Phylogeny and FISH probe analysis of the “Candidatus Competibacter”-lineage in wastewater treatment systems

Our understanding of the microbial ecology of enhanced biological phosphorus removal (EBPR) wastewater treatment systems has been greatly advanced through the application of molecular methods such as fluorescence in situ hybridization (FISH). Considerable attention has been directed at the identification and characterization of the glycogen accumulating organisms (GAO), a phenotypic group thought to compete with the polyphosphate accumulating organisms (PAO) for resources at the theoretical expense of EBPR efficiency. Demonstrated candidates for members of the GAO phenotype include the gammaproteobacterial “Candidatus Competibacter”-lineage. The group is currently delineated by 8 FISH probe defined phylotypes, although further undescribed phylogenetic diversity beyond what is covered by these probes is evident. Where studied, marked differences in physiology between members are observed, including varied abilities for denitrification, fermentation, glycolysis pathways and carbon storage polymers. An understanding of the ecology of the lineage in EBPR, and its importance in the competition with the PAO, will rely on in depth knowledge of the phylogenetic and ecophysiological diversity present. This study aimed at resolving the phylogeny and reassessing FISH probes for coverage of the diversity of the lineage in EBPR systems. Analysis of the level of 16S rRNA gene sequence diversity of lineage members (> 89%) suggests that it is more appropriately viewed as a family rather than a single genus; designated here as the Competibacteraceae. The family is also suggested to include the related genus Plasticicumulans, which like the “Ca. Competibacter”-lineage members, are predominantly reported in activated sludge related environments in which carbon is periodically available, where their high propensity for storing carbon as polyhydroxyalkanoates appears key to their success. The novel family was divided into 12 individual clades with new FISH probes designed to cover these. Interestingly, FISH analyses in full-scale EBPR plants reveal that clade members were always found to co-exist in EBPR, indicating niche partitioning. A true understanding of the ecology of the Competibacteraceae, and the competition between the PAO and GAO, requires an in depth understanding of the phylogenetic, and associated phenotypic, diversity of these phenotypes. The Competibacteraceae probes detailed here will allow characterization of the ecophysiological diversity of the family in EBPR, contributing to an ultimate understanding of their ecology.