CH4 isotopic ordering records ultra-slow hydrocarbon biodegradation in the deep subsurface

Investigation of the biology and biochemistry of the deep subsurface provides invaluable information regarding the limit of life in extreme environments and its role in the global carbon cycle. It has been observed that subsurface microbial CH4 can form in apparent isotopic equilibrium, both with respect to methane clumped isotopic species and D/H fractionation with respect to coexisting water. This observation fostered the suggestion that methanogenic metabolisms in energy-starved environments can operate through slow and reversible enzymatic reactions. Here we present isotopic data including a vertical profile of clumped isotopic indices of methane from Paleozoic-aged pore waters in an aquiclude system from the Michigan Basin. We show evidence of both internal isotopic equilibrium of methane and intermolecular H-isotopic equilibrium between methane and co-occurring non-gaseous n-alkanes. Various mixing and microbial metabolic models were tested and allowed us to identify the possibility of the production of methane at thermodynamic equilibrium from the syntrophic degradation of sedimentary n-alkanes at ultra-slow rates. Significance Statement The recent ability to measure the clumped isotopic composition of methane has fostered new ways of observing the deep subsurface biogeochemistry and has been proposed as a new independent geothermometer when methane is formed at or near isotopic equilibrium. In this manuscript, we present the first continuous Paleozoic sedimentary profile of clumped isotopes in pore methane (i.e. directly sampled from the pores of tight sedimentary rocks) from a paleo-bioreactor in the subsurface and show that a syntrophic metabolic network between fermentative bacteria and mainly acetotrophic methanogens can thrive in an energy limited environment.