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 [H(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 H 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 (-NO) versus with nitrate (+NO; 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 +NO. The main effect of +NO increased H(aq) by 11.0 µM (+117%) compared with -NO. The main effect of +NO also decreased daily CH production by 8.17 mmol CH/d (31%) compared with -NO. Because there were no treatment effects on neutral detergent fiber digestibility, the main effect of +NO also decreased CH production by 1.43 mmol of CH/g of neutral detergent fiber degraded compared with -NO. There were no effects of treatment on other nutrient digestibilities, N flow, or microbial N flow per gram of nutrient digested. The spike in H(aq) after feeding NO provides evidence that methanogenesis is inhibited by substrate access rather than concentration, regardless of defaunation, or by direct inhibition of NO. 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 NO to NH 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 NO as an avenue for greater sustainability of greenhouse gas emissions in US dairy production.