Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H is an important inter-species energy currency even under gaseous CO -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO saturation.