 This study investigated the age, geochemistry, and microbiology of 138 groundwater samples from 95 monitoring wells, less than 250m depth, located in 14 aquifers in Canada. The results showed that older groundwaters contained more cells and higher levels of dissolved oxygen than younger groundwaters, indicating that aerobic metabolism was occurring in these subsurface environments. Additionally, the presence of dark oxygen indicated that it was being generated locally through microbial dismutation. This study provides evidence for the existence of an underappreciated oxygen source in subsurface ecosystems, which could have implications for understanding how life has evolved on Earth. This article was authored by Esimil Ruff, Pauline Humers, Isabella Rabe de Angelis, and others.