A large fraction of Earth's biosphere lives in the deep ocean and within the oceanic subsurface. This “dark” biosphere thrives under conditions of high hydrostatic pressures (>10 MPa, 1000 meter depth) and often high-temperatures and is adapted not only to pressure but also to sharp temperature and redox gradients. Despite their contribution to the Earth's microbiome, deep ocean processes, and the evolution of early life, the functions and environmental responses of thermopiezophilic microbes remain surprisingly understudied. Here we present a review of recent experimental and field studies on the function of chemolithoautotrophic microorganisms, i.e., organisms that use inorganic molecules such as hydrogen (H2) as electron donor to drive carbon fixation from CO2 into biomass, under nutrient and physicochemical conditions relevant to their deep-sea habitat. More specifically, we assess the effect of chemolithoautotrophic nitrate reduction on the distribution of N compounds and on the evolutionary responses of microbial communities under the extreme conditions of deep-sea hydrothermal vents. Data compiled in this study comes from high-pressure, high-temperature experiments on microbial communities and isolates (e.g., Nautilia spp., PV-1 strain) collected from diffuse flow vent fluids at East Pacific Rise hydrothermal vents. Microbial H2 metabolism and homeoviscous adaptation are investigated through analysis of gene expression, proteomics, membrane lipids, and biomass 15N/14N isotopic signatures. This work is essential for elucidating the evolutionary and metabolic activities of microorganisms cultured under in situ deep-sea vent conditions of pressure, temperature, and nutrient levels, both as mixed (natural) and pure cultures.