Shifts in ruminant fermentation during inhibition of methanogenesis are reflected in the isotope compositions of volatile fatty acids.

Ruminant animals are a major source of the potent greenhouse gas methane, but they are also a tractable target for climate solutions. Several strategies have been developed to lower methane emissions from ruminants, including feed additives that inhibit methanogenic archaea. Sustainable solutions must eliminate methane emissions without hampering the microbial fermentation of plant material, which the animal host relies on for carbon and energy. However, current tools cannot directly quantify or characterize the metabolic pathways of in vivo ruminant fermentation. To fill this gap, we developed an electrospray (ESI) Orbitrap mass spectrometry technique to measure the stable isotope ratios 13C/12C and 2H/1H of volatile fatty acids (VFAs) at their natural isotopic abundances directly from rumen fluid. We tested this technique on in vitro incubations of rumen fluid fed three different substrates with and without the additive Asparagopsis taxiformis. We found that the isotope composition of VFAs changed and reflected a remodeling of microbial fermentation pathways. Specifically, acetate's d13C value increased when methanogens were inhibited, suggesting a lower relative rate of acetate synthesis and a lack of acetogenic activity. Furthermore, the d2H value of propionate decreased, which may indicate a change in the balance between the two pathways of propionate synthesis toward the less energetic acrylate pathway. Both signals were consistent across feed types. Taken together, our results provide evidence that fermentative metabolism is remodeled during methanogenesis inhibition and decreases relative fluxes through ATP-generating pathways. More broadly, this study demonstrates the utility of ESI-Orbitrap-based isotopic analysis for studying rumen microbial ecology.