Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge


In order to improve wastewater treatment processes, a need exists for tools that rapidly give detailed insight into the community structure of activated sludge, supplementary to chemical and physical data. In this study, the advantages of microarrays and quantitative polymerase chin reaction (PCR) methods were combined into a real-time PCR assay that allows the simultaneous quantification of phylogenetic and functional genes involved in nitrification and denitrification processes. Simultaneous quantification was possible along a 5-log dynamic range and with high linear correlation (R 2 > 0.98). The specificity of the assay was confirmed by cloning and sequencing analyses of PCR amplicons obtained from activated sludge. The real-time assay was validated on mixed liquid samples of different treatment plants, which varied in nitrogen removal rate. The abundance of ammonia oxidizers was in the order of magnitude of 106 down to 104 ml−1, whereas nitrite oxidizers were less abundant (103–101 order of magnitude). The results were in correspondence with the nitrite oxidation rate in the sludge types. As for the nirS, nirK, and nosZ gene copy numbers, their abundance was generally in the order of magnitude of 108–105. When sludge samples were subjected to lab-scale perturbations, a decrease in nitrification rate was reflected within 18 h in the copy numbers of nitrifier genes (decrease with 1 to 5 log units), whereas denitrification genes remained rather unaffected. These results demonstrate that the method is a fast and accurate tool for the analysis of the (de)nitrifying community structure and size in both natural and engineered environmental samples.

This is a preview of subscription content, log in to check access.

Fig. 1


  1. Boon N, De Windt W, Verstraete W, Top EM (2002) Evaluation of nested PCR–DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analyses of bacterial communities from different wastewater treatment plants. FEMS Microbiol Ecol 39:101–112

  2. Braker G, Tiedje JM (2003) Nitric oxide reductase (norB) genes from pure cultures and environmental samples. Appl Environ Microbiol 69:3476–3483

  3. Braker G, Fesefeldt A, Witzel KP (1998) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ Microbiol 64:3769–3775

  4. De Beer D, Schramm A (1999) Micro-environments and mass transfer phenomena in biofilms studied with microsensors. Water Sci Technol 39(7):173–178

  5. Degrange V, Bardin R (1995) Detection and counting of Nitrobacter populations in soil by PCR. Appl Environ Microbiol 61:2093–2098

  6. Devers M, Soulas G, Martin-Laurent F (2004) Real-time reverse transcription PCR analyses of expression of atrazine catabolism genes in two bacterial strains isolated from soil. J Microbiol Methods 56:3–15

  7. Dionisi HM, Layton AC, Harms G, Gregory IR, Robinson KG, Sayler GS (2002) Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR. Appl Environ Microbiol 68:245–253

  8. Fogel GB, Collins CR, Li J, Brunk CF (1999) Prokariotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microb Ecol 38:93–113

  9. Greenberg AE, Cleseri LS, Eaton AD (1992) Standard methods for the examination of water and wastewater, 18th edn. APHA, Washington

  10. Grommen R, Van Hauteghem I, Van Wambeke M, Verstraete W (2002) An improved nitrifying enrichment to remove ammonium and nitrite from freshwater aquaria systems. Aquaculture 211:115–124

  11. Hastings RC, Saunders JR, Hall GH, Pickup RW, McCarthy AW (1998) Application of molecular biological techniques to a seasonal study of ammonia oxidation in a eutrophic freshwater lake. Appl Environ Microbiol 64:3674–3682

  12. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. BioTechniques 23:504–511

  13. Heylen K, Vanparys B, Wittebolle L, Verstraete W, Boon N, De Vos P (2006) Cultivation of denitrifying bacteria: Optimization of isolation conditions and diversity study. Appl Environ Microbiol 72:2637–2643

  14. Kelly JJ, Siripong S, McCormack J, Janus LR, Urakawa H, El Fantroussi S, Noble PA, Sappelsa L et al (2005) DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples. Water Res 39:3229–3238

  15. Kloos K, Mergel A, Rosch C, Bothe H (2001) Denitrification within the genus Azospirillum and other associative bacteria. Aust J Plant Physiol 28:991–998

  16. Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: a model for molecular microbial ecology. Annu Rev Microbiol 55:485–529

  17. Kowalchuk GA, Stephen JR, De Boer W, Prosser JI, Embley TM, Woldendorp JW (1997) Analyses of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 63:1489–1497

  18. Lopez-Gutierrez JC, Henry S, Hallet S, Martin-Laurent F, Catroux G, Philippot L (2004) Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J Microbiol Methods 57:399–407

  19. Manser R, Muche K, Gujer W, Siegrist H (2005) A rapid method to quantify nitrifiers in activated sludge. Water Res 39:1585–1593

  20. Mateju V, Cizinska S, Krejci J, Janoch T (1992) Biological water denitrification—a review. Enzyme Microb Technol 14:170–183

  21. Morrison TB, Ma Y, Weis JH, Weis JJ (1999) Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR. J Clin Microbiol 37:987–992

  22. Norton JM, Alzerreca JJ, Suwa Y, Klotz MG (2002) Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria. Arch Microbiol 177:139–149

  23. Painter HA (1986) Nitrification in the treatment of sewage and wastewaters. In: Prosser JI (ed). Nitrification. IRL Press, Oxford, UK, pp 185–211

  24. Philippot L (2002) Denitrifying genes in bacterial and Archaeal genomes. Biochim Biophys Acta 1577:355–376

  25. Philippot L (2005) Tracking nitrate reducers and denitrifiers in the environment. Biochem Soc Trans 33:200–204

  26. Philippot L, Piutti S, Martin-Laurent F, Hallet S, Germon JC (2002) Molecular analyses of the nitrate-reducing community from unplanted and maize-planted soils. Appl Environ Microbiol 68:6121–6128

  27. Pynaert K, Sprengers R, Laenen J, Verstraete W (2002) Oxygen-limited nitrification and denitrification in a lab-scale rotating biological contactor. Environ Technol 23:353–362

  28. Pynaert K, Smets BF, Beheydt D, Verstraete W (2004) Start-up of autotrophic nitrogen removal reactors via sequential biocatalyst addition. Environ Sci Technol 38:1228–1235

  29. Rotthauwe JH, Boer W, Liesack W (1997) The ammonia monoxygenase structural gene amoA as a functional marker: molecular fine scale analyses of natural ammonia oxidizing populations. Appl Environ Microbiol 63:4704–4712

  30. Schmidt I, Gries T, Willuweit T (1999) Nitrification—Fundamentals of the metabolism and problems at the use of ammonia oxidizers. Acta Hydrochim Hydrobiol 27:121–135

  31. Stubner S (2002) Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SyberGreen detection. J Microbiol Methods 50:155–164

  32. Throback IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:407–417

  33. Vanparys B, Bodelier P, De Vos P (2006) Validation of the correct start codon of norX/nxrX and universality of the norAXB/nxrAXB gene cluster in Nitrobacter species. Curr Microbiol 53(3):255–257

  34. Wallenstein MD, Vilgalys RJ (2005) Quantitative analyses of nitrogen cycling genes in soils. Pedobiologia 49:665–672

  35. Wittebolle L, Boon N, Vanparys B, Heylen K, De Vos P, Verstraete W (2005) Failure of the ammonia oxidation process in two pharmaceutical wastewater treatment plants is linked to shifts in the bacterial communities. J Appl Microbiol 99:997–1006

  36. Wu L, Thompson DK, Liu X, Fields MW, Tiedje JM, Zhou J (2004) Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications. Environ Sci Technol 38:6775–6782

  37. Yuan Z, Blackall LL (2002) Sludge population optimisation: a new dimension for the control of biological wastewater treatment systems. Water Res 36:482–490

  38. Zehr JP, Mellon M, Braun S, Litaker W, Steppe T, Paerl HW (1995) Diversity of heterotrophic nitrogen-fixation genes in a marine cyanobacterial mat. Appl Environ Microbiol 61:2527–2532

  39. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616

Download references


This work was supported by project grant G.O.A. 1205073 (2003–2008) of the ‘Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek’ (Belgium). The authors thank Han Vervaeren and Robin Temmerman for their critical reading of the manuscript.

Author information

Correspondence to Nico Boon.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Geets, J., de Cooman, M., Wittebolle, L. et al. Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Appl Microbiol Biotechnol 75, 211–221 (2007).

Download citation


  • SYBR Green I real-time PCR
  • 16S rRNA gene
  • amoA
  • Nitrite oxidoreductase
  • nosZ
  • nirS
  • nirK
  • Activated sludge