Species-level microbiome composition of activated sludge and anaerobic digesters - introducing MiDAS 3 ecosystem-specific reference database and taxonomy.

The function of microbial communities in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of its members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain) high resolution taxonomic classification is a crucial towards understanding the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on high-quality full-length sequences derived from activated sludge and anaerobic digester systems in Denmark that proposes unique provisional names for all microorganisms down to species level. The MiDAS 3 database was used for analysis of microbial communities in 20 Danish wastewater treatment plants with nutrient removal, sampled over 12 years. The 50 most abundant species belonged to 41 genera, of which 18 have no known function in the system, emphasizing the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The new MiDAS 3 database also forms the backbone of the MiDAS Field Guide - an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (more than 1,800) and species (more than 4,200) found in activated sludge and anaerobic digesters in Denmark. The identity of the microbes is linked to functional information, where available, and the website provides the possibility to BLAST new sequences against the MiDAS 3 database. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository and facilitating understanding of wastewater treatment ecosystem function.