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**Methane Emissions Regulated by Microbial Community Response to the Addition of Monensin and Fumarate in Different Substrates**  
**[PERSON] 1,2,*()**, [PERSON] 1,2,3,* and [PERSON] 4  
Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration Bio-Diversity Conservation, Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China  
Zogie Peatland and Global Change Research Station, Chinese Academy of Sciences, Hongyuan 624400, China  
CAS Center for Excellence in Tibetan Plateau Earth Sciences, Chinese Academy of Sciences, Beijing 100101, China  
Sichuan Academy of Grassland Sciences, Chengdu 611731, China; [EMAIL_ADDRESS]  
Correspondence: [EMAIL_ADDRESS] (D.X.); [EMAIL_ADDRESS] (H.C.)  
**Featured Application: Runnian livestock contributes significantly to global methane (CH\({}_{4}\)) emissions. The CH\({}_{4}\) emissions from ruminants in the Qinghai-Tibet Plateau (QTP) are an important part of China's animal methane emissions. This study evaluated the in vitro effects of CH\({}_{4}\) mitigation additives with different mechanisms and their potential interactions with compounds regularly used in yak feed. Basal substrates (forage and vinasse) and methane mitigation additives (monensin sodium salt (MSS) and disodium fumarate (DF)) have interactions with the in vitro rumen fermentation and microbial composition. The different microbial population was critical to the variations in methane production. Comprehensive analyses of rumen microbiome may help further understand the diet-inhibitor interactions in mitigating methane emissions from ruminants in QTP.**  
Runinants contribute significantly to global methane (CH\({}_{4}\)) emissions. This study aimed to evaluate the in vitro effects of monensin sodium salt (MSS) and disodium fumarate (DF) on CH\({}_{4}\) production, rumen fermentation, and microbial community, with different substrates. The addition of MSS and DF, alone and in combination, significantly reduced the concentration and production of CH\({}_{4}\) (\(p<0.05\)), and while with vinasse as substrate, the CH\({}_{4}\) production was higher for forage. The highest propionate production and lowest acetate and propionic ratio (A:P) values were all observed in cultures added to the combination of 14 mmol/L DF and 80 mg/kg MSS in both substrates, suggesting that these additives improved the rumen fermentation efficiency. The diversity indexes of prokaryotic microbiota with forage as the substrate were significantly higher than vinasse, and there were different effects on diversity indexes with the addition of MSS and DF depending on the incubated substrate. Supplementation with MSS and DF increased the number of starch degradation and fumarate reducing bacteria, decreased the number of methanogens, but had no significant effect on the number of fibrotic bacteria. pH, NH\({}_{3}\)-N, and rumen volatile fatty acids (VFA) were the main factors influencing prokaryotic community structure. In conclusion, basal substrates (forage and vinasse) and CH\({}_{4}\) mitigation additives (MSS and DF) have interactions on the in vitro rumen fermentation and microbial composition.  
monensin; disodium fumarate; in vitro; rumen fermentation; microbial community