Diversity and physiology of the uncultured and abundant genera in the Saprospiraceae family across global wastewater treatment systems

The Saprospiraceae family is a very diverse family within the phylum Bacteroidetes, commonly present in high abundance in wastewater treatment plants (WWTPs) worldwide, but little is known about their function and importance. The genera described are characterized by rod-shaped or filamentous morphology and an aerobic heterotrophic metabolism with their potential involvement in nutrient removal in WWTPs. Here we used MiDAS4 global survey, including samples from 480 WWTPs located in 30 countries, to analyze the abundance and global distribution of members of the Saprospiraceae family. In addition, we retrieved 32 high-quality metagenome assembled genomes (MAGs) from Danish WWTPs for metabolic reconstruction, we designed novel fluorescence in situ hybridization (FISH) probes for visualization, and in combination with Raman microspectroscopy we detected and confirmed important physiological features. FISH revealed rod-shaped morphologies for all analyzed genera, present mostly inside the flocs. The genomic potential revealed diverse metabolism for several undescribed genera including genus OLB8, indicating possible degradation of polysaccharides, amino acids and other carbon substrate, and partial denitrification. FISH-Raman showed presence of glycogen and PHA, also confirmed by the metabolic potential of MAGs, while none of the genera contained polyphosphate. These results provide the first overview of some of the most abundant Saprospiraceae genera present in WWTPs across the world, revealing their potential involvement in degradation of complex macromolecules, such as polysaccharides and proteins, and in nutrient removal.