Microstructure-specific carbon isotopic signatures of organic matter from ∼3.5 Ga cherts of the Pilbara Craton support a biologic origin

•In situ carbon isotopic analysis of ∼3.5 Ga organic matter and coexisting carbonate.•δ13C values in specific organic microstructures range between −33.6‰ and −25.7‰.•The range of δ13C(OM) values represents primary variability of organic matter.•Correlation between δ13C values and microstructures argues against abiotic origin.•The range of δ13C(OM) and Δ13C(Carb-OM) values supports variable C-fixation paths. The ∼3.5 Ga Dresser Formation from the North Pole Dome of the Pilbara Craton (Western Australia) contains some of the oldest evidence for life on Earth. Here, we present a detailed study of microstructure-specific carbon isotopic composition of organic matter (OM) preserved in Dresser Formation bedded cherts and hydrothermal chert vein using in situ Secondary-Ion Mass Spectrometry (SIMS). The OM in these rocks occurs mainly as clots that, together with minor fine OM layers and laminae, are considered primary textures formed prior to host rock lithification. Other than rare OM-rich stylolites, no evidence was found for later OM migration beyond the micrometer scale. Average δ13C(OM) values in specific microstructural types range between −33.6‰ and −25.7‰. No correlation is seen between measured δ13C values and H/C ratios in the studied OM microstructures. This lack of correlation and the low metamorphic grade of the rocks studied argue against significant modification of OM isotopic composition by later metamorphic alteration. It is thus concluded that the range of δ13C values found in the samples represents primary OM isotopic variability. Within some individual samples variable δ13C(OM) values are correlated with specific microstructural types. This observation is not consistent with solely abiotic OM formation via Fisher-Tropsch type reactions. When compared with associated δ13C(ankerite) values, average δ13C(OM) values indicate C isotopic fractionation [Δ13C(Ank–OM)] of 25–33‰, which translates to dissolved CO2–OM isotopic fractionation [Δ13C(CO2–OM)] of 20–30‰. This range of Δ13C(CO2–OM) is consistent with enzymatic C fixation via the Calvin cycle utilized by photoautotrophs and the reductive acetyl-CoA pathway utilized by chemolithoautotrophs. Photosynthetic OM formation is supported by the relatively shallow water depth inferred for the Dresser environment and the restricted occurrence of stromatolites to shallow water deposits in this unit, whereas chemolithosynthesis is supported by the abundance of OM in sub-seafloor hydrothermal chert veins.