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

Abstract

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. ImportanceBiological wastewater treatment relies on complex microbial communities that assimilate nutrients and break down pollutants in the wastewater. Knowledge about the physiology and metabolism of bacteria in wastewater treatment plants (WWTPs) may therefore be used to improve the efficacy and economy of wastewater treatment. Our current knowledge is largely based on 16S rRNA gene amplicon profiling, fluorescence in situ hybridization studies, and predictions based on metagenome-assembled genomes. Bacterial isolates are often required to validate genome-based predictions as they allow researchers to analyze a specific species without interference from other bacteria and with simple bulk measurements. Unfortunately, there are currently very few pure cultures of microbes commonly found in WWTPs. To address this, we introduce an isolation strategy that takes advantage of state-of-the-art microbial profiling techniques to uncover suitable growth conditions for key WWTP microbes. We furthermore demonstrate that this information can be used to isolate key organisms representing global WWTPs.