Amplicon-guided isolation and cultivation of previously uncultured microbial species from activated sludge

Microbes are fundamental for biological wastewater treatment. However, most microbial species found in activated sludge (AS) from wastewater treatment plants (WWTPs) have never been isolated and grown as pure cultures, thus limiting our understanding of the underlying biological processes. To change this, we here introduce an experimental setup where the plating of dispersed AS bacteria is combined with 16S rRNA gene amplicon sequencing of the total plate biomass for rapid identification of growth conditions that allow for the isolation of key microbial species in AS. We show that agarose plates composed of AS fluid supplemented with various carbon sources support the growth of many previously uncultivated AS bacteria. To confirm that the approach can also be used to isolate previously uncultured species, we picked 200 colonies from the plates for growth in liquid medium. This resulted in 185 enriched cultures representing at least 76 strains based on unique 16S rRNA gene V1-V3 amplicon sequence variants (ASVs). Classification of the ASVs with the MiDAS 5.1 database revealed 39 distinct genera, including AAP99, Ca. Brachybacter, Ca. Proximibacter, Ellin6067, midas_g_12, and midas_29279, which all lack pure culture representatives. Among the ASVs that obtained species-level classification, we observed 26 unique species of which 16 were only classified based on the MiDAS placeholder taxonomy highlighting the potential for culturing many novel taxa. Purification by restreaking and preparation of glycerol stocks resulted in 10 pure cultures of species commonly found in WWTPs globally, including Rhodoferax midas_s_1744, Thauera midas_s_1356, Acidovorax midas_s_1484, Tessaracoccus midas_s_1151, and Sphingopyxis midas_s_983.