Hydrothermal vent systems provide access points to the extensive microbial communities of the rocky subseafloor. These subseafloor communities have the potential to influence ocean biogeochemistry and, in particular, the chemolithoautotrophic populations could potentially provide a large amount of new production to the deep sea. In this study, we used RNA-based stable isotope probing (RNA-SIP) metatranscriptomics to identify the active autotrophic players and genomic pathways present in venting fluids from Axial Seamount, a submarine volcano off the coast of Oregon, USA. Vent fluids for RNA-SIP were collected from a singular vent, Marker 113, and used in shipboard incubations with 13C labeled sodium bicarbonate at 30, 55, and 80ºC. Results from RNA-SIP incubations show enrichment of RNA at all three temperatures after 18-36 hours, indicating the presence and activity of subseafloor chemolithoautotrophic microbes in vent fluids. In RNA-SIP experiments across a range of temperatures, both taxonomic and functional diversity was reduced compared to un-manipulated diffuse fluids. At 30ºC and 55ºC, Epsilonproteobacteria were dominant, oxidizing hydrogen and primarily reducing nitrate. Methanogenic archaea were also present at 55ºC, and were the only autotrophs present at 80ºC. Correspondingly, the predominant CO2 fixation pathways changed from the reductive TCA cycle to the reductive acetyl-CoA pathway with increasing temperature. This study demonstrates the presence of an active autotrophic subseafloor community across geothermal gradients, lending insight into chemolithoautotrophic communities in the subseafloor at deep-sea hydrothermal vents
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