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

The majority of microbial species present in activated sludge (AS) from wastewater treatment plants (WWTPs) have never been isolated and grown as pure cultures in vitro. To address this we here introduce an experimental setup where plating of dispersed AS bacteria are combined with 16S rRNA gene amplicon sequencing for rapid identification of growth conditions that allows for isolation of key microbial community members in WWTPs. We show that agar plates composed of AS fluid supplemented with a variety of carbon sources promote the growth of many previously uncultivated AS bacteria. Subsequently, 200 colonies were picked from these plates and grown in liquid culture. The resulting cultures represented 103 different species of which 92 are novel based on 16S rRNA gene amplicon sequencing. Subsequent restreaking on plates resulted in a total of 10 stocked pure cultures of which six represent core or conditional abundant or rare bacterial species within WWTPs globally.