의 변화

Ahn, Johwan

  • Received : 2015.03.24
  • Accepted : 2015.07.21
  • Published : 2015.07.31


A sequencing batch reactor was operated under different pH conditions to see the influence of free ammonia (FA) and free nitrous acid (FNA) to microbial community on ammonium partial nitrification. Long-term influences of FA and FNA were evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis and fluorescence in situ hybridization. Nitrite accumulation was successfully achieved at pH 8.2 and 6.3. The shifts in the microbial community were observed when influent ammonia concentration increased to 1 g $NH_4$-N/L at pH 8.2, and then when pH was dropped to 6.3. Both Nitrosomonas and Nitrosospira were selected during the startup of the reactor, and eventually became dominant members as ammonia-oxidizing bacteria. The results of molecular microbiological analysis strongly suggested that the composition of microbial community was changed according to the method used to control nitrite-oxidizing bacteria.


Ammonia-oxidizing bacteria;Fluorescence in situ hybridization;Free ammonia;Free nitrous acid;Nitrite-oxidizing bacteria;Polymerase chain reaction-denaturing gradient gel electrophoresis


  1. Amann, R. I., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R., and Stahl, D. A. (1990). Combination of 16S rRNA-targeted Oligonucleotide Probes with Flow Cytometry for Analyzing Mixed Microbial Populations, Applied and Environmental Microbiology, 56, pp. 1919-1925.
  2. American Public Health Association, American Water Works Association and Water Environment Federation (APHA, AWWA, and AWEF). (1999). Standard Methods for the Examination of Water and Wastewater, Washington D.C.
  3. Andrews, J. H. and Harris, R F. (1986). r-and K-selection and Microbial Ecology, Advances in Microbial Ecology, 9, pp. 99-147.
  4. Anthonisen, A. C., Loehr, R. C., Prakasam, T. B., and Srinath, E. G. (1976). Inhibition of Nitrification by Ammonia and Nitrous Acid, Water Pollution Control Federation, 48, pp. 835-852.
  5. Bernhard, A. E., Donn, T., Giblin, A. E., and Stahl, D., A. (2005). Loss of Diversity of Ammonia-oxidizing Bacteria Correlates with Increasing Salinity in an Estuary System, Environmental Microbiology, 7, pp. 1289-1297.
  6. Blackburne, R., Yuan, Z., and Keller, J. (2008). Partial Nitrification to Nitrite using :Low Dissolved Oxygen Concentration as the Main Selection Factor, Biodegradation, 19, pp. 303-132.
  7. Chen, G. H., Wong, M. T., Okabe, S., and Watanabe, Y. (2003). Dynamic Response of Nitrifying Activated Sludge Batch Culture to Increased Chloride Concentration, Water Research, 37, pp. 3125-3135.
  8. Choi, E., Lee, Y., Gil, K., and Yun, Z. (2000). Nitrogen Removal from Anaerobic Digester Supernatant Via Nitritationdenitritation, Journal of Korean Society on Water Environment, 16, pp. 265-273. [Korean Literature]
  9. Cua, L. S. and Stein, L. Y. (2011). Effects of Nitrite on Ammonia-oxidizing Activity and Gene regulation in Three Ammonia-oxidizing Bacteria, FEMS Microbiology Letters, 319, pp. 169-175.
  10. Daims, H., Bruhl, A., Amann, R., Schleifer, K. H., and Wagner, M. (1999). The Domain-specific Probe EUB338 is Insufficient for the Detection of All Bacteria: Development and Evaluation of a More Comprehensive Probe Set, Systematic and Applied Microbiology, 22, pp. 434-444.
  11. De Boer, W., Gunnewiek, P. J. A., Veenhuis, M., Bock, E., and Laanbroek, H. J. (1991). Nitrification at Low pH by Aggregated Chemolithotrophic Bacteria, Applied and Environmental Microbiology, 57, pp. 3600-3604.
  12. Dincer, A. R., and Kargi, F. (1999). Salt Inhibition of Nitrification and Denitrification in Saline Wastewater, Environmental Technology, 20, pp. 1147-1153.
  13. Dytczak, M. A., Londry, K., Siegrist, H., and Oleszkiewicz, J. A. (2006). Extracellular Polymers in Partly Ozonated Return Activated Sludge: Impact on Flocculation and Dewaterability, Water Science and Technology, 54(9), pp. 155-164.
  14. Fdz-Polanco, F., Villaverde, S., and Garcia, P. A. (1994). Temperature Effect on Nitrifying Bacteria Activity in Biofilters: Activation and Free Ammonia Inhibition, Water Science and Technology, 30(11), pp. 121-130.
  15. Freitag, T. E., Chang, L., and Prosser, J. I. (2006). Changes in the Community Structure and Activity of Betaproteobacterial Ammonia-oxidizing Sediment Bacteria Along a Freshwatermarine Gradient, Environmental Microbiology, 8, pp. 684-696.
  16. Glass, C., Silverstein, J., and Oh, J. (1997). Inhibition of Denitrification in Activated Sludge by Nitrite, Water Environment Research, 69, pp. 1086-1093.
  17. Hanaki, K., Wantawin, C., and Ohgaki, S. (1990). Nitrification at Low Levels of Dissolved Oxygen with and Without Organic Loading in a Suspended-growth Reactor, Water Research, 24, pp. 297-302.
  18. Hellinga, C., Schellen, A. A. J. C., Mulder, J. W., van Loosdrecht, M. C. M., and Heijnen, J. J. (1998). The SHARON Process: an Innovative Method for Nitrogen Removal from Ammonium-rich Waste Water, Water Science and Technology, 37(9), pp. 135-142.
  19. Jetten, M. S. M., Logemann, S., Muyzer, G., and Robertson, L. A. (1997). Novel Principles in the Microbial Conversion of Nitrogen Compounds, Antonie van Leeuwenhoek, 71, pp. 75-93.
  20. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L., and Pace. N. R. (1985). Rapid Determination of 16S Ribosomal RNA Sequences for Phylogenetic Analyses, Proceedings of the National Academy of Sciences of the United States of America, 82, pp. 6955-6959.
  21. Limpiyakorn, T., Shinohara, Y., Kurisu, F., and Yagi, O. (2005). Communities of Ammonia-oxidizing Bacteria in Activated Sludge of Various Sewage Treatment plants in Tokyo, FEMS Microbiology Ecology, 54. pp. 205-217.
  22. Logemann, S., Schantl, J., Bijvank, S., van loosdrecht, M. C. M., Kuenen, J. G., and Jetten, M. (1998). Molecular Microbial Diversity in a Nitrifying Reactor System Without Sludge Retention, FEMS Microbiology Ecology, 27, pp. 239-249.
  23. Mobarry, B. K., Wagner, M., Urbain, V., Rittmann, B. E., and Stahl, D. A. (1996). Phylogenetic Probes for Analyzing Abundance and Spatial Organization of Nitrifying Bacteria, Applied and Environmental Microbiology, 62, pp. 2156-2162.
  24. Muyzer, G., de Waal, E. C., and Uitterlinden, A. G. (1993). Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-amplified Genes Coding for 16S rRNA, Applied and Environmental Microbiology, 59, pp. 695-700.
  25. Nicol, G. W., Leininger, S., Schleper, C., Prosser, J. I. (2008). The Influence of Soil pH on the Diversity, Abundance and Transcriptional Activity of Ammonia Oxidizing Archaea and Bacteria, Environmental Microbiology, 10, pp. 2966-78.
  26. Park, H. D. and Noguera, D. R. (2004). Evaluating the Effect of Dissolved Oxygen on Ammonia-oxidizing Bacterial Communities in Activated Sludge, Water Research, 38, pp. 3275-3286.
  27. Rittmann, B. and McCarty, P. L. (2001). Environmental Biotechnology: Principle and Applications, McGraw-Hill, New York.
  28. Stein, L. Y. and Arp, D. J. (1998). Loss of Ammonia Monooxygenase Activity in Nitrosomonas Europaea Upon Exposure to Nitrite, Applied and Environmental Microbiology, 64, pp. 4098-4102.
  29. Stempfhuber, B., Engel, M., Fischer, D., Neskovic-Prit, G., Wubet, T., Schoning, I., Gubry-Rangin, C., Kublik, S., Schloter-Hai, B., Rattei, T., Welzl, G., Nicol, G. W., Schrumpf, M., Buscot, F., Prosser, J. I., and Schloter, M. (2015). pH as a Driver for Ammonia-Oxidizing Archaea in Forest Soils, Microbial Ecology, 69, pp. 879-883.
  30. Tan, N. C. G., Kampschreur, M. J., Wanders, W., van der Pol, W. L. J., van de Vossenberg, J., Kleerebezem, R., van Loosdrecht, M. C. M., and Jetten, M. S. M. (2008). Physiological and Phylogenetic Study of an Ammonium-oxidizing Culture at High Nitrite Concentrations, Systematic and Applied Microbiology, 31, pp. 114-125.
  31. Terada, A., Sugawara, S., Yamamoto, T., Zhou, S., Koba, K., Hosomi, M. (2013). Physiological Characteristics of Predominant Ammonia-oxidizing Bacteria Enriched from Bioreactors with Different Influent Supply Regimes, Biochemical Engineering Journal, 79, pp. 153-161.
  32. Vadivelu, V. M., Keller, J., and Yuan, Z. (2007). Effect of Free Ammonia on the Respiration and Growth Processes of an Enriched Nitrobacter Culture, Water Research, 41, pp. 826-834.
  33. Vejmelkova, D., Sorokin, D. Y., Abbas, B., Kovaleva, O. L., Kleerebezem, R., Kampschreur, M. J., Muyzer, G., and van Loosdrecht, M. C. (2012). Analysis of Ammonia-oxidizing Bacteria Dominating in Lab-scale Bioreactors with High Ammonium Bicarbonate Loading, Applied Microbiology and Biotechnology, 93, pp. 401-410.
  34. Wagner, M., Rath, G., Amann, R., Koops, H. P., and Schleifer, K. H. (1995). In Situ Identification of Ammonia-oxidizing Bacteria, Systematic and Applied Microbiology, 18, pp. 251-264.
  35. Wang, X., Wen, X., Criddke, C., Wells, G., Zhang, J., and Zhao, Y. (2010). Community Analysis Ammonia-oxidizing Bacteria in Activated Sludge of Eight Wastewater Treatment Systems, Journal of Environmental Sciences, 22, pp. 627-634.
  36. Wilen, B. M., Gapes, D., Blackall, L. L., and Keller, J. (2004). Structure and microbial composition of nitrifying microbial aggregates and their relation to internal mass transfer effects, Water Science and Technology, 50(10), pp. 213-220.
  37. Yang, L. and Alleman, J. E. (1992). Investigation of batchwise nitrite build-up by an enriched nitrification culture, Water Science and Technology, 26, pp. 997-1005.