ARTICLES

Metagenomic analysis of two enhanced biological
phosphorus removal (EBPR) sludge communities
Héctor Garcı́a Martı́n1,5, Natalia Ivanova1,5, Victor Kunin1, Falk Warnecke1, Kerrie W Barry1,
© 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology

Alice C McHardy4, Christine Yeates2, Shaomei He3, Asaf A Salamov1, Ernest Szeto1, Eileen Dalin1,
Nik H Putnam1, Harris J Shapiro1, Jasmyn L Pangilinan1, Isidore Rigoutsos4, Nikos C Kyrpides1,
Linda Louise Blackall2, Katherine D McMahon3, and Philip Hugenholtz1

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.

Excessive inorganic phosphate (Pi) supply to freshwater negatively reliable industrial process. Typically, EBPR is studied in lab-scale
affects water quality and ecosystem balance through a process known sequencing batch reactors (SBRs) where the microbial community
as eutrophication1. Limitations on allowable Pi discharges from can be better monitored and perturbed, and PAOs can be enriched to
municipal and industrial sources through wastewater treatment have much higher levels than in full scale systems7.
proven effective in reducing Pi levels in many waterways2. Increasingly For 30 years, the bacterial genus Acinetobacter was incorrectly
stringent Pi limits for effluent wastewater are expected in the future assumed to be primarily responsible for EBPR based on cultivation
and hence efficient and reliable Pi removal methods are required. studies8–10. Only recently have culture-independent methods pointed
Because of the massive quantity of wastewater treated daily (more to A. phosphatis, a member of the order Rhodocyclales, as the principal
than 120 billion liters in the US alone3), any improvement in existing agent in acetate-fed EBPR11–13. A. phosphatis has yet to be grown in
methods should have tangible economic and ecological consequences. axenic culture despite continuing efforts but can be enriched to up to
EBPR is a treatment process in which microorganisms remove Pi 85% of the community in lab-scale bioreactors14. Shotgun sequencing
from wastewater by accumulating it inside their cells as polypho- applied directly to environmental samples has recently demonstrated
sphate. These polyphosphate-accumulating organisms (PAOs) are that near-complete genomes can be obtained for dominant popula-
then allowed to settle in a separate tank (clarifier), leaving the effluent tions in a community without the need for cultivation15,16. Therefore,
water largely depleted of Pi. EBPR is more economical in the long it was anticipated that a near-complete genome could be obtained for
term2 and has a lower environmental impact4 than traditional A. phosphatis from lab-scale EBPR enrichment cultures, allowing a
(chemical) Pi removal5, but is prone to unpredictable failures due comprehensive metabolic reconstruction. Comparative analysis of two
to loss or reduced activity of microbial populations responsible for Pi sludge samples should also lend insights into recent evolutionary
removal6. This is primarily because the design process is highly dynamics of this important PAO. Moreover, shotgun sequencing gives
empirical due to an incomplete understanding of sludge microbial a metagenomic context for dominant organisms by providing low-
ecology. Environmental engineers and microbiologists have been level genomic coverage of many other community members suitable
studying EBPR since its introduction in municipal wastewater treat- for a gene-centric analysis that could highlight habitat-specific meta-
ment plants over 30 years ago5 with the goal of making it a more bolic traits17.

1DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598, USA. 2Advanced Wastewater Management Centre, University of Queensland,

St. Lucia, 4072, Queensland, Australia. 3Civil and Environmental Engineering Department, University of Wisconsin-Madison, 1415 Engineering Drive, Madison,
Wisconsin 53706, USA. 4IBM Research Division, Thomas J. Watson Research Center, P.O. Box 218, Yorktown Heights, New York 10598, USA. 5These authors contributed
equally to this work. Correspondence should be addressed to P.H. (phugenholtz@lbl.gov).
Received 23 February; accepted 26 July; published online 24 September 2006; doi:10.1038/nbt1247

NATURE BIOTECHNOLOGY ADVANCE ONLINE PUBLICATION 1

Wisconsin (US) and the other Chlorobi β from Brisbane. However. in aerobically) via low affinity transporters encoded by two sets in lab-scale systems or whether the low abundance strains may be of transporter genes and high affinity Pi transporters encoded by three dominant at other sampling times or in other lab-scale EBPR sludges. for the US and OZ assemblies. OZ sludge-derived sequences. The Pi Flavobacteriales and Rhizobiales (Fig. The consensus Arguably. phosphatis scaffolds of the US JAZZ assem- bly. suggesting the possibility of transported into the cell during the aerobic period can be synthesized common functional themes in related flanking populations. clustered into phylotypes shown as circles on the tree. phosphatis strains pathways likely to be used by A. polyphosphate can be used directly to synthesize ATP or be degraded Metabolic reconstruction of A. NAD(P)H production via These PAOs then break the phosphodiester bonds of the stored glycogen degradation is insufficient to explain the observed levels of polyphosphate to provide an energy source for taking up PHA in acetate-fed systems20–22.may be closely enough related to share common metabolic traits. Circle sizes indicate relative abundance of phylotypes in the metagenomic data sets based on the number of reads comprising each contig on A near-complete set (97. respectively. 2a) in the absence of electron community for subsequent growth and replication in the aerobic acceptors26. However. provide the extra reducing power23–25. suggesting that the possibility of incor- rectly inferring the absence of pathways in this organism was low. indicating that they are closely (Supplementary Table 2). 1). sets of transporter genes (Fig. because of feedback Thirteen 16S rRNA phylotypes were identified on contigs of two or inhibition19. phosphatis populations Interestingly. Xanthomonadales. PHA production in the anaerobic phase. Green shading indicates clusters of US and OZ phylotypes that genes (Supplementary Table 2 online). ARTICLES RESULTS Proteobacteria EBPR community structure Caulobacter Sludge samples were obtained from two lab. chrome b/b6. We propose that quinone is reoxidized by a novel cyto- period. comprising B80% (US) and Rhodothermus B60% (OZ) of the biomass. Flavobacteriales Cytophaga Approximately 98 and 78 Mbp of shotgun 0. levels of Pi may contribute to superior EBPR performance. Blue circles indicate US sludge-derived respectively. Low abundance A.6 ± 0. A. phosphatis genomes are 495% was sufficiently complete to confidently infer presence and absence of identical at the nucleotide level over 79% of the reconstructed US pathways and thereby allow a comprehensive metabolic reconstruction genome (Supplementary Fig. US-acetate. as determined by fluorescence in situ hybridization7. gross biochemical measurements of lab-scale systems. we anticipate that the high affinity transporters should more reads in each Phrap assembly with the only overlap being be active only at the end of the aerobic period (Fig. at the nucleotide level from the dominant strains. sludge volume and Firmicutes solids residence time (Supplementary Table © 2006 Nature Publishing Group http://www. the least well understood component of EBPR meta- of these models is that Pi is removed from wastewater by uptake into bolism is the source of the reducing power (NAD(P)H) required for PAOs and conversion into polyphosphate during the aerobic period. It has been suggested that the and storing available VFAs (mostly acetate and propionate) as poly. Figure 2 highlights the major metabolic related strains of the same species.2%) of essential which a 16S rRNA gene was identified. for example. However. 2) during the anaerobic period. 2b). typi. phosphatis into Pi for ATP production via V. In the anaerobic phase. phosphatis during the anaerobic and were also detected in both metagenomes that were up to 15% divergent aerobic phases of the EBPR cycle. The US Bacteroidetes A. into polyphosphate via ATP (Fig. many US and OZ phylotypes clustered concentrations are at their lowest. Clostridium Accumulibacter phosphatis’’ OZ-propionate) feeds. including Verruco– microbia Prosthecobacter ‘‘Candidatus different volatile fatty acid (VFA. presented in Supplementary Figure 3 online. This protein appears to be a fusion of a cytochrome b/b6 2 ADVANCE ONLINE PUBLICATION NATURE BIOTECHNOLOGY . phosphatis genome is estimated to be 5. which includes all 16S rRNAs identified in the was identified in the higher read depth metagenomic data sets. 1 Figure 1 Maximum-likelihood tree based on partial and complete 16S rRNA genes identified on and 2 online) with an average GC content of metagenomic contigs comprising at least two reads. 2).com/naturebiotechnology 1 online). allowing them to dominate lab-scale EBPR sludges.and F-type ATPases.2 Mbp in size (Supplementary Figs. Rhizobiales Thiothrix Xanthomonadales scale SBRs that had been performing EBPR Acidovorax Rhodospirillum successfully for several months. Sludge sequences with 497% identity are 63% and average read depth of 8 and 5. one from Dechloromonas Madison.18. 2b) when Pi A.nature. Australia (OZ).10 sequence data were obtained from the US Chryseobacterium and OZ sludge. 1). phosphatis shuttles Pi across its plasma membrane (out anaero- tion as to whether the dominant strains have become EBPR specialists bically. The genome coverage of the dominant A. phosphatis (Fig. The ability to scavenge relatively low into broader phylogenetic groups. no explanation has Efficiently sequestering VFA during the anaerobic period is thought to been proposed for the necessary reoxidation of reduced quinones pro- give the PAOs a selective advantage over other members of the duced by succinate dehydrogenase (Fig. the US and OZ A. 1). Despite significant differences in operating conditions. sequences and red circles. Each SBR Chlorobium γ Rhodocyclus was independently seeded from a local waste. see Fig. α water treatment plant. The ATP Several metabolic models for EBPR have been proposed based on generated is then used in PHA production. This raises the ques. A.tricarboxylic acid (TCA) cycle operates in the anaerobic phase to hydroxyalkanoates (PHAs. An expanded phylogenetic tree is cally not colocalized in bacterial genomes. Accumulibacter species dominated both sludges.

pccAB ibd2. Gene identification numbers (gene oids) can be found in Supplementary Fig. polyhydroxybutyrate (PHB.C H+ Quinol reductase b F-ATPases Flipase Polymerase H + H + V-ATPase EPS H +.N H + a + Acetate H Glycogen + EM ED (high aff. polyhydroxyvalerate (PHV. glycogen degradation.T. PHA production requires energy (ATP) and reducing power (NAD(P)H). ADP H+ O2 – – H2O NO3 NO2 Figure 2 EBPR-relevant metabolism inferred from the A. from acetate and propionate). Na PhaB. NATURE BIOTECHNOLOGY ADVANCE ONLINE PUBLICATION 3 .meaB H +.) CS Pi Oxalo PhaZ acetate Citrate MalDH acn Pit H+ 2+ Malate Isocitrate (low affinity) + H Mg Propionyl 2+ fumC IDH CoA Mg + H 2+ AMP Fumarate OGDC Oxo complex glutarate Mg (synthesis) G-STK + A-STK NAD(P)H NAD(P) H 2+ pap Polyphosphate adk ATP Succinate Succinyl Mg SDH CoA K+ 2+ Ca Mg2+ ADP QH2 Q PPi Pi mcm. phosphatis ensures its dominance in the SBR microbial ecosystem. ATP (in red) is supplied by polyphosphate degradation and. A possible alternative use of the TCA cycle splits it in two branches through the use of fumarate reductase (dashed line). to a lesser degree. 4 online.B. phosphatis composite genome. meaC (high affinity) PhaZ meaA Pyruvate Acetyl.) PH2MB PH2MV H yjcG Mg 2+ cs phaA Oxalo phaB phaA acetate Citrate phaC phaB Proton transport H+ H+ Pi MalDH phaC acn pyrophosphatase © 2006 Nature Publishing Group http://www. acetate is not present in the medium for other species and the PHA reserves of A. Na Oligomers H Glycogen + + H + Glucose6P Nucleotide EM ED sugars Fructose6P H +. from acetate only).CoA PHB Mg 2 te.nature.C.CoA PHB POR pyruvate synthase PHV MDH PhaZ PH2MB PH2MV (OA dec. polyhydroxy-2-methylbutyrate (PH2MB. Na GDP Methyl + Malonyl CoA H+ 2 QH Q H +.N + acs (low affinity) pta a + PDC Symporter H+ Pyruvate Acetyl. The restoration of polyphosphate reserves via ATP depletes the water of Pi.meaB pccAB Pyrophosphate Orthophosphate ppk1 Methyl malonyl CoASH CoA Unacetylated Membrane coenzyme A bound ATP AMP E. (b) In the aerobic phase. NAD(P)H (in blue) is provided by glycogen degradation and the TCA cycle (enabled by a novel quinol reductase). Acetaionate H+ + phaA Mg 2+ MDH phaB phaC PHV prop + + . The dotted lines leading from the high affinity phosphate transporters (Pst) indicate that these transporters are unlikely to be active for most of the aerobic phase. when oxygen is available for respiration. thus giving rise to EBPR. from propionate only). The dashed line represents an alternative pathway for PHB degradation36 for which not all genes have been characterized.) H+ H Mg 2 + Pit ackA . croR Pst FDox FDred + ccR. Na + mmdA.com/naturebiotechnology AMP ppx Malate Isocitrate Propionyl fumC IDH CoA prpE pap adk ATP Polyphosphate OGDC Fumarate G-STK complex Oxo K+ 2+ frd A-STK glutarate Ca Mg2+ (degradation) ADP Propionate SDH Succinate Succinyl CoA ppk2 QH2 Q GTP mcm. from acetate and propionate) and polyhydroxy-2-methylvalerate (PH2MV. ARTICLES a H + F-ATPases H + V-ATPase H + . Na H+ (OA dec.) (low aff. (a) In the anaerobic phase acetate and propionate are stored as four types of PHA.

. which is Not all of the inferred metabolic capabilities of A. although it is possible that other strains encode respiratory quinol-NAD(P) reductase. EPS bind A. phosphatis. increase in relative abundance if the sludges were operated under An alternative scenario to full anaerobic TCA function is the anaerobic/anoxic conditions with nitrate.and propionate-fed EBPR sludges dominated by A. rapid adaptation to local conditions. ing that A. 2a. PHV (5–20% of PHAs) usually observed in acetate-fed EBPR. Another contentious point in EBPR metabolic models is whether Because wastewater contains high levels of fixed nitrogen and readily the Embden Meyerhof (EM) or Entner Doudoroff (ED) pathway is available organic carbon. phosphatis to outcompete other species for VFA storage and may explain absent from A. 3b). phosphatis genome © 2006 Nature Publishing Group http://www. which are e– Cytosol necessary for settling in the clarifier. This implies the existence of found as well as enzymes typically feeding into this pathway. phosphatis also has genes for flagella biosynthesis. phosphatis to (o2 kbp). We predict that these populations would A. We speculate that EPS clusters are modular structures hypothesized quinol reductase function (b).and glycoprotein-containing Q EPS types are produced. Full anaerobic functioning of the TCA cycle enabled by the novel cytochrome would allow It is interesting that respiratory nitrate reductase appears to be A. with five transmembrane helices and a soluble NAD(P)– and flavin.30. at the expense of the proton motive force. EbN1 FAD NAD also lack the key ED genes. or that other A. the key genes for the ED pathway were not function in phosphorus-limited habitats. NAD(P)+. 3a).and nitrogen-limited habitats. If the pathway may be dominant29. phosphatis likely only has the EM pathway available to A. A similar uphill electron transfer through a bc1 and flanking EBPR species must perform this essential task under anoxic NADH-Q oxidoreductase complex has been experimentally shown conditions. Gene expression or to be related to its EBPR lifestyle. It is a fusion of one gene with a that are interchangeable by nonhomologous recombination to allow cytochrome b/b6 domain and another gene with soluble ferrodoxin-. although no degrade glycogen. These reservoirs may serve as sources for 4 ADVANCE ONLINE PUBLICATION NATURE BIOTECHNOLOGY . indicat. Because conventional membrane to function as part of the electron transport chain.nature. 2a) and may explain the small amount of for respiration. phosphatis appear not accounted for in most EBPR models28. dashed line). phosphatis dominates EBPR communities. ferrodoxin and flavin groups to reduce EPS clusters are discussed elsewhere (V. nitrate reductase was identified on small contigs cycle enabled by the novel cytochrome would allow A. This apparent contradiction between NAD(P) the genomic evidence and the NMR data will need to be addressed by b NAD(P)H further experimental work such as proteomics. 1). a domain configuration that is currently unique in functions as a quinol reductase. Interestingly. phosphatis population with nitrite methylmalonyl-CoA (Fig. it is unlikely that these genes will be used for glycogen degradation. Although the EBPR sludges in the present study were in Thiobacillus ferrooxidans27. phosphatis strain to reduce genome. This would result in PHV accumulation via supplying the dominant A. This has a substantial impact on the expressed in EBPR sludge. an energetically which pathway or pathways are being used by A. However.b) Domain structure (a) and and OZ sludges. as fumarate reductase is also present would occupy an important ecological niche under these conditions by (Fig. phosphatis in the wastewater treatment FD environment31. Consistent with the vital role of EPS. the EBPR sludge studied Cytochrome_b__N fer2 binding_6 binding_1 by Hesselmann et al. (Fig. Nitrate reducing populations operation of a split TCA cycle. Dechloromonas aromatica and Azoarcus sp. was dominated by A. phosphatis strains these habitats to facilitate the ability of A. phosphatis can denitrify32. phosphatis to move toward or other Accumulibacter species may have the ED pathway. such as varying influent composi- and flavin-binding domains. then flagella may be expressed in did not contain A. the presence All EM pathway genes are present in the dominant A. (a. at the expense of the proton gradient nitrate reductase genes. Furthermore. derived from low abundance community members in both outcompete other species for VFA storage and may explain why the US and OZ sludges. Kunin et al. the extra reducing power. phosphatis. phosphatis as experimental evidence indicates that both why A. Other possible ecological implications of volatile reduced quinone via the cytochrome. The sources of limiting nutrients. phosphatis of the high affinity Pi transporters would allow this bacterium to strains.30. Full anaerobic functioning of the TCA not grown on nitrate. A. the EPS clusters are conspicuously variable Figure 3 A novel cytochrome encoded in the A.com/naturebiotechnology bh (Supplementary Fig. The gene complements of the clusters bL strongly suggest that exopolysaccharide. the nitrate reductase appears to lack the subunit that usually binding domain. This casts doubt and soluble NAD(P)H-quinone dehydrogenases are present in the on the ability of the dominant A. suggesting that the sludges analyzed environmental reservoir is water. FAD The production of extracellular polymeric substances (EPS) is essential for the survival of A. However. This suggests that A. phosphatis reservoirs in nutrient-limited habitats such as freshwater. phosphatis cells in dense ‘flocs’. The genome does encode the rest of the denitrification pathway from nitrite onwards and a dissimilatory nitrate reductase. there are at least two QH2 EPS gene clusters (25–38 kbp) in the US A. suggesting that it may not be able public sequence databases (Fig. phosphatis. implicating ED as the dominant pathway. each with distinct physical and chemical Periplasm properties. We predict that electrons are passed from the tion and temperature. phosphatis to generate very expensive process33. to carbon. phosphatis genome that between the two otherwise closely related dominant strains in the US would allow anaerobic use of the TCA cycle. If A. phosphatis does not reduce nitrate. phosphatis is adapted cellular energy budget because the EM pathway yields more ATP. acetate. we suggest that this fusion protein functions in reverse as a nitrate. Nonsettling cells are washed out of the system. One of the most surprising findings proteomic data that leverage the metagenomic data will determine is a full complement of genes for nitrogen fixation. in preparation). The key genes for fixing CO2 are also present. phosphatis dominates EBPR communities. NAD(P). NMR studies of EBPR sludges indicate that the ED flagella have been observed for this organism in EBPR. In contrast. ARTICLES latter explanation is less likely as the closest sequenced relatives of a1 100 200 300 400 500 600 637 A. including phosphoribulokinase and the large subunit of rubisco.

VFA handling (VFA sodium sympor.g. The present analysis provides a possible mechanism for the Thiothrix is the dominant flanking species in the propionate-fed OZ generation of the unaccounted fraction of reducing power required for sludge. respectively.. phosphatis suggests that operating conditions broadly determine some niches also appears to have an unusual cobalt dependence: it has only that may be occupied by multiple species. and use it as a food source. phosphatis representatives were overrepresented in both sludges data not shown). Despite the two EBPR communities having minimal overlap at the libacter-specific PCR confirms the hypothesis that this organism is species level (apart from A. This may explain why of EBPR. one of them (nitrate transporters) complementary to the therefore be able to degrade this component of the A. and their abundance in overrepresented families In addition to A. to local conditions. for glycolysis and proposes that A. This demonstrated for Leptospirillum ferrodiazotrophum34. A. phosphatis encodes a gene (UDP-glucose dehydrogenase) for with no clear role in the present model (iron transporters and DNA the production of the precursor of glucoronic acid.7% of reads in US families in EBPR have no annotation. We speculate that they are part of EPS. soil and three whale. tigations of the EBPR transcriptome. although not necessarily Thiothrix. relative to the other habitats. biosynthesis. phosphatis EPS hypothesized functioning of A. phosphatis. respectively. We fall samples17 were classified in gene families according to sequence believe this will lead to breakthroughs in metabolic modeling and our similarity and the relative representation of each family was deter. suggesting the exchange genes). A.4% and 2. given the affinity of EBPR-related metabolism of dominant flanking populations EPS for cations. Xylella-like (glucuronic acid) and environment (e. supporting the hypothesis that the selection strategy for isolating this bacterium. In the The determination of the near complete genome of A. respectively) and Thiothrix. phosphatis in serious consideration.nature. around 15% of the overrepresented Xylella. that is. phosphatis is adapted to nutrient- Conversely. mined. Availability of the complete genome sequence will further allow targeted studies of the enzymes involved in carbon and phosphorus Gene-centric analysis transformation pathways. phosphatis under anoxic conditions. ARTICLES reseeding of EBPR communities. In short. Treatment Plant in Queensland. several flanking species were relatively suggests an important role. through nonhomologous recombination to allow for rapid adaptation such as the presence of a given species in the seeding sludge. determined by conserved gene analysis. phosphatis. a family of nitrate The inferred ability of A. as has recently been flanking communities are responsible for nitrate reduction. anaerobic operation of the TCA cycle cycles of 6 h and the hydraulic residence time and solids residence time were (cytochrome b/b6) and cobalt uptake (cobalt transporters). we limited habitats and that it may be obtained in pure culture by use of a would expect that one or more propionate-using flanking populations nitrogen-free selective growth medium supplemented with cobalt or would be enriched. VFAs. for NATURE BIOTECHNOLOGY ADVANCE ONLINE PUBLICATION 5 . The dominant flanking populations in model. 1). A sizable fraction of the families believed to be important for survival in the EBPR environment from the metabolic reconstruction METHODS were overrepresented in the sludge data sets. phosphatis strains from two different we speculate that the composition of the flanking communities is EBPR sludges suggests that EPS clusters are frequently exchanged determined by a combination of operating conditions and chance. This suggests the existence of common functional presence of this sugar in the EPS. and Thornside Sewage and high affinity transporters). We We performed a gene-centric analysis of the metagenomic data17 to can now study how gene expression is regulated in response to en- determine overrepresented gene families in EBPR communities relative vironmental factors such as concentrations of dissolved oxygen. toxins) with representatives in A. 24 gene families lacking indeed present in freshwater and associated sediments (V. These include genes Both SBRs were seeded from local wastewater treatment plants (Nine Springs required for phosphate transport (specific components of both low Wastewater Treatment Plant in Madison. OZ sludge. This level of representation in the data sets allows the DISCUSSION presence but not absence of metabolic pathways to be inferred. in conjunction with other families the US A. and PHA synthetases). Wisconsin. nitrate and phosphate. phosphatis Thiothrix-like population. include two iron transport families and a family involved in the US and OZ sludges. several gene families corresponding to pathways for subsequent monomer degradation were identified in the the metabolic capabilities expected to be important in the EBPR Flexibacter-like (beta-galactose). This hypothesis could be tested by changing the OZ sludge to PHA synthesis.com/naturebiotechnology a nitrogen-free. like species (13. A nitrogen-free selective growth medium would there. 12 h and 4 d. phosphate transporters and VFA handling genes) Thiothrix-like (xylose) populations. Preliminary studies using Accumu. Most conspicuous among these are 11 families © 2006 Nature Publishing Group http://www. ally important genes in EBPR remain to be characterized. Comparison of A.. to other habitats. phosphatis). as well as flux through these pathways. The above examples suggest that other overrepresented gene fore need to be supplemented with cobalt or cobalamin to support families with no obvious fit in the present metabolic model merit growth of A. Unfortunately. redundancy appears to be present in the two phylogenetically different cleotide reductase and a full complement of genes for cobalamin flanking communities. suggests the use of the EM instead of the ED pathway an acetate feed and monitoring the effect on the Thiothrix population. resulting in genomic intriguing annotated functions. acid mine drainage biofilm15. Genes annotated in the two US sludge assem. we identified a complete methylcitrate represents a turning point in our understanding of the genetic basis pathway used for propionate degradation. phosphatis to fix nitrogen suggests a transporters is overrepresented. some functional cobalamin-dependent versions of methionine synthase and ribonu. The Xylella-like population may themes. One of the EPS gene cassettes in are overrepresented in both sludges. Other overrepresented families with well represented in the metagenomic data sets. For instance. Despite an apparent lack of compositional overlap Genes encoding enzymes for carbohydrate polymer hydrolysis and in the flanking communities.and Flexibacter-like species (0. colbalt-rich medium have resulted in enrichment but annotated as Ca2+-binding proteins related to RTX toxins (repeat not isolation (data not shown). The SBRs were operated in four ter. proteome and metabolome. phosphatis and other flanking species. Kunin et al. if the US sludge was switched to a propionate feed. Preliminary efforts to isolate A. ability to predict when and where EBPR will be operating effectively. Australia). the metagenome will facilitate inves- blies. were a DNA exchange. suggesting that many function- Phrap contigs containing 16S rRNA genes. This is because cobalamin.8% of reads in OZ Phrap contigs containing 16S rRNA genes) (Fig. but no clear role in the present fragments up to 64 kbp in size. for the US SBR and 24 h and 8 days.

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Golecki. contributed to the planning and design of the project. © 2006 Nature Publishing Group http://www. E.M. Bryce Shepherd. A.S. R. contributed to the binning of the Water Res. contributed to the genome completeness estimation. Some physiological character- the phrap assemblies has been deposited at DDBJ/EMBL/GenBank under the istics of Acinetobacter spp. P.H. Microbiology and biochemistry of the E. N. 66–74 (2004). Comparative metagenomics of microbial communities. N. phosphatis. C. 2128–2138 (1996). First evidence for existence of an uphill N. 34 (1999). contributed to metabolic reconstruction.A.. Appl. contributed to community structure Chromatography/Mass Spectrometry to Study Poly-b-Hydroxyalkanoates metabolism analysis. Appeldoorn. contributed to the metabolic reconstruction of 23.nature. The polyphosphate and glycogen accumulating organisms in enhanced biological phos- National Science Foundation (BES 0332136) supported the efforts of K. F. Khrisna Palaniappan. The versions Technol. Noordwijkerhout. M.W. contributed to the A.J. Mino. 3193–3207 (1998).K. M. D. Res. http://www. 8 and 40 kb. & Bond.. and Chris Detter. Kortstee. analysis and writing. DNA was extracted at the end of the anaerobic period. Appl. Crocetti. M. Reis. 22. M. US EPA/OW Clean Water Needs Survey (CWNS) for the United States and US Territories gene predictions (fgenesb.T. L. M. This work was performed under the auspices of the DOE’s Office 123. Appl. A. F.J. Jay Keasling and Eddy kinase from activated sludge performing enhanced biological phosphorus removal. Biotechnol. the study.F. Tyson. van der Meer.M. New York. K.W. no.. W.L. S. Recent developments in the biochemistry and ecology of enhanced biological phosphorus DE-AC03-76SF00098.S. Biochemistry (Mosc. M. A. bacteria in enhanced biological phosphorus activated sludge. Bonting. Community structure and metabolism through reconstruction of Lapidus. 139. & McMahon..P. The largest JAZZ scaffold (contigs linked by end pair information) for the US sludge was over 5 Mbp (second JAZZ 1..softberry. The sequencing for the project was provided by the DOE Community 18. He. & Burton.W.phrap. & Keller.Y. Gu. 34. G. L.nature. FEMS Microbiol. representing low abundance populations in the EBPR sludges.G. The genomic fragments (US EPA/Office of Water.C. Use of Metabolic Inhibitors and Gas A. Seviour. Polyphosphate Daniel Noguera.W. Ecol. H. J. H. Development of an rRNA-targeted oligonucleotide probe specific for AATO01000000. D. However.D. 6 ADVANCE ONLINE PUBLICATION NATURE BIOTECHNOLOGY . in Neurospora crassa. M. W. and JAZZ35 as a Published online at http://www. and I. Appl. ACKNOWLEDGMENTS Environ. were created The authors declare that they have no competing financial interests. G. R.. G. 6. 3602–3606 (2000). 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