Ruminal Fermentation of Anti-Methanogenic Nitrate- and Nitro-Containing Forages In Vitro

Abstract

Nitrate, 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) can accumulate in forages and be poisonous to animals if consumed in high enough amounts. These chemicals are also recognized as potent anti-methanogenic compounds, but plants naturally containing these chemicals have been studied little in this regard. Presently, we found that nitrate-, NPA-, or NPOH-containing forages effectively decreased methane production, by 35–87%, during in vitro fermentation by mixed cultures of ruminal microbes compared to fermentation by cultures incubated similarly with alfalfa. Methane production was further decreased during the incubation of mixed cultures also inoculated with Denitrobacterium detoxificans, a ruminal bacterium known to metabolize nitrate, NPA, and NPOH. Inhibition of methanogens within the mixed cultures was greatest with the NPA- and NPOH-containing forages. Hydrogen accumulated in all the mixed cultures incubated with forages containing nitrate, NPA or NPOH and was dramatically higher, exceeding 40 μmol hydrogen/mL, in mixed cultures incubated with NPA-containing forage but not inoculated with D. detoxificans. This possibly reflects the inhibition of hydrogenase-catalyzed uptake of hydrogen produced via conversion of 50 μmol added formate per milliliter to hydrogen. Accumulations of volatile fatty acids revealed compensatory changes in fermentation in mixed cultures incubated with the nitrate-, NPA-, and NPOH-containing forages as evidenced by lower accumulations of acetate, and in some cases, higher accumulations of butyrate and lower accumulations of ammonia, iso-buytrate, and iso-valerate compared to cultures incubated with alfalfa. Results reveal that nitrate, NPA, and NPOH that accumulate naturally in forages can be made available within ruminal incubations to inhibit methanogenesis. Further research is warranted to determine if diets can be formulated with nitrate-, NPA-, and NPOH-containing forages to achieve efficacious mitigation in ruminant methane emissions without adversely affecting fermentative efficiency or risking toxicity to animals.

Long-Term Encapsulated Nitrate Supplementation Modulates Rumen Microbial Diversity and Rumen Fermentation to Reduce Methane Emission in Grazing Steers

“…The reduction in the proportion of ruminal acetate 12 h after supplementation and higher butyrate concentrations by ENS is in line with results from trials of mixed cultures of rumen microbes incubated with nitrate and nitro-compounds (Zhou et al, 2012; Mamvura et al, 2014; Anderson et al, 2016). It may be a compensatory route for the dispensing of reducing equivalents during the inhibition of rumen methanogenesis, shifting electron transfer mainly to more reduced fatty acids such as propionate and butyrate, as this subsequently results in less acetate accumulation (Van Nevel and Demeyer, 1996).…”

The rumen microbiome inhibits methane formation through dietary choline supplementation

“…Furthermore, D. detoxificans gains energy by oxidizing nitrogenous compounds such as nitroethane, 2-nitroalcohol and 3-nitro-1-propionate, using trimethylamine from choline metabolism as electron acceptor 38 . These nitrogenous compounds can be accumulated in forages, in particular legumes such as the alfalfa used in the basal diet in the present study and be made available within the rumen 39 . The N compounds mentioned are known to act as methanogen inhibitors 40 , and thus may have contributed to the CH 4 inhibition observed in the present study.…”

Inhibited Methanogenesis in the Rumen of Cattle: Microbial Metabolism in Response to Supplemental 3-Nitrooxypropanol and Nitrate

“…Ruminal conditions that control the favorability of NO -2 reduction may determine the occurrence of the H 2 sink mechanism and the methanogenic inhibition by NO -2 mechanism. A mixed culture in vitro experiment by Anderson et al (2016) indicated a decreased acetate to propionate ratio and an increased headspace p H 2 in response to increased NO -3 supplementation, whereas these changes were impaired when the mixed culture was also inoculated with Denitrobacterium detoxificans, despite a more pronounced decrease of headspace CH 4 partial pressure. This inoculation may have stimulated the reduction of NO -2 and alleviated methanogenic inhibition and H 2 accumulation, and next affected the production of the different VFA.…”