Combinations of nitrate, saponin, and sulfate additively reduce methane production by rumen cultures in vitro while not adversely affecting feed digestion, fermentation or microbial communities

•Nitrate–sulfate–saponin combination additively inhibited methanogenesis (by 46%).•This combination did not adversely affect feed digestion or fermentation.•This combination also reduced abundances of protozoa, but not methanogens.•This combination increased abundances of select cellulolytic bacterial populations.•Combinations of the compounds altered communities of archaea and bacteria.

This study investigated the effects of saponin (0.6 g/L), nitrate (5 mM) and sulfate (5 mM), alone and in combinations, on methanogenesis, rumen fermentation, microbial community, and abundances of select microbial populations using in vitro rumen culture. Combinations of nitrate with saponin and/or sulfate additively suppressed methane production, with the lowest reduction (nearly 46%) observed for the combination of all the three inhibitors. None of the treatments adversely affected feed digestion or rumen fermentation. All the inhibitors, either alone or in combinations, did not alter the abundances of total bacteria, Ruminococcus albus, or archaea. However, saponin, alone and together with nitrate and/or sulfate, increased the abundance of Fibrobacter succinogenes and Ruminococcus flavefaciens, but decreased that of protozoa. DGGE analyses revealed limited changes in both bacterial and archaeal communities by the treatments. The nitrate–saponin–sulfate combination may be an effective and practical strategy to mitigate methane emission from ruminants.