Liu, L.; Singleton, C. M.; Kirkegaard, R. H.; Sereika, M.; Riisgaard-Jensen, M.; Knudsen, K. S.; Mussig, A. J.; Petersen, J. F.; Kondrotaite, Z.; Peces, M.; MiDAS Global Consortium, ; Hugenholtz, P.; Albertsen, M.; Nielsen, P. H.; Dueholm, M. K. D.

Wastewater treatment relies on complex microbial communities, yet existing genome-resolved references for this essential engineered ecosystem remain dominated by short-read assemblies, limiting genome contiguity and linkage between taxonomic and metabolic function. We applied long-read sequencing to activated sludge from 83 globally distributed plants, reconstructing 53,501 metagenome-assembled genomes to establish the Microbial Database of Activated Sludge (MiDAS) global genome catalog. The catalog encompasses high-quality genomes for 12,047 prokaryotic species, 82% of which are not represented in GTDB release 226, and provides a median of 32 high-quality genomes for each of the 250 core prokaryotic genera previously defined in our MiDAS global 16S rRNA gene survey. This enables analyses of predicted functional traits and their ecological context, for example, we identified two sparsely represented Nitrospiraceae genera with conserved nitrite-oxidation genes that are abundant in higher-temperature wastewater treatment plants. In summary, the MiDAS genome catalog provides a framework for linking taxonomy, metabolism and ecological roles in wastewater treatment systems globally.