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 are combined with 16S rRNA gene amplicon sequencing of 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 growing cultures representing 102 strains based on unique 16S rRNA gene V1-V3 amplicon sequence variants (ASVs). Classification of the ASVs with the MiDAS 4 database revealed 48 distinct genera, including the previously uncultured AAP99, Ca. Propionivibrio, Ellin6067, midas_g_12, and Ca. Brachybacter. Among the ASVs that obtained species-level classification, we observed 43 unique species of which 29 were only classified based on the MiDAS placeholder taxonomy highlighting the potential for culturing many novel taxa. Preparation of glycerol stocks and subsequent validation by restreaking on plates resulted in 10 pure cultures of which six represent core or conditional rare or abundant (CRAT) species observed within the MiDAS global survey of WWTPs.