The microbial dark matter and “wanted list” in worldwide wastewater treatment plants

BACKGROUND: Wastewater treatment plants (WWTPs) are one of the largest biotechnology applications in the world and are of critical importance to modern urban societies. An accurate evaluation of the microbial dark matter (MDM, microorganisms whose genomes remain uncharacterized) proportions in WWTPs is of great value, while there is no such research yet. This study conducted a global meta-analysis of MDM in WWTPs with 317,542 prokaryotic genomes from the Genome Taxonomy Database and proposed a “wanted list” for priority targets in further investigations of activated sludge. RESULTS: Compared with the Earth Microbiome Project data, WWTPs had relatively lower genome-sequenced proportions of prokaryotes than other ecosystems, such as the animal related environments. Analysis showed that the median proportions of the genome-sequenced cells and taxa (100% identity and 100% coverage in 16S rRNA gene region) in WWTPs reached 56.3% and 34.5% for activated sludge, 48.6% and 28.5% for aerobic biofilm, and 48.3% and 28.5% for anaerobic digestion sludge, respectively. This result meant MDM had high proportions in WWTPs. Besides, all of the samples were occupied by a few predominant taxa, and the majority of the sequenced genomes were from pure cultures. The global-scale “wanted list” for activated sludge contained four phyla that have few representatives and 71 operational taxonomic units with the majority of them having no genome or isolate yet. Finally, several genome mining methods were verified to successfully recover genomes from activated sludge such as hybrid assembly of the second- and third-generation sequencing. CONCLUSIONS: This work elucidated the proportion of MDM in WWTPs, defined the “wanted list” of activated sludge for future investigations, and certified potential genome recovery methods. The proposed methodology of this study can be applied to other ecosystems and improve understanding of ecosystem structure across diverse habitats. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40168-023-01503-3.