Axial Seamount RNA-SIP experiments from Marker 33 vent, via shipboard and seafloor incubator

Diverse microbial communities drive biogeochemical cycles in the ocean and account for ~70% of the total marine biomass, yet studying these organisms and processes is limited by technological and instrument capabilities, particularly in the deep ocean. To address this knowledge gap, we developed an ROV-powered incubator instrument to carry out and compare results from an in situ and shipboard RNA Stable Isotope Probing (RNA-SIP) experiment to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All RNA-SIP experiments with sodium bicarbonate at 55ºC showed successful uptake of the label, and comparison of transcripts from shipboard and seafloor incubations showed that nitrate reduction coupled to hydrogen oxidation was carried out by thermophilic Epsilonbacteraeota in all experiments. However, shipboard incubations had a higher abundance of transcripts annotated for anaerobic processes while the seafloor incubations showed a higher abundance of transcripts annotated for aerobic processes, suggesting oxygen was lost before the experiment began shipboard. Microbial communities were also under more stress during the shipboard incubations. Results provide new insight into the activities of natural microbial assemblages under near-native conditions in the ocean, and the development of this novel incubator will allow for future experimentation with marine microbes.