Application of antibiotics for the selective isolation of previously uncultured species from activated sludge

The microbial communities in activated sludge (AS) drive pollutant degradation and nutrient transformation into biomass and gaseous products, while also enabling resource recovery processes. In these systems, microorganisms grow as flocs, whose aggregation properties are essential for retaining active biomass while producing a clarified effluent. Understanding the microbial composition of AS and the functions of individual taxa is crucial for improving wastewater treatment practices and developing new treatment technologies. Although DNA-based studies have identified abundant taxa and inferred their metabolic roles, many of these organisms remain uncultured, limiting experimental validation of genome-based predictions. Here, we investigated whether antibiotics can transiently reduce community complexity and alleviate competitive exclusion during cultivation, thereby facilitating isolation of previously uncultured activated sludge bacteria. Dispersed single cells from AS were cultivated on agarose plates containing filter-sterilized AS fluid and 1 of 11 antibiotics at three concentrations. Full-length 16S rRNA gene amplicon sequencing indicated that antibiotics reduced microbial diversity and altered community composition in an antibiotic- and concentration-dependent manner. Two antibiotic conditions were selected for pure-culture isolation, resulting in 74 isolates that represented 28 different species based on genomic average nucleotide identity. These include 13 putatively novel species based on GTDB classification, and 19 species belonging to nine globally abundant AS core genera. Although several isolates belonged to genera with cultured representatives, they likely represent distinct species with potentially different ecological functions and physiological traits. These findings demonstrate that antibiotics can function as ecological selectors during cultivation and aid the targeted isolation of ecosystem-relevant activated sludge bacteria.IMPORTANCEBiological wastewater treatment relies on diverse microbial communities to degrade pollutants and drive nutrient transformations. Understanding the physiology and metabolism of these microorganisms is essential for improving the efficiency and cost-effectiveness of treatment processes. Much of our current knowledge is derived from 16S rRNA gene amplicon sequencing and metagenomic analyses. However, validating these sequencing- and genome-based insights requires bacterial species as pure cultures, and only a limited number of taxa common in wastewater treatment plants are currently available in culture. Here, we present an isolation strategy that uses antibiotics as a selective pressure to reduce microbial complexity and alleviate competitive exclusion during cultivation, while full-length 16S rRNA gene amplicon sequencing is used to monitor enrichment and guide targeted isolation, thereby facilitating the recovery of process-relevant activated sludge bacteria, including potentially uncultured taxa. These isolates can serve as model organisms for experimental validation of genome-based predictions.