Adaptive thermogenesis and fermentation as survival strategies of vegetative cells of Bacillus subtilis under simulated Martian stressors

Abstract

<title>Abstract</title> Microbial responses to Mars‑analog environments are key to understanding the limits of life and improving life‑detection strategies. Here, we investigated the survival and physiological adaptation of <italic>Bacillus subtilis</italic> exposed for five sols to an upgraded MARTE simulation chamber, redesigned to enhance environmental stability and sample retrieval. Unlike previous investigations that focused on other bacterial species, shorter exposure times, or spore‑based survival, this work specifically evaluates the response of vegetative cells. Survival decreased significantly, but proteomic analysis revealed a persistent metabolic activity, with upregulation of proteins involved in carbohydrate metabolism, stress adaptation, and protein turnover. Low‑temperature responses were dominated by thermogenic pathways, including increased abundance of glucose‑6‑phosphate dehydrogenase and glycerol‑3‑phosphate dehydrogenase, while anaerobic fermentation contributed to heat generation and gas release. Enhanced pigment biosynthesis and the formation of structured biofilms further indicated coordinated protective strategies. These results demonstrate that <italic>B. subtilis</italic> deploys multifactorial mechanisms to withstand prolonged Mars‑analog conditions and highlight the value of improved simulation platforms for advancing experimental astrobiology.