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.

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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.


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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

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.

Correspondence to Philip Hugenholtz.

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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)

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