Triple-oxygen isotopic evidence of prolonged direct bioleaching of pyrite with O2

• Unequivocal evidence of O2 directly oxidizing pyrite sulfur. • The proportion of air-O2 oxygen in pyrite-derived sulfate is ca. 90 % at maximum. • Microbial pyrite oxidation can be ca. 10x faster than abiotic pyrite oxidation, even during initial stage leaching. • The Rio Tinto area in SW Spain hosts a unique environment where high flow of very low pH water maintains oxidative conditions dominated by O2 with solutions containing >50 % Fe2+. Sulfate is often touted as containing atmospheric oxygen whose isotopic signature can constrain redox, environmental conditions, and biological activity. Yet, the amount and isotopic fractionation associated with air-O 2 incorporation during sulfate formation is still debated, making its verification difficult. In this study, we identify a distinct, microbially dominated environment with the potential to preserve maximum signals of air-O 2 in sulfate. We report triple-oxygen isotope data for sulfate produced from pyrite oxidation in microbial and abiotic experiments, and from natural dissolved sulfate from the Rio Tinto, Spain, an acid mine drainage site. The oxygen isotope systematics of sulfate in these environments define a unique kinetic isotope effect associated with initial stage pyrite oxidation by Acidithiobacillus ferrooxidans that preserves >80 % oxygen from air-O 2 in sulfate. Unlike experiments, which evolve toward water-oxygen dominated sulfate on short time scales, Rio Tinto, Spain hosts a microbe rich environment with distinct geochemistry that maintains high O 2 -oxygen in sulfate. Therefore, in addition to containing isotopic records from water and air, sulfates can also contain a biosignature that is promising for understanding conditions on Mars and early Earth. One Sentence Summary: Sulfate can contain > 80 % dissolved O 2 -oxygen and triple-oxygen isotopes show kinetic effects associated with initial stage leaching by microbes.