Triple-oxygen isotopic evidence of a biosignature from direct bioleaching of pyrite with O2

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-O2 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-O2 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-O2 in sulfate. This microbial triple-oxygen isotopic fractionation is statistically distinct from abiotic controls and the microbial main stage. Therefore, in addition to containing signals from water and air, sulfates can also contain a biosignature that is promising for understanding conditions on Mars and early Earth.