High diversity, abundance and expression of hydrogenases in groundwater

Hydrogen may be the most important electron donor available in the subsurface. Here we analyze the diversity, abundance and expression of hydrogenases in 5 proteomes, 25 metagenomes and 265 amplicon datasets of groundwaters with diverse geochemistry. A total of 1,772 new [NiFe]-hydrogenase gene sequences were recovered, which almost doubled the number of sequences in a widely used database. [NiFe]-hydrogenases were highly abundant, almost as abundant as the DNA-directed RNA polymerase. The abundance of hydrogenase genes increased with depth from 0 to 129 m. Hydrogenases were present in 502 out of 1,245 metagenome-assembled-genomes. The populations with hydrogenases accounted for [~]50% of all populations. Hydrogenases were actively expressed, making up as much as 5.9% of methanogen proteomes. Most of the newly discovered diversity of hydrogenases was in "Group 3b", which was linked to sulfur metabolism. "Group 3d" was the most abundant, which was previously linked to fermentation, but we observed this group mainly in methanotrophs and chemoautotrophs. "Group 3a", associated with methanogenesis, was the most active in proteomes. Two newly discovered groups of [NiFe]-hydrogenases further expanded the biodiversity. Our results highlight the vast diversity, abundance and expression of hydrogenases in the sampled groundwaters, suggesting a high potential for hydrogen oxidation in subsurface habitats.