Metatranscriptomic changes in the propionate pathway and carbohydrate enzyme revealed the rebalancing mechanism of rumen microbiota after CH4 inhibition by 3-NOP

Background Inhibition of methanogen by 3-NOP will produce excess hydrogen gas, which affects the nutrient digestion and fermentation functions of rumen microbiota. Certain hydrogenotrophic microorganisms contribute to a fraction of the hydrogen sink via processes such as propionate production, acetogenesis, and anion reduction. However, these mechanisms alone are insufficient to fully account for the sustained methane generation and the decline in hydrogen gas levels. A more comprehensive understanding is needed on the rebalancing process of rumen microbiota, the temporal dynamics and microbial drivers of 3-NOP resistance. Results We opted for a fully-automated fermentation system in vitro to closely monitor the process by which 3-NOP inhibits methane production. At the onset of net hydrogen consumption (24h), we collected fermentation fluid samples (n = 12) for metatranscriptomic sequencing, along with conducting a quantitative analysis of key microorganisms. The inhibition experiments of pure culture were also conducted on three different nutritional types of methane bacteria. 3-NOP significantly reduced the transcripts of the methane metabolism pathway, but significantly increased the transcripts of two propionate pathways(lactate and succinate). Based on annotating gene sets and mapping the transcriptomic reads to the assembled genome database, the transcripts of Methanomassiliicoccales and a portion of Methanobrevibacter were significantly elevated. Analysis of carbohydrate enzymes showed that 3-NOP significantly increased the transcription of cellulase in fungi ( Neocallimastigaceae ) and Fibrobacter succinogenes , but significantly reduced the transcription of Ruminococcus flavefaciens and Ruminococcus albus . Methanogens that form symbiotic relationships with fungi ( Neocallimastigaceae ) remain unaffected by 3-NOP through a coupled mechanism involving cellulose degradation and hydrogenosome activity in the rumen. These adaptive strategies of methanogenesis—non-hydrogenotrophic and the symbiotic partnership with rumen fungi—enable methanogens to attenuate the inhibitory effects of 3-NOP, potentially leading to resistance following prolonged exposure. Conclusion These findings underscore the need for caution in the sustained use of 3-NOP as a standalone methane mitigation strategy and highlight critical targets for developing next-generation inhibitors.