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

ABSTRACT 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 CH 4 production by 100%, whereas NO₃⁻ tested at 12 and 24 mM reduced CH 4 production by 72%. However, the amount of spared H 2 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 H 2 sink without changing fermentation end products. The overall methanogenic community was not altered, but each NC differentially reduced the abundance of Methanobrevibacter ruminantium M1, Methanosphaera stadtmanae , 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 M. stadtmanae cultures at 24 and 48 h post-incubation. The findings aligned with phase 1, confirming reduced CH 4 production and H 2 flux dynamics. ENA and ENP inhibited M. stadtmanae growth and CH 4 production at all tested doses, whereas NO₃⁻ was effective at concentrations above 0.75 mM. These results highlight distinct mechanisms for CH 4 mitigation, warranting further studies on additional methanogenic species to refine mitigation strategies. IMPORTANCE EME 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 H 2 and VFA accumulations and archaeal populations. Although ENP completely inhibited CH 4 production, it resulted in low H 2 accumulations, suggesting an indirect effect and a dose-dependent modulation of fermentation pathways. NO₃⁻ produced a moderate reduction in CH 4 output by diverting H 2 toward NH 3 production while maintaining fermentation stability. M. stadtmanae 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 CH 4 reduction, underscoring the need for in vivo validation alongside detailed multi-omics analyses to fully understand their effects on rumen microbiota and metabolic networks.

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