A methylamine-driven long term groundwater enrichment containing members of the Candidate Phyla Radiation

Bacteria of the recently discovered and widespread Candidate Phyla Radiation (CPR) metabolically depend on host species. As a consequence, CPR are typically lost once removed from their natural environment for enrichment and cultivation approaches. Only few CPR are currently available as isolates, and even initially successful enrichments showed a tendency to crash for unknown reasons after prolonged incubations.

Here, we report on an aerobic groundwater-derived enrichment containing CPR that has been continuously maintained for more than two years, and has been monitored using 16S rRNA gene amplicon sequencing, quantitative PCR, BONCAT and metagenomics. Fed with methylamine (MA) as carbon and energy source, the activity of methylotrophs affiliated with Methylobacterium and Aminobacter fueled a community of 500 to 700 bacteria based on ASV analysis, including 28 ASVs of the CPR. In total, CPR contributed up to 15% of the community based on relative abundance of 16S rRNA genes. As CPR usually have less 16S rRNA gene copies than other bacteria, their actual abundance might be even higher. From a total of 132 high quality metagenome-assembled genomes (MAGs), 18 MAGs of CPR were recovered, affiliated with Cand. Magasanikbacteria of the ABY1 group and Cand. Kaiserbacteria of the Cand. Paceibacteria. Both BONCAT-activated cell sorting and 16S rRNA gene copy numbers suggested proliferation of the CPR. Following passaging of the enrichment, CPR typically increased after 4 to 6 weeks, when nutrients were depleted and oxygen consumption ceased, suggesting their growth to benefit from conditions where other microbes are starving. Apart from CPR and MA degraders, also MAGs implicated in nitrogen cycling like Nitrosomonas and Nitrospira were recovered, housing ammonia monooxygenase and nitrite oxidoreductase genes. This indicated that MA degradation fueled a network of nitrogen based metabolic interactions and thus supported the complex community observed, potentially also benefitting CPR. The enrichment culture represents a valuable model system to investigate the role of CPR for the rest of the community and the potential functional interplay among these organisms.