Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities


Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, “Candidatus Accumulibacter phosphatis.” The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Maximum-likelihood tree based on partial and complete 16S rRNA genes identified on metagenomic contigs comprising at least two reads.
Figure 2: EBPR-relevant metabolism inferred from the A. phosphatis composite genome.
Figure 3: A novel cytochrome encoded in the A. phosphatis genome that would allow anaerobic use of the TCA cycle.


  1. Harper, D. Eutrophication of Freshwaters (Chapman and Hall, London, 1991).

    Google Scholar 

  2. Farmer, A.M. (ed.) “Implementation of the 1991 EU Urban Waste Water Directive and its Role in Reducing Phosphate Discharges” (Summary of Report). Scope Newsletter No. 34 (1999).

  3. US EPA/OW Clean Water Needs Survey (CWNS) for the United States and US Territories (US EPA/Office of Water, Washington, DC, 1996).

  4. CEEP. in Second International Conference on the recovery of phosphorus from sewage and animal wastes. Noordwijkerhout, Netherlands, (2001).

  5. Tchobanoglous, G. & Burton, F.L. . Wastewater Engineering: Treatment, Disposal, and Reuse. (McGraw-Hill, New York, 1991).

    Google Scholar 

  6. Blackall, L.L., Crocetti, G.R., Saunders, A.M. & Bond, P.L. A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants. Antonie Van Leeuwenhoek 81, 681–691 (2002).

    CAS  Article  Google Scholar 

  7. He, S., Gu, A.Z. & McMahon, K.D. Fine-scale differences between Accumulibacter-like bacteria in enhanced biological phosphorus activated sludge. Water Sci. Technol. 54, 111–117 (2006).

    CAS  Article  Google Scholar 

  8. Fuhs, G.W. & Chen, M. Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microb. Ecol. 2, 119–138 (1975).

    CAS  Article  Google Scholar 

  9. Streichan, M., Golecki, J.R. & Schon, G. Polyphosphate-accumulating bacteria from sewage treatment plants with different processes for biological phosphorus removal. FEMS Microbiol. Ecol. 73, 113–124 (1990).

    CAS  Article  Google Scholar 

  10. Deinema, M.H., van Loosdrecht, M.C.M. & Scholten, A. Some physiological characteristics of Acinetobacter spp. accumulating large amounts of phosphate. Water Sci. Technol. 17, 119–125 (1985).

    CAS  Article  Google Scholar 

  11. Wagner, M. et al. Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol. 60, 792–800 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hesselmann, R.P., Werlen, C., Hahn, D., van der Meer, J.R. & Zehnder, A.J. Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Syst. Appl. Microbiol. 22, 454–465 (1999).

    CAS  Article  Google Scholar 

  13. Crocetti, G.R. et al. Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation. Appl. Environ. Microbiol. 66, 1175–1182 (2000).

    CAS  Article  Google Scholar 

  14. McMahon, K.D., Dojka, M.A., Pace, N.R., Jenkins, D. & Keasling, J.D. Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal. Appl. Environ. Microbiol. 68, 4971–4978 (2002).

    CAS  Article  Google Scholar 

  15. Tyson, G.W. et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428, 37–43 (2004).

    CAS  Article  Google Scholar 

  16. Venter, J.C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304, 66–74 (2004).

    CAS  Article  Google Scholar 

  17. Tringe, S.G. et al. Comparative metagenomics of microbial communities. Science 308, 554–557 (2005).

    CAS  Article  Google Scholar 

  18. Oehmen, A., Saunders, A.M., Vives, M.T., Yuan, Z. & Keller, J. Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources. J. Biotechnol. 123, 22–32 (2005).

    Article  Google Scholar 

  19. Burns, D.J. & Beever, R.E. Mechanisms controlling the two phosphate uptake systems in Neurospora crassa. J. Bacteriol. 139, 195–204 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kortstee, G.J., Appeldoorn, K.J., Bonting, C.F., van Niel, E.W. & van Veen, H.W. Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal. Biochemistry (Mosc.) 65, 332–340 (2000).

    CAS  Google Scholar 

  21. Seviour, R.J., Mino, T. & Onuki, M. The microbiology of biological phosphorus removal in activated sludge systems. FEMS Microbiol. Rev. 27, 99–127 (2003).

    CAS  Article  Google Scholar 

  22. Schuler, A.J. & Jenkins, D. Enhanced biological phosphorus removal from wastewater by biomass with different phosphorus contents, Part III: Anaerobic sources of reducing equivalents. Water Environ. Res. 75, 512–522 (2003).

    CAS  Article  Google Scholar 

  23. Pereira, H. et al. Model for carbon metabolism in biological phosphorus removal processes based on in vivo 13C-NMR labelling experiments. Water Res. 30, 2128–2138 (1996).

    CAS  Article  Google Scholar 

  24. Louie, T.M., Mah, T.J., Oldham, W. & Ramey, W.D. Use of Metabolic Inhibitors and Gas Chromatography/Mass Spectrometry to Study Poly-β-Hydroxyalkanoates metabolism involving Cryptic Nutrients in Enhanced Biological Phosphorus Removal Systems. Water Res. 34, 1507–1514 (2000).

    CAS  Article  Google Scholar 

  25. Lemos, P.C., Serafim, L.S., Santos, M.M., Reis, M.A. & Santos, H. Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: 13C labeling and in vivo nuclear magnetic resonance. Appl. Environ. Microbiol. 69, 241–251 (2003).

    CAS  Article  Google Scholar 

  26. Mino, T., Van Loosdrecht, M.C.M. & Heijnen, J.J. Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Res. 32, 3193–3207 (1998).

    CAS  Article  Google Scholar 

  27. Elbehti, A., Brasseur, G. & Lemesle-Meunier, D. First evidence for existence of an uphill electron transfer through the bc(1) and NADH-Q oxidoreductase complexes of the acidophilic obligate chemolithotrophic ferrous ion-oxidizing bacterium Thiobacillus ferrooxidans. J. Bacteriol. 182, 3602–3606 (2000).

    CAS  Article  Google Scholar 

  28. Schuler, A.J. & Jenkins, D. Enhanced biological phosphorus removal from wastewater by biomass with different phosphorus contents, Part I: Experimental results and comparison with metabolic models. Water Environ. Res. 75, 485–498 (2003).

    CAS  Article  Google Scholar 

  29. Maurer, M., Gujer, W., Hany, R. & Bachmann, S. Intracellular carbon flow in phosphorus accumulating organisms from activated sludge systems. Water Res. 31, 907–917 (1997).

    CAS  Article  Google Scholar 

  30. Hesselmann, R.P.X., Von Rummell, R., Resnick, S.M., Hany, R. & Zehnder, A.J.B. Anaerobic metabolism of bacteria performing enhanced biological phosphate removal. Water Res. 34, 3487–3494 (2000).

    CAS  Article  Google Scholar 

  31. Wilen, B.M., Jin, B. & Lant, P. Relationship between flocculation of activated sludge and composition of extracellular polymeric substances. Water Sci. Technol. 47, 95–103 (2003).

    CAS  Article  Google Scholar 

  32. Zeng, R.J., Lemaire, R., Yuan, Z. & Keller, J. Simultaneous nitrification, denitrification, and phosphorus removal in a lab-scale sequencing batch reactor. Biotechnol. Bioeng. 84, 170–178 (2003).

    CAS  Article  Google Scholar 

  33. White, D. The Physiology and Biochemistry of Prokaryotes. (Oxford University Press, New York, 1995).

    Google Scholar 

  34. Tyson, G.W. et al. Genome-directed isolation of the key nitrogen fixer Leptospirillum ferrodiazotrophum sp. nov. from an acidophilic microbial community. Appl. Environ. Microbiol. 71, 6319–6324 (2005).

    CAS  Article  Google Scholar 

  35. Aparicio, S. et al. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, 1301–1310 (2002).

    CAS  Article  Google Scholar 

  36. Korotkova, N., Lidstrom, M.E. & Chistoserdova, L. Identification of genes involved in the glyoxylate regeneration cycle in Methylobacterium extorquens AM1, including two new genes, meaC and meaD. J. Bacteriol. 187, 1523–1526 (2005).

    CAS  Article  Google Scholar 

Download references


We thank Edward Berry for expert advice on cytochromes and Greg Crocetti, Daniel Noguera, Suzan Yilmaz, Paul Wilmes, Phil Bond, Jay Keasling and Eddy Rubin for helpful discussions. We also thank Aaron Saunders, Huabing Liu, Daniel Gall, Eugene Goltsman, Inna Dubchak, Matt Nolan, Steve Lowry, Alla Lapidus, Bryce Shepherd, Thomas Huber, Khrisna Palaniappan, Frank Korzeniewski and Sam Pitluck for technical assistance and additional analyses, and Chris Detter, Paul Richardson, Tijana Glavina del Rio, Susan Lucas, Alex Copeland, Dan Rokhsar, Igor Grigoriev and Victor Markowitz for facilitating the study. The sequencing for the project was provided by the DOE Community Sequencing Program at JGI ( The National Science Foundation (BES 0332136) supported the efforts of K.D.M. and S.H. This work was performed under the auspices of the DOE's Office of Science, Biological and Environmental Research Program; the University of California, Lawrence Livermore National Laboratory, under contract no. W-7405-Eng-48; Lawrence Berkeley National Laboratory under contract no. DE-AC03-76SF00098; and Los Alamos National Laboratory under contract no. W-7405-ENG-36.

Author information

Authors and Affiliations



H.G.M. contributed to metabolic reconstruction, binning and size estimation of A. phosphatis, comparison of the US and OZ genomes, gene-centric analysis and manuscript writing. N.I. contributed to the metabolic reconstruction of A. phosphatis, analysis of metabolism of the flanking community, gene-centric analysis and writing. V.K. contributed to the gene-centric analysis and A. phosphatis strain discrimination. F.W. contributed to community structure analysis. K.W.B. performed the Phrap assemblies and contributed to A. phosphatis binning. A.C.M. and I.R. contributed to the binning of the EBPR community. C.Y. and S.H. contributed to operation of and extraction of genomic DNA from the OZ and US EBPR reactors respectively. A.A.S. annotated the metagenomic assemblies. E.S. loaded the annotated assemblies into IMG/M and implemented tools specific for visualization of the data sets. E.D. constructed the shotgun libraries. N.H.P. and H.J.S. performed the JAZZ assemblies. J.L.P. contributed to the A. phosphatis genome size estimation. N.C.K. contributed to the genome completeness estimation. L.L.B., K.D.M. and P.H. contributed to the planning and design of the project, data analysis and manuscript writing.

Corresponding author

Correspondence to Philip Hugenholtz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Overview of US and OZ draft composite genomes. (DOC 202 kb)

Supplementary Fig. 2

Histogram of the number of reads with a given coverage. (DOC 252 kb)

Supplementary Fig. 3

Expanded maximum likelihood 16SrRNA tree. (DOC 201 kb)

Supplementary Fig. 4

EBPR-relevant metabolism with gene OIDs included. (DOC 412 kb)

Supplementary Table 1

SBR operational differences between the US & OZ SBS sludges. (DOC 41 kb)

Supplementary Table 2

Estimate of completeness of the dominant US A. phosphatis genome. (DOC 209 kb)

Supplementary Methods (DOC 126 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Martín, H., Ivanova, N., Kunin, V. et al. Metagenomic analysis of two enhanced biological phosphorus removal (EBPR) sludge communities. Nat Biotechnol 24, 1263–1269 (2006).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing