Investigating the antimethanogenic effects of selected nitro-compounds on methane production, rumen fermentation, and methanogenic archaea <i>in vitro</i>

Enteric methane emissions (EME) cause adverse environmental effects and energy losses to the host. Given the potent antimethanogenic effects of the nitro compounds (NC) ethyl-nitroacetate (ENA), ethyl-2-nitropropionate (ENP), and nitrate (NO₃⁻), we hypothesized that they could effectively mitigate EME. Phase 1 determined dose responses of each NC, identified optimal doses, and evaluated their effects on gaseous composition, rumen fermentation, and bacterial-archaea populations. ENA and ENP tested at 8 and 16 mM inhibited CH₄ production by 100%, whereas NO₃⁻ tested at 12 and 24 mM reduced CH₄ production by 72%. However, the amount of spared H₂ differed among NC, revealing differential effects on fermentation pathways. ENA increased propionate and butyrate concentrations at the expense of acetate, whereas ENP slightly reduced acetate and modestly increased propionate at 24 h post-incubation. NO₃⁻ acted as an alternative H₂ sink without changing fermentation end products. The overall methanogenic community was not altered, but each NC differentially reduced the abundance of <i>Methanobrevibacter ruminantium</i> M1, <i>Methanosphaera stadtmanae</i>, and the methanogenic archaeon ISO4-H5. ENA greatly altered the bacterial profiles, followed by ENP and NO₃⁻. Phase 2 investigated the impact of NC (0-1.25 mM) on <i>M. stadtmanae</i> cultures at 24 and 48 h post-incubation. The findings aligned with phase 1, confirming reduced CH₄ production and H₂ flux dynamics. ENA and ENP inhibited <i>M. stadtmanae</i> growth and CH₄ production at all tested doses, whereas NO₃⁻ was effective at concentrations above 0.75 mM. These results highlight distinct mechanisms for CH₄ mitigation, warranting further studies on additional methanogenic species to refine mitigation strategies.IMPORTANCEEME signify detrimental environmental impacts and constitutes an energy loss for the host. ENA, ENP, and NO₃⁻ showed distinct antimethanogenic effects, resulting in varied impacts on gas composition, rumen fermentation, and bacterial-archaea populations. ENA exerted the strongest and most direct inhibitory effect on methanogenesis, leading to notable changes in H₂ and VFA accumulations and archaeal populations. Although ENP completely inhibited CH₄ production, it resulted in low H₂ accumulations, suggesting an indirect effect and a dose-dependent modulation of fermentation pathways. NO₃⁻ produced a moderate reduction in CH₄ output by diverting H₂ toward NH₃ production while maintaining fermentation stability. <i>M. stadtmanae</i> cultures verified that ENA, ENP, and NO₃⁻ have distinct mechanisms of action, thereby affecting methanogenesis differently. These findings highlight the potential of nitro-compounds for CH₄ reduction, underscoring the need for <i>in vivo</i> validation alongside detailed multi-omics analyses to fully understand their effects on rumen microbiota and metabolic networks.