Feeding Strategies for Enrichment and Characterization of Anammox Biomass in a Sequencing Batch Reactor


Anammox bacteria represent a promising alternative for treating ammonium-rich wastewater. In this work reported, biomass performing anaerobic oxidation of ammonium was enriched in a sequencing batch reactor, from sludge used for the treatment of high-nitrogen waste from an amino acid-producing industry. After 89 days of operation, both ammonium and nitrite were consumed. During operation under a 24-hour cycle, the applied nitrogen load (ANL) was increased from 155 to 802 mg N/L·d. This strategy resulted in efficiencies of nitrogen removal and nitrogen conversion rate of 91.7% and 98.5%, respectively. Specific anammox activity increased proportionally to ANL and it was partially inhibited at 802 mg N/L·d. Sequencing analysis using 16S rRNA anammox primers, after 170 days of operation, showed that 21 clones were grouped into two OTUs (operational taxonomic units). The identity of the 16S rRNA gene of OTU esp 1 showed similarity to Brocadia species, and OTU esp 2 displayed 99% similarity to Anammoxoglobus propionicus. After 450 days of operation, sequencing analysis using universal primers showed that 48 clones were grouped into 19 OTUs representing six major groups of bacteria: Planctomycetes, beta-Proteobacteria, green sulfur bacteria of the Chlorobi phylum, Nitrospira, Chloroflexi and OP 11. Brocadia sp. was the only anammox bacteria in the biomass at this time.

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Martins, T. , Souza, T. and Varesche, M. (2014) Feeding Strategies for Enrichment and Characterization of Anammox Biomass in a Sequencing Batch Reactor. American Journal of Analytical Chemistry, 5, 891-900. doi: 10.4236/ajac.2014.514097.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Mulder, A., Van 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.
[2] Jetten, M.S.M., van Dongen, L.G.J.M. and van Loosdrecht, M.C.M. (2001) The Combined Sharon/Anammox Process. IWA Publishing London, UK.
[3] Strous, M., Fuerst, J.A., Kramer, E.H.M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K.T., Webb, R., Kuenen, J.G. and Jetten, M.S.M. (1999) Missing Lithotroph Identified as New Planctomycete. Nature, 400, 446-449.
[4] Egli, K., Fanger, U., Alvarez, P.J.J., Siegrist, H., van der Meer, J.R. and Zehnder, A.J.B. (2001) Enrichment and Characterization of an Anammox Bacterium from a Rotating Biological Contactor Treating Ammonium-Rich Leachate. Archives of Microbiology, 175, 198-207.
[5] de Araujo, M.M. and Zaiat, M. (2009) An Upflow Fixed-Bed Anaerobic-Aerobic Reactor for Removal of Organic Matter and Nitrogen from L-Lysine Plant Wastewater. Canadian Journal of Civil Engineering, 36, 1085-1094.
[6] Jetten, M.S.M., Wagner, M., Fuerst, J., van Loosdrecht, M., Kuenen, G. and Strous, M. (2001) Microbiology and Application of the Anaerobic Ammonium Oxidation (“anammox”) Process. Current Opinion in Biotechnology, 12, 283-288.
[7] van Graaf, A.A.V., de Bruijn, P., Robertson, L.A., Jetten, M.S.M. and Kuenen, J.G. (1996) Autotrophic Growth of Anaerobic Ammonium-Oxidizing Micro-Organisms in a Fluidized Bed Reactor. Microbiology, 142, 2187-2196.
[8] Strous, M., Kuenen, J.G. and Jetten, M.S.M. (1999) Key Physiology of Anaerobic Ammonium Oxidation. Applied and Environmental Microbiology, 65, 3248-3250.
[9] APHA, AWWA and WPCF (2005) Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington DC.
[10] Schmid, M., Twachtmann, U., Klein, M., Strous, M., Juretschko, S., Jetten, M., Metzger, J.W., Schleifer, K.H. and Wagner, M. (2000) Molecular Evidence for Genus Level Diversity of Bacteria Capable of Catalyzing Anaerobic Ammonium Oxidation. Systematic and Applied Microbiology, 23, 93-106.
[11] Egli, K., Bosshard, F., Werlen, C., Lais, P., Siegrist, H., Zehnder, A.J.B. and van der Meer, J.R. (2003) Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon. Microbial Ecology, 45, 419-432.
[12] Griffiths, R.I., Whiteley, A.S., O’Donnell, A.G. and Bailey, M.J. (2000) Rapid Method for coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition. Applied and Environmental Microbiology, 66, 5488-5491.
[13] Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. and Higgins, D.G. (2007) Clustal W and Clustal X Version 2.0. Bioinformatics, 23, 2947-2948.
[14] Schloss, P.D. and Handelsman, J. (2005) Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness. Applied and Environmental Microbiology, 71, 1501-1506.
[15] Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S., Jobb, G., Forster, W., Brettske, I., Gerber, S., Ginhart, A.W., Gross, O., Grumann, S., Hermann, S., Jost, R., Konig, A., Liss, T., Lussmann, R., May, M., Nonhoff, B., Reichel, B., Strehlow, R., Stamatakis, A., Stuckmann, N., Vilbig, A., Lenke, M., Ludwig, T., Bode, A. and Schleifer, K.H. (2004) ARB: A Software Environment for Sequence Data. Nucleic Acids Research, 32, 1363-1371.
[16] Thuan, T.H., Jahng, D.J., Jung, J.Y., Kim, D.J., Kim, W.K., Park, Y.J., Kim, J.E. and Ahn, D.H. (2004) Anammox Bacteria Enrichment in Upflow Anaerobic Sludge Blanket (UASB) Reactor. Biotechnology and Bioprocess Engineering, 9, 345-351.
[17] Araujo, J.C., Campos, A.C., Correa, M.M., Silva, E.C., Matte, M.H., Matte, G.R., Von Sperling, M. and Chernicharo, C.A. (2011) Anammox Bacteria Enrichment and Characterization from Municipal Activated Sludge. Water Science and Technology, 64, 1428-1434.
[18] Lopez, H., Puig, S., Ganigue, R., Ruscalleda, M., Balaguer, M.D. and Colprim, J. (2008) Start-Up and Enrichment of a Granular Anammox SBR to Treat High Nitrogen Load Wastewaters. Journal of Chemical Technology and Biotechnology, 83, 233-241.
[19] Martins, T.H. and Varesche, M.B.A. (2013) Specific Anammox Activity under Different Conditions (I): The Influence of Temperature and Initial Nitrogen Concentration. Proceedings of the 13th World Congress on Anaerobic Digestion: Recovering (bio) Resources for the World, IWA, Santiago de Compostela.
[20] Strous, M., Heijnen, J.J., Kuenen, J.G. and Jetten, M.S.M. (1998) The Sequencing Batch Reactor as a Powerful Tool for the Study of Slowly Growing Anaerobic Ammonium-Oxidizing Microorganisms. Applied Microbiology and Biotechnology, 50, 589-596.
[21] Magrí, A., Vanotti, M.B. and Szogi, A.A. (2012) Anammox Sludge Immobilized in Polyvinyl Alcohol (PVA) Cryogel Carriers. Bioresource Tech-nology, 114, 231-240.
[22] Cho, S., Takahashi, Y., Fujii, N., Yamada, Y., Satoh, H. and Okabe, S. (2010) Nitrogen Removal Performance and Microbial Community Analysis of an Anaerobic Up-Flow Granular Bed Anammox Reactor. Chemosphere, 78, 1129-1135.
[23] Kartal, B., Kuypers, M.M.M., Lavik, G., Schalk, J., den Camp, H., Jetten, M.S.M. and Strous, M. (2007) Anammox Bacteria Disguised as Denitrifiers: Nitrate Reduction to Dinitrogen Gas via Nitrite and Ammonium. Environmental Microbiology, 9, 635-642.
[24] Strous, M., Pelletier, E., Mangenot, S., Rattei, T., Lehner, A., Taylor, M.W., Horn, M., Daims, H., Bartol-Mavel, D., Wincker, P., Barbe, V., Fonknechten, N., Vallenet, D., Segurens, B., Schenowitz-Truong, C., Medigue, C., Collingro, A., Snel, B., Dutilh, B.E., Op den Camp, H.J.M., van der Drift, C., Cirpus, I., van de Pas-Schoonen, K.T., Harhangi, H.R., van Niftrik, L., Schmid, M., Keltjens, J., van de Vossenberg, J., Kartal, B., Meier, H., Frishman, D., Huynen, M.A., Mewes, H.W., Weissenbach, J., Jetten, M.S.M., Wagner, M. and Le Paslier, D. (2006) Deciphering the Evolution and Metabolism of an Anammox Bacterium from a Community Genome. Nature, 440, 790-794.
[25] Daims, H., Nielsen, P.H., Nielsen, J.L., Juretschko, S. and Wagner, M. (2000) Novel Nitrospira-Like Bacteria as Dominant Nitrite-Oxidizers in Biofilms from Wastewater Treatment Plants: Diversity and in Situ Physiology. Water Science and Technology, 41, 85-90.
[26] Daims, H., Nielsen, J.L., Nielsen, P.H., Schleifer, K.H. and Wagner, M. (2001) In Situ Characterization of Nitrospira-Like Nitrite Oxidizing Bacteria Active in Wastewater Treatment Plants. Applied and Environmental Microbiology, 67, 5273-5284.
[27] Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb, R.I., Schouten, S., Fuerst, J.A., Damste, J.S.S., Jetten, M.S.M. and Strous, M. (2007) Candidatus “Anammoxoglobus Propionicus” a New Propionate Oxidizing Species of Anaerobic Ammonium Oxidizing Bacteria. Systematic and Applied Microbiology, 30, 39-49.

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