Microbial communities in an anammox reactor treating municipal wastewater at mainstream conditions: Practical implications of different molecular approaches

Biases on molecular techniques used to study microbial communities in the anammox process are not always considered. This work aimed to evaluate different molecular approaches to study bacterial communities in an anammox reactor treating real urban wastewater at mainstream conditions. 16S rRNA amplicon sequencing with different primer sets, metagenomics, and qPCR were evaluated. An enriched anammox reactor feed with synthetic wastewater was used as a positive control. We reported that, despite its in silico coverage, the universal primers 515F-909R had, in fact, very low coverage (1%) of the Planctomycetota phylum. The functional genes approach showed a taxonomic resolution at the species level, although genomic database updates are a bias to be considered. From the phylogenetic analysis, coherence among the approaches was obtained. Primers targeting anammox 16S rRNA and hydrazine synthase genes in qPCR showed consistency in the quantification, making them useful for comparing similar investigations. According to the results, recommendations on the biomolecular approach to be used were outlined.