Hector Garcia Martin2, Natalia Ivanova2, Victor Kunin2, Linda Blackall3, Katherine ... that historically could only be obtained using pure cultures (McMahon et al.
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Genetic Blueprints for Enhanced Biological Phosphorus Removal (EBPR) Based on Environmental Shotgun Sequencing Hector Garcia Martin2, Natalia Ivanova2, Victor Kunin2, Linda Blackall3, Katherine D. McMahon1,*, and Philip Hugenholtz2 1
Department of Civil and Environmental Engineering, University of Wisconsin at Madison, Madison, WI, 53706, USA. 2Joint Genome Institute, Walnut Creek, CA, 94598, USA. 3Advanced Wastewater Treatment Centre, University of Queensland, St. Lucia, 4072, QLD, Australia. *Corresponding author KEYWORDS: Enhanced biological phosphorus removal, Rhodocyclus, Accumulibacter, metagenome The EBPR process is widely used throughout the world to sequester phosphate during wastewater treatment with activated sludge. It is a biochemically complex process, involving the cycling of at least three biopolymers (polyphosphate, polyhydroxyalkanoates (PHA), and glycogen) as the microbes present in the sludge are exposed alternately to anaerobic and aerobic conditions (Blackall et al. 2002). The biphasic nature of the process is thought to be key to both the microbial ecology and metabolism of EBPR organisms. The dominant organism that is repeatedly enriched in lab-scale acetate-fed EBPR sequencing batch reactors (SBR) was recently identified as a member of the Betaproteobacteria in the Rhodocyclus group, and was named Accumulibacter phosphatis (Hesselmann et al. 1999; Crocetti et al. 2000). No pure culture of this organism is yet available, though powerful culture-independent molecular techniques are providing much of the essential information that historically could only be obtained using pure cultures (McMahon et al. 2002). Here we report on another significant advance in the quest to understand the genetic and biochemical basis for EBPR: the sequencing of the EBPR metagenome. Sludge samples were obtained from a lab-scale SBRs that had been running EBPR successfully for several months; one from Madison, Wisconsin (US) and the other from Brisbane, Australia (OZ). Each SBR was independently seeded from a local wastewater treatment plant. Despite significant differences in operating conditions, including different volatile fatty acid (VFA; US-acetate, OZpropionate) feeds, sludge volume and solids retention time, Accumulibacter species dominated both sludges, comprising ~80% of the biomass as determined by fluorescence in situ hybridization (FISH). Approximately 94 Mbp and 87 Mbp of shotgun sequence data were obtained from the US and OZ sludge respectively. Both datasets readily assembled into contiguous stretches of DNA sequence of up to 3 Mbp. Over 30000 coding sequences were predicted. As expected, most of the large fragments originated from A. phosphatis. The A. phosphatis genome is estimated to be 5.5 Mbp in size with an average GC content of 63%. Interestingly, the US and OZ A. phosphatis genomes are >95% identical at the nucleotide level over 83% of the reconstructed US genome indicating that they are closely related strains of the same species. Lower abundance Accumulibacter species were also detected in both sludges that were on average 15% divergent at the nucleotide level from the dominant species. Twenty seven and twenty one partial to complete 16S rRNA gene sequences were identified in the US and OZ metagenomes respectively, together representing 30 phylotypes. In only three cases Copyright ©2006 Water Environment Foundation. All Rights Reserved
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were the same flanking phylotypes found in the two sludges, possibly indicating the presence of the same species. However, many US and OZ phylotypes clustered into broader phylogenetic groups, e.g. Xanthomonadales and Flavobacteriales, suggesting the possibility of common functional themes in related flanking populations. The genome coverage of the dominant A. phosphatis populations was sufficiently complete to confidently infer presence or absence of pathways and thereby allow a comprehensive metabolic reconstruction (Figures 1 and 2). This enabled us to identify the key pathways necessary for EBPR, hypothesize why A. phosphatis is the primary agent of lab-scale SBRs, and propose conditions required for its cultivation. Figure 1 highlights the major metabolic pathways likely to be used by A. phosphatis during the anaerobic phases of the EBPR cycle. Figure 2 illustrates the proposed aerobic metabolism. The least well understood component of EBPR metabolism is the source of the reducing power (NAD(P)H) required for PHA production in the anaerobic phase. NAD(P)H production via glycogen degradation is insufficient to explain the observed levels of PHA in acetate-fed systems (Seviour et al. 2003). It has been suggested that the tricarboxylic acid (TCA) cycle operates in the anaerobic phase to provide the extra reducing power (Pereira et al. 1996). However, no explanation has been proposed for the necessary reoxidation of reduced quinones produced by succinate dehydrogenase (Figure 1) in the absence of electron acceptors (Mino et al. 1998). We propose that the quinone is reoxidized by a novel cytochrome. This protein appears to be a fusion of a cytochrome and a soluble NAD(P)-binding domain, a configuration that is unique among bacteria. Full anaerobic functioning of the TCA cycle enabled by the novel cytochrome would allow A. phosphatis to outcompete other species for VFA storage and may explain why A. phosphatis dominates EBPR communities. An alternative scenario to full anaerobic TCA function is the operation of a split TCA cycle since fumarate reductase is also present (Fig. 1, dotted line). Another contentious point in EBPR metabolic models is the pathway used for glycogen degradation, Embden Meyerhof (EM) or Entner Doudoroff (ED). This has a substantial impact on the cellular energy budget because the EM pathway yields more ATP. All EM pathway genes are present in A. phosphatis. In contrast, the key genes for the ED pathway are absent as well as enzymes typically feeding into this pathway, therefore confirming that A. phosphatis uses the EM pathway to degrade glycogen. The production of extracellular polymeric substances (EPS) is essential for the survival of A. phosphatis in the wastewater treatment environment. EPS bind A. phosphatis cells in dense “flocs”, which are necessary for settling in the clarifier. Non-settling cells are washed out of the system. Consistent with the vital role of EPS, there are at least two large EPS gene clusters in the US A. phosphatis genome. The gene complements of the clusters strongly suggest that exopolysaccharideand glycoprotein- containing EPS types are produced, each with distinct physical and chemical properties. Interestingly, the EPS clusters are conspicuously volatile between the two otherwise closely related dominant strains in the US and OZ sludges. We speculate that EPS clusters are modular structures that are interchangeable among A. phosphatis populations, to allow rapid adaptation to local conditions, such as varying influent composition and temperature. It is remarkable that respiratory nitrate reductase (nar) appears to be absent from A. phosphatis since experimental evidence indicates that both acetate- and propionate-fed EBPR sludges dominated by A. phosphatis can denitrify. The genome does encode the rest of the denitrification pathway from nitrite onwards. If A. phosphatis does not reduce nitrate, flanking EBPR species must Copyright ©2006 Water Environment Foundation. All Rights Reserved
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perform this essential task. Although the EBPR sludges in the present study were not grown on nitrate, nar was identified on small contigs, derived from low abundance community members in both the US and OZ sludges. We would predict that these populations would increase in relative abundance if the sludges were operated under anaerobic/anoxic conditions with nitrate. Nitrate reducing populations would occupy an important ecological niche under these conditions by supplying the dominant A. phosphatis population with nitrite for respiration. The determination of the EBPR metagenome represents a turning point in our effort to understand why EBPR occurs in activated sludge, and how to optimize treatment processes to maximize EBPR performance. It will enable targeted studies of the enzymes involved in carbon and phosphorus transformation pathways, as well as flux through these pathways. We can now easily study how gene expression is regulated in response to environmental factors such as concentrations of dissolved oxygen, volatile fatty acids, nitrate, and phosphate. In short, the metagenome will facilitate investigations of the EBPR transcriptome, proteome, and metabalome. This will undoubtedly lead to breakthroughs in metabolic modeling and our ability to predict when and where EBPR will be operating effectively.
Figure 1. Reconstruction of the anaerobic metabolism of A. phosphatis based on the genes identified in the metagenome sequence.
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Figure 2. Reconstruction of the aerobic metabolism of A. phosphatis based on the genes identified in the metagenome sequence. References cited Blackall, L. L., Crocetti, G. R., Saunders, A. M. and Bond, P. L. (2002) A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants. Antonie Van Leeuwenhoek, 81(1-4): 681-91. Crocetti, G. R., Hugenholtz, P., Bond, P. L., Schuler, A., Keller, J., Jenkins, D. and Blackall, L. L. (2000) Identification of polyphosphate accumulating organisms and the design of 16s rRNAdirected probes for their detection and quantitation. Appl Environ Microbiol, 66(3): 1175-1182. Hesselmann, R. P. X., Werlen, C., Hahn, D., van der Meer, J. R. and Zehnder, A. J. B. (1999) Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Syst. Appl. Microbiol., 22(3): 454-465. McMahon, K. D., Dojka, M. A., Pace, N. R., Jenkins, D. and Keasling, J. D. (2002) Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal. Appl Environ Microbiol, 68(10): 4971-8. Mino, T., Van Loosdrecht, M. C. M. and Heijnen, J. J. (1998) Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Res., 32(11): 3193-3207. Pereira, H., Lemos, P. C., Reis, M. A. M., Crespo, J. P. S. G., Carrondo, M. J. T. and Santos, H. (1996) Model for carbon metabolism in biological phosphorus removal process based on in vivo 13C-NMR labelling experiments. Water Res., 30(9): 2128-2138. Seviour, R. J., Mino, T. and Onuki, M. (2003) The microbiology of biological phosphorus removal in activated sludge systems. FEMS Microbiol Rev, 27(1): 99-127. Copyright ©2006 Water Environment Foundation. All Rights Reserved
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