Single-cell analysis in hypersaline brines predicts a water-activity limit of microbial anabolic activity

Hypersaline brines provide excellent opportunities to study extreme microbial life. Here, we investigated anabolic activity in nearly 6000 individual cells from solar saltern sites with water activities (a_w) ranging from 0.982 to 0.409 (seawater to extreme brine). Average anabolic activity decreased exponentially with a_w, with nuanced trends evident at the single-cell level: The proportion of active cells remained high (>50%) even after NaCl saturation, and subsets of cells spiked in activity as a_w decreased. Intracommunity heterogeneity in activity increased as seawater transitioned to brine, suggesting increased phenotypic heterogeneity with increased physiological stress. No microbial activity was detected in the 0.409-a_w brine (an MgCl₂-dominated site) despite the presence of cell-like structures. Extrapolating our data, we predict an a_w limit for detectable anabolic activity of 0.540, which is beyond the currently accepted limit of life based on cell division. This work demonstrates the utility of single-cell, metabolism-based techniques for detecting active life and expands the potential habitable space on Earth and beyond.