Inhibition of methanogenesis by nitrate, with or without defaunation, in continuous culture

Within the rumen, nitrate can serve as an alternative sink for aqueous hydrogen [H₂(aq)] accumulating during fermentation, producing nitrite, which ideally is further reduced to ammonium but can accumulate under conditions not yet explained. Defaunation has also been associated with decreased methanogenesis in meta-analyses because protozoa contribute significantly to H₂ production. In the present study, we applied a 2 × 2 factorial treatment arrangement in a 4 × 4 Latin square design to dual-flow continuous culture fermentors (n = 4). Treatments were control without nitrate (-NO₃^-) versus with nitrate (+NO₃^-; 1.5% of diet dry matter), factorialized with normal protozoa (faunated, FAUN) versus defaunation (DEF) by decreasing the temperature moderately and changing filters over the first 4 d of incubation. We detected no main effects of DEF or interaction of faunation status with +NO₃^-. The main effect of +NO₃^- increased H₂(aq) by 11.0 µM (+117%) compared with -NO₃^-. The main effect of +NO₃^- also decreased daily CH₄ production by 8.17 mmol CH₄/d (31%) compared with -NO₃^-. Because there were no treatment effects on neutral detergent fiber digestibility, the main effect of +NO₃^- also decreased CH₄ production by 1.43 mmol of CH₄/g of neutral detergent fiber degraded compared with -NO₃^-. There were no effects of treatment on other nutrient digestibilities, N flow, or microbial N flow per gram of nutrient digested. The spike in H₂(aq) after feeding NO₃^- provides evidence that methanogenesis is inhibited by substrate access rather than concentration, regardless of defaunation, or by direct inhibition of NO₂^-. Methanogens were not decreased by defaunation, suggesting a compensatory increase in non-protozoa-associated methanogens or an insignificant contribution of protozoa-associated methanogens. Despite adaptive reduction of NO₃^- to NH₄⁺ and methane inhibition in continuous culture, practical considerations such as potential to depress dry matter intake and on-farm ration variability should be addressed before considering NO₃^- as an avenue for greater sustainability of greenhouse gas emissions in US dairy production.