Candidatus accumulibacter population structure in enhanced biological phosphorus removal Sludges as revealed by polyphosphate kinase genes

We investigated the fine-scale population structure of the Candidatus Accumulibacter lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase I gene (ppk1) as a genetic marker. We retrieved fragments of Candidatus Accumulibacter 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the Candidatus Accumulibacter lineage. Sequences from at least five clades of Candidatus Accumulibacter were recovered by ppk1]-targeted PCR, and subsequently, specific primer sets were designed to target the ppkl gene for each clade. Quantitative real-time PCR (qPCR) assays using Candidatus Accumulibacter-specific 16S rRNA and Candidatus Accumulibacter clade-specific ppk1 primers were developed and conducted on three laboratoryscale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total Candidatus Accumulibacter lineage and the relative distributions and abundances of the five Candidatus Accumulibacter clades. The qPCR-based estimation of the total Candidatus Accumulibacter fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppkl, demonstrating the power of ppkl as a genetic marker for detection of all currently defined Candidatus Accumulibacter clades. The relative distributions of Candidatus Accumulibacter clades varied among different EBPR systems and also temporally within a system. Our results suggest that the Candidatus Accumulibacter lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.