Metagenomics and in situ analyses reveal Propionivibrio spp. to be abundant GAO in biological wastewater treatment systems

Enhanced biological phosphorus removal (EBPR) is widely applied for phosphorus removal from wastewater. The process relies on polyphosphate-accumulating organisms (PAOs) that are able to take up phosphorus in excess of what is needed for growth. However, glycogen-accumulating organisms (GAOs) may reduce the EBPR efficiency as they compete for substrates with PAOs, but do not store excess polyphosphate. “Candidatus Accumulibacter” is widely considered to be the important PAO. In this study a laboratory scale sequencing batch reactor was operated for EBPR for the enrichment of “Ca. Accumulibacter”. Applying the PAOmix probe set, routinely applied to target the “Ca. Accumulibacter”, suggested a PAO enrichment of 70% of the biovolume by FISH. Known GAOs were detected in low abundance with FISH (< 2%). However, reactor stoichiometry (P release/VFA-molar uptake rate of 0.3) suggested PAO and GAO to be present at equal levels. Metagenomics was employed to elucidate the identity and recover genomes from the abundant community members. Phylogenetic analyses revealed closely related “Ca. Accumulibacter” and Propionivibrio genera were co-dominant and were both targeted by the PAOmix probes. In situ staining and genome annotation supported that the “Ca. Accumulibacter” and Propionivibrio were behaving as PAO and GAO, respectively. FISH analyses of full-scale EBPR systems revealed that Propionivibrio spp. can be abundant. The discovery of Propionivibrio, a putative GAO closely related to “Ca. Accumulibacter” and targeted by the PAOmix probes, means that the results generated from studies applying this probe set should be interpreted with great care and has important implications for EBPR research.