Monodeuterated Methane, an Isotopic Tool To Assess Biological Methane Metabolism Rates

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 these metabolic rates presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for investigating microbial methane metabolism based on hydrogen atom dynamics, which is complementary to carbon-focused assessments of methanotrophy. The method uses monodeuterated methane (CH₃D) as a metabolic substrate, quantifying the aqueous D/H ratio over time using off-axis integrated cavity output spectroscopy. This approach represents a nontoxic, comparatively rapid, and straightforward approach that supplements existing radiotopic and stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional dimension for examining rates and pathways of methane metabolism. We provide direct comparisons between the CH₃D procedure and the well-established ¹⁴CH₄ radiotracer method for several methanotrophic systems, including type I and II aerobic methanotroph cultures and methane-seep sediment slurries and carbonate rocks under anoxic and oxic incubation conditions. In all applications tested, methane consumption values calculated via the CH₃D method were directly and consistently proportional to ¹⁴C radiolabel-derived methane oxidation rates. We also employed this method in a nontraditional experimental setup, using flexible, gas-impermeable bags to investigate the role of pressure on seep sediment methane oxidation rates. Results revealed an 80% increase over atmospheric pressure in methanotrophic rates the equivalent of ~900-m water depth, highlighting the importance of this parameter on methane metabolism and exhibiting the flexibility of the newly described method. IMPORTANCE Microbial methane consumption is a critical component of the global carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon exploitation. Nonetheless, quantifying methane metabolism typically involves logistically challenging methods and/or specialized equipment; these impediments have limited our understanding of methane fluxes and reservoirs in natural systems, making effective management difficult. Here, we offer an easily implementable, precise method using monodeuterated methane (CH₃D) that advances three specific aims. First, it allows users to directly compare methane consumption rates between different experimental treatments of the same inoculum. Second, by empirically linking the CH₃D procedure with the well-established ¹⁴C radiocarbon approach, we determine absolute scaling factors that facilitate rate measurements for several aerobic and anaerobic systems of interest. Third, CH₃D represents a helpful tool in evaluating the relationship between methane activation and full oxidation in methanotrophic metabolisms. The procedural advantages, consistency, and novel research questions enabled by the CH₃D method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.