Blind spots of universal primers and specific FISH probes for functional microbe and community characterization in EBPR systems

Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon sequencing are commonly used for microbial ecological analyses in biological enhanced phosphorus removal (EBPR) systems, the successful application of which was governed by the oligonucleotides used. We performed a systemic evaluation of commonly used probes/primers for known polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). Most FISH probes showed blind spots and covered nontarget bacterial groups. Ca. Competibacter probes showed promising coverage and specificity. Those for Ca. Accumulibacter are desirable in coverage but targeted out-group bacteria, including Ca. Competibacter, Thauera, Dechlorosoma, and some polyphosphate-accumulating Cyanobacteria. Defluviicoccus probes are good in specificity but poor in coverage. Probes targeting Tetrasphaera or Dechloromonas showed low coverage and specificity. Specifically, DEMEF455, Bet135, and Dech453 for Dechloromonas covered Ca. Accumulibacter. Special attentions are needed when using these probes to resolve the PAO/GAO phenotype of Dechloromonas. Most species-specific probes for Ca. Accumulibacter, Ca. Lutibacillus, Ca. Phosphoribacter, and Tetrasphaera are highly specific. Overall, 1.4% Ca. Accumulibacter, 9.6% Ca. Competibacter, 43.3% Defluviicoccus, and 54.0% Dechloromonas in the MiDAS database were not covered by existing FISH probes. Different 16S rRNA amplicon primer sets showed distinct coverage of known PAOs and GAOs. None of them covered all members. Overall, 520F-802R and 515F-926R showed the most balanced coverage. All primers showed extremely low coverage of Microlunatus (below 36.0%), implying their probably overlooked roles in EBPR systems. A clear understanding of the strength and weaknesses of each probe and primer set is a premise for rational evaluation and interpretation of obtained community results.