Metagenomics reveals rumen residues as a superior inoculum for volatile fatty acid production in vitro.

Rumen microbiome is widely recognized as an efficient system for lignocellulose degradation. Rumen fluid (RF) has been often used as the inoculum in previous study of bioprocesses, however, the potential of rumen solids (RS) to enhance volatile fatty acid (VFA) production remains underexplored. The anaerobic fermentation performance of RS, RF, and RF + RS mixture as inocula was compared using corn stover as substrate, with RS addition at 5%, 10%, or 20% (w/w) to corn stover and RF addition at 1:2 (v/v) to buffer solution. At a corn stover content of 2.5% (w/v, based on final working volume), the highest VFA concentration of 10.05 g/L was achieved with 20% RS as inoculum, outperforming those with both RF and RF + RS. Metagenomic analysis revealed significant differences in bacterial, fungal, and archaeal community structures with 20% RS and RF. With 20% RS, hydrolytic bacteria (e.g., Enterobacter) dominated the ecological niche, microbial co-occurrence network analysis of the 2,000 most abundant genera revealed a simpler network with fewer negative associations, and functional analysis demonstrated a notable increase in relative abundance of glycosyltransferase (GT) families within carbohydrate-active enzymes (CAZymes). With 20% RS, enrichment in glycolysis-related genes was observed, indicating a preference for carbohydrate degradation, while acid-producing pathways were enriched such as pyruvate metabolism with RF. Rumen solids, as a superior inoculum for VFA production, effectively enhanced lignocellulose bioconversion by enriching specific low-abundance microbial taxa, forming a microbial network with fewer negative associations, increasing the relative abundance of GT families, and strengthening the systemic hydrolysis capacity.