Groundwater Enrichment Experiments Raw sequence reads

The Candidate Phyla Radiation (CPR) represents a widespread but poorly understood group of bacteria. So far, very few cultured representatives exist, due to metabolic dependencies on microbial host species. When transferring samples like groundwater, where CPR constitute over 50% of the microbiome, to laboratory conditions, typically most CPR are lost, and suitable incubation conditions and hosts remain elusive. Here, we examined bacterial 16S rRNA gene amplicon datasets from 397 groundwater enrichments under treatments stimulating chemolithoautotrophy, methylotrophy or heterotrophy, to determine how CPR diversity and abundance can be retained and which groups of CPR are susceptible to enrichment. Despite the initial decrease, CPR abundance increased over longer incubation periods, reaching significant proportions of up to 11-30%. Across enrichments, 1410 CPR ASVs were observed, covering six classes dominant in groundwater. Notably, prevalent CPR groups varied depending on treatments, with Cand. Saccharimonadia dominating (up to 31.4%) in treatments with polysaccharides, while Cand. Parcubacteria (>23%) thrived in treatments stimulating methylotrophs and autotrophs. ASV-specific growth rates based on quantitative PCR showed that some CPR doubled within 1-2 days, in line with faster growing groundwater bacteria, while most CPR had doubling times of ~14 days. Similarities in growth rates of CPR and non-CPR matched with correlations based on co-occurrence network analysis, potentially reflecting interactions of CPR with their hosts. Metabolic network analysis based on metagenome-assembled genomes showed that surprisingly, up to 62% of genes of CPR were affiliated with functions occurring under oxic conditions, although CPR are considered to have an anaerobic, fermentative metabolism. In fact, in 25 CPR genomes, enzymes directly employing oxygen as substrate were encoded, challenging previous assumptions about their preferences for anoxic conditions. Overall, this study sheds light on incubation conditions favorable for CPR enrichment and provides insights into their metabolic capabilities, ecological roles, and interactions with other microbes in groundwater ecosystems.