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

Biological 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 representing the 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.