Biogenic ore formation has long required clarification of depositional mechanisms and textural characteristics such as carbonate dissolution and ‘snow on the roof’. This research explored evidence for microbial sulfate reduction and biogenic mineralisation associated with sulfide ore deposits, using sphalerite from eight locations. Sulfur isotopic and microtextural analyses were conducted on sphalerites from Navan (Ireland), Wiesloch (Germany), Broken Hill supergene, Century and Cadjebut (Australia), Pomorzany (Poland), Balmat (USA) and Geco (Canada), using Sensitive High-Resolution Ion Microprobe, X-ray Fluorescence Microscopy, fluorination, cathodoluminescence, and atom probe tomography. Results correlated microbial sulfate reduction to stromatolitic zonation accompanied by strongly negative δ34S (−10 to −40) and δ33S (−5 to −20). Mississippi Valley-type sphalerites were classified as either MVTa (abiotic) or MVTb (biotic). The colloform banding suggest cyclic ore deposition, which led to the construction of pH dependent precipitation pathways that begin with acidic fluids and evolve into alkaline conditions with ongoing microbial activity. While sulfate reducing bacteria are the most likely organisms associated with sulfide mineralisation, syntrophic microbial communities are also suggested as avenues to process organic compounds such as hydrocarbons and aid sulfate reduction. The distinct isotopic signatures indicate that sphalerite from Navan, Wiesloch and the supergene Broken Hill ores formed from biogenic sulfate reduction. Rayleigh-type closed system isotopic fractionation was identified in abiotic sphalerite from Pomorzany and mass-independent fractionation (resemblant of pre-Great Oxidation Event conditions) in sphalerite from Geco.
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