Applied Microbiology and Biotechnology

, Volume 97, Issue 19, pp 8795–8804 | Cite as

Microbial activity of suspended biomass from a nitritation–anammox SBR in dependence of operational condition and size fraction

  • Eva Marianne GilbertEmail author
  • Elisabeth Müller
  • Harald Horn
  • Susanne Lackner
Environmental biotechnology


Single-stage nitritation–anammox combines the growth of aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AnAOB) in one reactor. The necessary compromise of their milieu conditions often leads to the growth of nitrite-oxidizing bacteria (NOB). For this study, a sequencing batch reactor (SBR) for nitritation–anammox was operated for 180 days with sewage sludge reject water (removal capacity, 0.4 kg N m−3 day−1). The growth of NOB was favored by enhanced oxygen supply rather than extended aerobic phases. Suspended-type biomass from this SBR was taken regularly and sieved into three size fractions (all of them <1,000 μm). Batch experiments as well as fluorescence in situ hybridization were performed to study the distribution and activity of AnAOB, AOB, and NOB within those size fractions. Both the measured conversion rates and detected abundances decreased with increasing size fraction. The highest anammox conversion rates (15 g NH4 +–N per kilogram VSS per hour) and the highest abundances of Brocadia fulgida were found in the medium size fraction (100–315 μm). The batch experiments proved to be accurate tools for the monitoring of multiple processes in the reactor. The results were representative for reactor performance during the 6 months of reactor operation.


Nitritation–anammox Nitratation Suspended biomass Particle size distribution Microbial distribution Batch experiments Activity measurements 



This research was funded by the Bavarian Environment Agency. The authors would like to thank Susanne Thiemann (Institute of Water Quality Control, Technical University Munich) for FISH analyses and Steffen Krause (Sanitary Engineering and Waste Management, Bundeswehr Universty Munich) for supply and assistance with the particle size analyses.

Supplementary material

253_2012_4591_MOESM1_ESM.docx (315 kb)
ESM 1 (DOCX 315 kb)


  1. Amann R, Krumholz L, Stahl D (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172(2):762–770PubMedGoogle Scholar
  2. Anthonisen A, Loehr R, Prakasam T, Srinath E (1976) Inhibition of nitrification by ammonia and nitrous acid. J Water Pollut Con F 48(5):835–852Google Scholar
  3. APAH (2005) Standard methods for the examination of water and wastewater, 21st edn. American Public Health Association, Washington, DCGoogle Scholar
  4. Bassin JP, Pronk M, Kraan R, Kleerebezem R, van Loosdrecht MCM (2011) Ammonium adsorption in aerobic granular sludge, activated sludge and anammox granules. Water Res 45(16):5257–5265PubMedCrossRefGoogle Scholar
  5. Blackburne R, Vadivelu VM, Yuan Z, Keller J (2007) Kinetic characterisation of an enriched Nitrospira culture with comparison to Nitrobacter. Water Res 41(14):3033–3042PubMedCrossRefGoogle Scholar
  6. Blackburne R, Yuan Z, Keller J (2008) Partial nitrification to nitrite using low dissolved oxygen concentration as the main selection factor. Biodegradation 19(2):303–312PubMedCrossRefGoogle Scholar
  7. Dapena-Mora A, Fernández I, Campos JL, Mosquera-Corral A, Méndez R, Jetten MSM (2007) Evaluation of activity and inhibition effects on anammox process by batch tests based on the nitrogen gas production. Enzyme Microb Tech 40(4):859–865CrossRefGoogle Scholar
  8. de Kreuk MK, Kishida N, van Loosdrecht MCM (2007) Aerobic granular sludge—state of the art. Water Sci Technol 55(8–9):75PubMedGoogle Scholar
  9. Egli K (2003) On the use of anammox in treating ammonium-rich wastewaterGoogle Scholar
  10. Gieseke A, Bjerrum L, Wagner M, Amann R (2003) Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm. Environ Microbiol 5(5):355–369PubMedCrossRefGoogle Scholar
  11. Ginestet P, Audic J, Urbain V, Block J (1998) Estimation of nitrifying bacterial activities by measuring oxygen uptake in the presence of the metabolic inhibitors allylthiourea and azide. Appl Environ Microb 64(6):2266–2268Google Scholar
  12. Gresch M, Armbruster M, Braun D, Gujer W (2011) Effects of aeration patterns on the flow field in wastewater aeration tanks. Water Res 45(2):810–818PubMedCrossRefGoogle Scholar
  13. Grunditz C, Dalhammar G (2001) Development of nitrification inhibition assays using pure cultures of Nitrosomonas and Nitrobacter. Water Res 35(2):433–440PubMedCrossRefGoogle Scholar
  14. Hawkins S, Robinson K, Layton A, Sayler G (2010) Limited impact of free ammonia on Nitrobacter spp. inhibition assessed by chemical and molecular techniques. Bioresource Technol 101(12):4513–4519Google Scholar
  15. Helmer C, Kunst S, Juretschko S, Schmid M, Schleifer K-H, Wagner M (1999) Nitrogen loss in a biofilm system. Water Sci Technol 39(7):13–21CrossRefGoogle Scholar
  16. Helmer-Madhok C, Schmid M, Filipov E, Gaul T, Hippen A, Rosenwinkel K-H, Seyfried C, Wagner M, Kunst S (2002) Deammonification in biofilm systems: population structure and function. Water Sci Technol 46(1–2):223–231PubMedGoogle Scholar
  17. Jetten MSM, Strous M, Pas-Schoonen KT, Schalk J, van Dongen U, Graaf AA, Logemann S, Muyzer G, van Loosdrecht MCM, Kuenen JG (1998) The anaerobic oxidation of ammonium. Fems Microbiol Rev 22(5):421–437Google Scholar
  18. Joss A, Derlon N, Cyprien C, Burger S, Szivak I, Traber J, Siegrist H, Morgenroth E (2011) Combined nitritation–anammox: advances in understanding process stability. Environ Sci Technol 45(22):9735–9742PubMedCrossRefGoogle Scholar
  19. Katsogiannis A, Kornaros M, Lyberatos G (2003) Enhanced nitrogen removal in SBRs bypassing nitrate generation accomplished by multiple aerobic/anoxic phase pairs. Water Sci Technol 47(11):53–59PubMedGoogle Scholar
  20. Kim D, Lee D, Keller J (2006) Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH. Bioresource Technol 97(3):459–468CrossRefGoogle Scholar
  21. Kowalchuk G, Stephen JR (2001) Ammonia-oxidizing bacteria: a model for molecular microbial ecology. Annu Rev Microbiol 55:485–529PubMedCrossRefGoogle Scholar
  22. Laanbroek HJ, Gerards S (1993) Competition for limiting amounts of oxygen between grown in mixed continuous cultures. Arch Microbiol 159(5):453–459CrossRefGoogle Scholar
  23. Lackner S, Horn H (2012) Evaluating operation strategies and process stability of a single stage nitritation–anammox SBR by use of the oxidation-reduction potential (ORP). Bioresource Technol 107:70–77CrossRefGoogle Scholar
  24. Lotti T, Kleerebezem R, Loosdrecht MCM van (2012) The MBR as superior tool for enrichment and kinetic characterization of anammox bacteria. IWA NRR 2012: Trends in NRR. Harbin, ChinaGoogle Scholar
  25. Müller E (2006) Bacteria and extracellular polymeric substances in activated sludge scum formation. PhD thesis, TU Munich, GermanyGoogle Scholar
  26. Nielsen M, Bollmann A, Sliekers AO, Jetten MSM, Schmid M, Strous M, Schmidt I, Larsen L, Nielsen LP, Revsbech N (2005) Kinetics, diffusional limitation and microscale distribution of chemistry and organisms in a CANON reactor. FEMS Microbiol Ecol 51(2):247–256PubMedCrossRefGoogle Scholar
  27. Nogueira R, Melo L (2006) Competition between Nitrospira spp. and Nitrobacter spp. in nitrite-oxidizing bioreactors. Biotechnol Bioeng 95(1):169–175PubMedCrossRefGoogle Scholar
  28. Schramm A, Beer D, de Wagner M, Amann R (1998) Identification and Activities in situ of Nitrosospira and Nitrospira spp. as dominant populations in a nitrifying fluidized bed reactor. Appl Environ Microb 64(9):3480–3485Google Scholar
  29. Simm RA, Mavinic DS (2006) A targeted study on possible free ammonia inhibition of Nitrospira. J Environ Eng Sci 5(5):365–376CrossRefGoogle Scholar
  30. Sliekers AO, Haaijer SCM, Stafsnes MH, Kuenen JG, Jetten MSM (2005) Competition and coexistence of aerobic ammonium- and nitrite-oxidizing bacteria at low oxygen concentrations. Appl Microbiol Biot 68(6):808–817CrossRefGoogle Scholar
  31. Strous M, Gerven E, van Kuenen JG, Jetten MSM (1997) Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (anammox) sludge. Appl Environ Microb 63(6):2446–2448Google Scholar
  32. Strous M, Kuenen JG, Jetten MSM (1999) Key physiology of anaerobic ammonium oxidation. Appl Environ Microb 65(7):3248–3250Google Scholar
  33. Terada A, Lackner S, Kristensen K, Smets B (2010) Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter-diffusion geometry. Environ Microbiol 12(10):2858–2872PubMedGoogle Scholar
  34. Third K, Paxman J, Schmid M, Strous M, Jetten MSM, Cord-Ruwisch R (2005) Enrichment of anammox from activated sludge and its application in the CANON process. Microbial Ecol 49(2):236–244CrossRefGoogle Scholar
  35. 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 Res 44(14):4195–4203PubMedCrossRefGoogle Scholar
  36. Turk O, Mavinic D (1989) Maintaining nitrite build-up in a system acclimated to free ammonia. Water Res 23(11):1383–1388CrossRefGoogle Scholar
  37. van de Graaf AA, de Bruijn P, Robertson LA, Jetten MSM, Kuenen JG (1996) Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142(8):2187–2196CrossRefGoogle Scholar
  38. Villaverde S, Fdz-Polanco F, Garcia PA (2000) Nitrifying biofilm acclimation to free ammonia in submerged biofilters. Start-up influence. Water Res 34(2):602–610CrossRefGoogle Scholar
  39. Vlaeminck SE, Garcia Encina PA, Lacalle ML, Fdz-Polanco F (2000) New operational strategy for SBR technology for total nitrogen removal from industrial wastewaters highly loaded with nitrogen. Water Sci Technol 41(12):85–93Google Scholar
  40. Vlaeminck SE, Cloetens L, Carballa M, Boon N, Verstraete W (2008) Granular biomass capable of partial nitritation and anammox. Water Sci Technol 58(5):1113–1120PubMedCrossRefGoogle Scholar
  41. Vlaeminck SE, Terada A, Smets B, Clippeleir HD, Schaubroeck T, Bolca S, Demeestere L, Mast J, Boon N, Carballa M, Verstraete W (2010) Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox. Appl Environ Microb 76(3):900–909CrossRefGoogle Scholar
  42. Yoo H, Ahn K-H, Lee H-J, Lee K-H, Kwak Y-J, Song K-G (1999) Nitrogen removal from synthetic wastewater by simultaneous nitrification and denitrification (SND) via nitrite in an intermittently-aerated reactor. Water Res 33(1):145–154CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Eva Marianne Gilbert
    • 1
    Email author
  • Elisabeth Müller
    • 2
  • Harald Horn
    • 1
  • Susanne Lackner
    • 1
  1. 1.Engler-Bunte-Institute, Water Chemistry and Water TechnologyKarlsruhe Institute of TechnologyKarlsruheGermany
  2. 2.Institute of Water and Environment, Water Quality ControlTechnische Universität MünchenGarchingGermany

Personalised recommendations