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The deep continental crust represents a vast potential habitat for microbial life where its activity remains poorly constrained. Organic acids like acetate are common in these ecosystems, but their role in the subsurface carbon cycle - including the mechanism and rate of their turnover - is still unclear. Here, we develop an isotope-exchange 'clock' based on the abiotic equilibration of H-isotopes between acetate and water, which can be used to define the maximum in situ acetate residence time. We apply this technique to the fracture fluids in Birchtree and Kidd Creek mines within the Canadian Precambrian crust. At both sites, we find that acetate residence times are <1 million years and calculated a rate of turnover that could theoretically support microbial life. However, radiolytic water-rock reactions could also contribute to acetate production and degradation, a process that would have global relevance for the deep biosphere. More broadly, our study demonstrates the utility of isotope-exchange clocks in determining residence times of biomolecules with possible applications to other environments., (© 2024. The Author(s).)

Stable hydrogen isotope compositions ( 2 H/ 1 H ratios) have been an invaluable tool for studying biogeochemical processes in nature, but the diversity of molecular targets amenable to such analysis is limited. Here, we demonstrate a new technique for measuring δ 2 H of biomolecules via Orbitrap mass spectrometry (MS) using acetate as a model analyte. Acetate was chosen as a target molecule because its production and consumption are central to microbial carbon cycling, yet the mechanisms behind acetate turnover remain poorly understood. δ 2 H of acetate could provide a useful constraint on these processes; however, it remains uncharacterized in nature due to analytical challenges. Electrospray ionization (ESI)-Orbitrap MS circumvents these challenges and delivers methyl-specific H-isotope compositions of acetate with nanomole sensitivity, enough to enable analyses of environmental samples. This approach quantifies the methyl-specific δ 2 H and molecular-average δ 13 C of acetate simultaneously while achieving <3 and <0.5‰ uncertainty, respectively. Using optimized ionization and Orbitrap parameters, this level of precision is obtained within 15 min using only 15 nmol of acetate. As a demonstration of our analytical approach, we cultured three acetogenic bacteria and found a large 2 H-fractionation between acetate and water (>310‰ depletion) associated with the Wood-Ljungdahl pathway, while fermentation expressed a muted (∼80‰) fractionation. With its high precision and sensitivity, Orbitrap MS is a promising tool for investigating these signals in nature after offline purification. Furthermore, the ESI-Orbitrap method presented here could be applied to other molecules amenable to ESI, including central metabolites and sugars, greatly expanding the molecular targets used in hydrogen isotope biogeochemistry.