Methane formation is the final product of methanogenic archaea that use hydrogen or other substrates during the anaerobic fermentation of lignocellulosic biomass. Meanwhile, production and accumulation of VFA during incubation can reflect microbial activities and methanogenesis function. In the present study, the correlation matrix between the relative abundance of methanogen and fermentation parameters and methane production were examined by Spearman’s correlation heatmap at the species level (
Figure 6). The correlation analysis showed that
M. ruminantium was positively correlated with acetate, butyrate, total VFA concentration, and methane production when incubated at 6 or 24 h. Previous studies showed that
Methanobrevibacter is the most abundant rumen archaea, with sequences mainly being associated with
M. ruminantium (28%) and
M. gottschalkii (33.6%) [
27]. As the main hydrogen-utilizing methanogen, the abundance and activity of
M. ruminantium is significantly affected by the production of acetate and butyrate, which is accompanied by the hydrogen formation during incubation. In addition, based on the complete genomic sequence analysis of
M. ruminantium conducted by Leahy et al [
27] it was suggested that
M. ruminantium may be one of the protozoa-associated methanogens, and it can effectively obtain hydrogen for methanogenesis through hydrogen transfer from protozoa [
26]. Meanwhile, positive correlation was also observed between butyrate concentration and
Methanobrevibacter sp. D5 and
Methanobrevibacter sp.
SM9, which may be explained by the similar methanogenesis pathway and central metabolism to
M. ruminantium.
Methanosphaera sp.
ISO3-F5 was reported at a low relative abundance accounting for an average abundance of 8.2%±6.7% in the rumen of cattle and sheep [
26]. As one of the main methylotrophic methanogens,
Methanosphaera sp.
ISO3-F5 can utilize hydrogen to convert methanol into methane instead of reducing carbon dioxide to methane. In the present study, acetate was positively correlated with
Methanosphaera sp.
ISO3-F5, which would result from the increased availability of hydrogen when acetate formed during lignocellulosic degradation. Moreover, acetate concentration was positively related to the abundance of
Methanosarcina barkeri and
Methanosarcina Mazeiin in the present study, which was in agreement with the previous studies that acetate and other fermentation products (e.g., methanol and methylamines) were main substrates for the methane production by these two methanogen strains [
24]. In conclusion, this study demonstrated that ensiling and SE pretreatments have a great potential for effective degradation of
B. papyrifera. Compared with ensiling pretreatment, SE pretreatment decreased the ADF and NDF contents by 39.4% and 10.6%, respectively, after 72 h incubation. These results were closely related to the dynamic variations of methanogenic diversity and relative abundance. It is concluded that SE pretreatment may be a promising technique for the efficient utilization of
B. papyrifera, contributing to sustainable livestock production systems