Biological Validation and Agnostic Experiments for Extant and Extinct Microbial Life within the Martian Subsurface

Over the last several decades, investigations of Earth's subsurface and other extremely low-biomass systems have refined our understanding of the environmental limits of life, driven by methodological advances that permit agnostic life detection of biology and their respective physical biosignatures and chemical biomarkers. These advances enable mission concepts centered on microbiological processes that facilitate identification of both active life and preserved biosignatures through measurements of metabolism and associated biochemical markers that, on Mars, are more likely to be retained below the surface. Terrestrially, although biological processes can exert a significant influence on Earth's crust, the presence of habitable conditions does not necessarily imply the existence of cellular life. The Viking missions constituted the first direct life-detection experiments on Mars but produced equivocal outcomes, prompting subsequent exploration strategies to emphasize surface habitability rather than direct biological testing. Leveraging progress in subsurface microbiology and planetary exploration, we contend that Mars missions are now poised to shift toward direct tests for extant microbial activity in the subsurface, with metabolic processes serving as a broadly applicable indicator of life.