Reevaluation of the Phylogenetic Diversity and Global Distribution of the Genus Candidatus Accumulibacter

Candidatus Accumulibacter was the first microorganism identified as a polyphosphate-accumulating organism (PAO) important for phosphorus removal from wastewater. Members of this genus are diverse, and the current phylogeny and taxonomic framework appear complicated, with most publicly available genomes classified as Candidatus Accumulibacter phosphatis, despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer-scale differentiation into different types and clades; nevertheless, taxonomic assignments remain inconsistent across studies. Therefore, a comprehensive reevaluation is needed to establish a common understanding of this genus, in terms of both naming and basic conserved physiological traits. Here, we provide this reassessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive data sets. We identified 15 novel species, along with Candidatus Accumulibacter phosphatis, Candidatus Accumulibacter delftensis, and Candidatus Accumulibacter aalborgensis. To compare the species in situ, we designed new speciesspecific fluorescence in situ hybridization (ASH) probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, Ca. Accumulibacter species were widespread in wastewater treatment plants (WWTPs) with phosphorus removal, indicating process design as a major driver for their abundance. Genome mining for PAO-related pathways and FISH-Raman microspectroscopy confirmed the potential for PAO metabolism in all Co. Accumulibacter species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal. IMPORTANCE Candidatus Accumulibacter is the most studied PAO, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing approaches. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to phylogeny based on the 16S rRNA gene and other phylogenetic markers, we obtained a higher-resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of genes and pathways of interest, validated in situ by application of a new set of FISH probes and Raman microspectroscopy, provided additional high-resolution metabolic insights into these organisms.

Automated summary

Candidatus Accumulibacter is an important polyphosphate-accumulating organism (PAO) in wastewater phosphorus removal. However, its taxonomic classification at the species level is complicated. This study provides a comprehensive reassessment of the phylogeny using genome, ppk1, and 16S rRNA gene-based approaches, resulting in the identification of novel species and clarification of the taxonomy. Additionally, the metabolic capabilities of Candidatus Accumulibacter species were explored using genome mining and in situ validation, providing insights into their niche differentiation and contribution to nutrient removal.