Abstract. Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring the rates of these metabolisms presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for measuring rates of microbial methane metabolism that is non-toxic, rapid, and relatively high throughput, alleviating some of the current methodological challenges. Specifically, we tested the potential for using monodeuterated methane (CH₃D) as a metabolic substrate for measuring the rate of methane activation by quantifying the change in the aqueous D / H ratio over time using a water isotope analyzer. This method represents a non-toxic, comparatively rapid and straightforward approach that is complementary to existing radio (¹⁴C)- and stable (¹³C) carbon isotopic methods; by probing hydrogen atom dynamics, it offers an additional dimension through which to examine the rates and pathways of methane metabolism. We provide direct comparisons between the CH₃D procedure and the well-established ¹⁴CH₄ radiotracer approach for several methanotrophic systems, including type I and type II aerobic methanotroph cultures, and methane seep sediment and carbonate rocks under anoxic and oxic incubation conditions. We also employ this method to investigate the role of pressure on methane oxidation rates in anoxic seep sediment, revealing an 80 % increase at the equivalent of ~ 900 m water depth (40 MPa).

The monodeuterated methane approach offers a procedurally straightforward, reliable method that advances three specific aims: 1) the direct comparison of methane oxidation rates between different experimental treatments of the same inoculum; 2) the determination of an absolute scaling factor using paired CH₃D and ¹⁴C-radiocarbon procedures for new systems of interest; and 3) a continued evaluation of C- and H-atom tracking through methanotrophic metabolisms, with specific foci on enzyme reversibility and anabolic/catabolic branch points. The procedural advantages, consistency, and novel research questions enabled by the monodeuterated methane method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.