Inhibiting methanogenesis by targeting thermodynamics and enzymatic reactions in mixed cultures of rumen microbes .

Mitigation of enteric methane (CH) emissions from ruminant livestock represents an opportunity to improve the sustainability, productivity, and profitability of beef and dairy production. Ruminal methanogenesis can be mitigated via two primary strategies: (1) alternative electron acceptors and (2) enzymatic inhibition of methanogenic pathways. The former utilizes the thermodynamic favorability of certain reactions such as nitrate/nitrite reduction to ammonia (NH) while the latter targets specific enzymes using structural analogs of CH and methanogenic cofactors such as bromochloromethane (BCM). In this study, we investigated the effects of four additives and their combinations on CH production by rumen microbes in batch culture. Sodium nitrate (NaNO), sodium sulfate (NaSO), and 3-nitro-1-propionate (3NPA) were included as thermodynamic inhibitors, whereas BCM was included as a enzymatic inhibitor. Individual additives were evaluated at three levels of inclusion in experiments 1 and 2. Highest level of each additive was used to determine the combined effect of NaNO + NaSO (NS), NS + 3NPA (NSP), and NSP + BCM (NSPB) in experiments 3 and 4. Experimental diets were high, medium, and low forage diets (HF, MF, and LF, respectively) and consisted of alfalfa hay and a concentrate mix formulated to obtain the following forage to concentrate ratios: 70:30, 50:50, and 30:70, respectively. Diets with additives were placed in fermentation culture bottles and incubated in a water bath (39°C) for 6, 12, or 24h. Microbial DNA was extracted for 16S rRNA and ITS gene amplicon sequencing. In experiments 1 and 2, CH concentrations in control cultures decreased in the order of LF, MF, and HF diets, whereas in experiments 3 and 4, CH was highest in MF diet followed by HF and LF diets. Culture pH and NH in the control decreased in the order of HF, MF, to LF as expected. NaNO decreased ( < 0.001) CH and butyrate and increased acetate and propionate ( < 0.03 and 0.003, respectively). Cultures receiving NaNO had an enrichment of microorganisms capable of nitrate and nitrite reduction. 3NPA also decreased CH at 6h with no further decrease at 24 h ( < 0.001). BCM significantly inhibited methanogenesis regardless of inclusion levels as well as in the presence of the thermodynamic inhibitors ( < 0.001) while enriching succinate producers and assimilators as well as propionate producers ( < 0.05). However, individual inclusion of BCM decreased total short chain fatty acid (SCFA) concentrations ( < 0.002). Inhibition of methanogenesis with BCM individually and in combination with the other additives increased gaseous H concentrations ( < 0.001 individually and 0.028 in combination) while decreasing acetate to propionate ratio ( < 0.001). Only the cultures treated with BCM in combination with other additives significantly (p < 0.05) decreased the abundance of expressed as log fold change. Overall, the combination of thermodynamic and enzymatic inhibitors presented a promising effect on ruminal fermentation , inhibiting methanogenesis while optimizing the other fermentation parameters such as pH, NH, and SCFAs. Here, we provide a proof of concept that the combination of an electron acceptor and a methane analog may be exploited to improve microbial efficiency via methanogenesis inhibition.