## 4 Discussion  
### Methane Emissions Regulated by Microbial Community under Different Substrates  
Methane production is affected by the substrate, microbial communities, rumen pH, etc. [25]. Different substrates induce different conditions during rumen fermentation. [PERSON] et al. [26] found the production of methane was affected by substrates with different forage:concentrate ratios, as 0.3:0.7 (LF) was higher than forage 0.7:0.3 (HF). [PERSON] et al. [27] found that high substrate concentrations produced more CO\({}_{2}\) and CH\({}_{4}\) than low concentrations. [PERSON] et al. [28] reported that in vitro methane production was increased by decreasing the forage:concentrate (F:C) ratio. This study identified that substrate (F vs. V) had the most significant impact on methane production. Compared to F substrate, V had lower NDF and ADF, and higher CP and EE (Table S1), and methane production with V as the substrate was higher than with F. This may be due to the higher fermentation rates of high-concentrate substrates than those of high-forage ones. Enteric methane is formed during the microbial degradation of food in the rumen, and any variation in its production level relies on changes in the microbial ecosystem [29]. The diversity indexes of prokaryotic microbiota, with F as the substrate were significantly higher than V, and the microbial community structure (e.g., Euryarchaeota and Tenericutes) varied under the two substrates. [PERSON] et al. [30] revealed that feeding ruminants a high-concentrate diet changed the diversity and community structure of methanogens but did not affect the abundance of rumen methanogens. Other studies also found that the type of diet can significantly affect the abundance and community of methanogens [31,32], while [PERSON] and [PERSON] (2015) showed that substrate can cause changes in rumen microbial community in vitro fermentation systems, using the denaturing gradient gel electrophoresis (DGGE) profiles of bacterial and archaeal communities [33]. The structure and/or activity of the microbial community determines the methane production and, partially, the utilization of H\({}_{2}\)[34]. [PERSON] et al. [35] reported that the variable forage:concentrate ratios changed the abundances of cellulolytic bacteria, total fungi and counts of protozoa in the in vitro experiment, however did not change the abundance of methanogens and total bacteria. Consistent with the results of this study, the number of _R. flavefaciens, F. succinogenes_ and _R. albus_ were higher with F as substrate than V, but total bacteria, methanogenic archaea, ciliate protozoa and _S. ruminantstium_ were lower with F as substrate than with V. F can provide more material for fibrolytic bacteria growth. Therefore, the variation of rumen methane production may be due to the differences of rumen microbiome caused by different substrates. Effect of Methane Mitigation Additives on In Vitro Methane Production, Rumen fermentation, and Microbes  
Supplementation with MSS and DF, alone and in combination, enhanced propionate production compared to the control and thus decreased methane production and A:P (Table 2). For all substrates, the mean CH\({}_{4}\) production decreased by 6.8 and 14.0% for concentrations of 7 and 14 mmol/L DF, respectively, while MSS (40 and 80 mg/kg) reduced the methane production by 15.2 and 46.3%, respectively. The effects of MSS and DF on methane production were not consistent. [PERSON] et al. [36] reported that supplementation with fumarate to cultures that were fermenting hay powder and concentrate, significantly decreased methane production and increased propionate production. Some studies have also found that the addition of fumarate can decrease methane production during rumen fermentation [37,38]. [PERSON] et al. [39] reported that fumarate can also be decomposed into acetate stoichiometrically. The addition of fumarate decreased the ratio of acetate:propionate, but increased propionate and acetate production.