The environmental history of Earth's ocean, atmosphere and related biosphere is commonly tracked through evaluating carbon and sulfur isotope records in sedimentary rocks. Unlike carbon, sulfur isotopes reflect a balance of various microbial processes. The reductive side of the S cycle is dominated by sulfate reducing bacteria – a metabolism that is putatively quite old. In complement to sulfate reduction, the oxidative sulfur cycle is driven by a broad suite of (a)biological oxidation and microbial disproportionation reactions. The goal of this study is the isotopically characterize microbial sulfur disproportionation, as this process is poorly understood and capable of producing enormous, mass-dependent isotope fractionations. Given recent findings of large isotope effects as a result of sulfate reduction (a well-characterized process), further calibrating the isotopic consequences of disproportionation is even timelier. Here, the inclusion of the minor sulfur isotope – 33S – will allow for the isotopic fingerprint of disproportionation and sulfate reduction to be uniquely differentiated from one another, even when the conventional 34S/32S is the same. This project will place new isotopic constraints on the microbial disproportionation of sulfite, thiosulfate and elemental sulfur. The proposed work will target growth of the genetically tractable pure culture Desulfocapsa sulfexigens (already in culture in the Johnston Lab). Using chemostats, we will establish the characteristic 32S - 33S - 34S fractionation imposed by disproportionation on thiosulfate, sulfite and elemental S across a range of environmentally reasonable metabolic rates. Chemostat experiments will be underpinned by batch experiments (on each intermediate) that will serve to establish maximum growth rates (needed for proper chemostat calibration). In addition to surveying rate, the role of Fe, which is proposed to modulate sulfur isotope fractionation in these systems, will be more thoroughly assessed. In complement to multiple sulfur isotope work, 18O/16O measurements will be made on sulfate as a means of further probing the oxidative branch of the disproportionation metabolism. Along with ancillary geochemical data, all isotope data will then be modeled with the goal of developing a calibrated fractionation model for sulfur disproportionation. This will bring our quantitative understanding of disproportionation to a level that is then sufficient to revisit paleo-environmental records. This increased calibration will allow for more refined reconstructions of paleo-environments. Of particular interest is better understanding the Paleozoic, where current calibrations are insufficient to explain sedimentary records. However, this general calibration will also inform a range of questions at the heart of NASA's broader goals, as it relates to better understanding the 'early evolution of life and the biosphere' through to the development of early metabolisms and proliferation of the biosphere through the entire Precambrian.