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Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphateaccumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were ␤-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the ␤-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the ␤-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.

Enhanced Biological Phosphorus Removal (EBPR) is not well understood at the metabolic level despite being one of the best-studied microbially-mediated industrial processes due to its ecological and economic relevance. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, "Candidatus Accumulibacter phosphatis". This 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. 3Excessive inorganic phosphate (Pi) supply to freshwater negatively affects water quality and ecosystem balance through a process known as eutrophication 1 . Limitations on allowable Pi discharges from municipal and industrial sources via wastewater treatment have proven effective in reducing Pi levels in many waterways 2 . Increasingly stringent Pi limits for effluent wastewater are expected in the future and hence efficient and reliable Pi removal methods are required. Due to the massive quantity of wastewater treated daily (more than 120 billion liters in the US alone 3 ), any improvement in existing methods should have tangible economic and ecological consequences.Enhanced Biological Phosphorus Removal (EBPR) is a treatment process in which microorganisms remove Pi from wastewater by accumulating it inside their cells as polyphosphate. These polyphosphate-accumulating organisms (PAOs) are then allowed to settle in a separate tank (clarifier), leaving the effluent water largely Pi-depleted. EBPR is more economical in the long term 2 and has a lower environmental impact 4 than traditional (chemical) Pi removal 5 , but is prone to unpredictable failures due to loss or reduced activity of microbial populations responsible for Pi removal 6 . This is primarily because the design process is highly empirical due to an incomplete understanding of sludge microbial ecology. Environmental engineers and microbiologists have been studying EBPR since its introduction in municipal wastewater treatment plants over thirty years ago 5 with the goal of making it a more reliable industrial process. Typically, EBPR is studied in lab-scale sequencing batch reactors (SBRs) where the microbial community can be better monitored and perturbed, and PAOs can be enriched to much higher levels than in full scale systems 7 .For th...

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G؉C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with ␦-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the ␦-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the ␦-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.Siroheme dissimilatory sulfite reductases (EC 1.8.99.3) catalyze the reduction of sulfite to sulfide, an essential step in the anaerobic sulfate-respiration pathway. Consequently, this enzyme has been found in all dissimilatory sulfate-reducing prokaryotes (SRPs) investigated so far. Furthermore, siroheme dissimilatory sulfite reductase-like enzymes have been detected in the hyperthermophilic archaeon Pyrobaculum islandicum capable of using sulfite as terminal electron acceptor (23), the phototrophic bacterium Allochromatium vinosum (10, 12), and the obligate chemolithotrophic species Thiobacillus denitrificans (32). In the latter two organisms the dissimilatory sulfite reductase has a proposed function in sulfide oxidation.Siroheme sulfite reductases consist of at least two different polypeptides in an ␣ 2 ␤ 2 structure. The genes encoding the two subunits are found adjacent to each other in the respective genomes (see, for example, references 3, 15, 17, 18, and 35) and probably arose from duplication of an ancestral gene (3). Comparative amino acid sequence analysis of the dissimilatory sulfite reductase genes (dsrAB) has recently been used to investigate the evolutionary history of anaerobic sulfate (sulfite) respiration (...

A primary-structure analysis of the 16s rRNA gene was performed with 10 strains representing five described and one unidentified species of the genus Microcystis. The phylogenies determined illustrate the evolutionary affiliations among Microcystis strains, other cyanobacteria, and related plastids and bacteria. A cluster of 10 strains that included hepatotoxic isolates identified as Microcystis aeruginosa formed a monophyletic group. However, the genus Microcystis appeared to be polyphyletic and contained two strains that clustered with unicellular cyanobacteria belonging to the genus Synechococcus. The clustering of related Microcystis strains, including strains involved in the production of the cyclic peptide toxin microcystin, was consistent with cell morphology, gas vacuolation, and the low G+C contents of the genomes. The Microcystis lineage was also distinct from the lineage containing the unicellular genus Synechocystis and the filamentous, heterocystforming genus Nostoc. The secondary structure of a Microcystis 16s rRNA molecule was determined, and genus-specific sequence signatures were used to design primers that permitted identification of the potentially toxic cyanobacteria belonging to the genus Microcystis via DNA amplification.The Cyanobacteria is a diverse bacterial phylum with respect to form, function, and habitat. On the basis of microfossil and geochemical evidence the origin of cyanobacterium-like organisms has been dated to the late Precambrian era. On the basis of the results of phylogenetic studies workers have inferred that the cyanobacterial phylum is 1 of the 11 bacterial phyla (32,42). The possibility that eukaryotic chloroplasts arose from a cyanobacterial ancestor by a symbiotic event has also been inferred on the basis of molecular data (8, 38).Members of the genus Microcystis are a major cause of freshwater noxious cyanobacterial blooms, which have a broad geographical distribution. The microcystins, a family of cyclic heptapeptide toxins that are not synthesized ribosomally (3) and are produced by most members of this genus, cause acute hepatotoxicity in agricultural livestock. Recent epidemiological data have also linked chronic subacute consumption of microcystins to human liver tumor promotion (5). The increasing occurrence of Microcystis blooms in major sources of human drinking water makes identification and prediction of these toxic blooms very important.Due to the variably expressed and minor morphological and developmental characteristics used for identification, classification of cyanobacterial strains at the genus or species level may be ambiguous (29), particularly when laboratory cultures and environmental isolates are compared (7). The current cyanobacterial taxonomy does not provide an unequivocal system for identification of the toxigenic and bloom-forming genus Microcystis (20). Depending on the taxonomic treatises used for classification, which differ in their emphasis on the cell size, shape, buoyancy, and toxicity of the planktonic, freshwater cyanobacteria, d...