Rumen-Derived Consortia Shaped by Substrate-Specific Enrichment Show Specialized Lignocellulose Utilization, Diversified Hydrogen Metabolism, and Cryopreservation Stability

Efficient utilization of lignocellulosic biomass by the rumen microbiome is critical for improving feed efficiency in ruminants, yet the development of stable, functionally specialized microbial consortia remains limited. This study aimed to assemble substrate-adapted rumen microbial consortia using an ecology-guided enrichment approach. Rumen fluid collected from cannulated Angus × Hereford heifers was sequentially enriched over 10 generations on four substrates with distinct cell wall characteristics: alfalfa, barley straw, carboxymethyl cellulose (CMC), and xylan. Fermentation parameters, including gas production and volatile fatty acids (VFAs), and bacterial community dynamics were analyzed, and selected consortia (alfalfa and xylan) were evaluated for stability following one month of cryopreservation. Across enrichments, total VFA concentrations declined (e.g., xylan: 109.8 mM (G0) to 56.37 mM (G10)), accompanied by reduced gas production and decreased alpha diversity, indicating substrate-driven selection. Distinct functional profiles emerged, including increased propionate in alfalfa consortia, higher acetate in barley straw, lactate–propionate cross-feeding with CMC, and caproate production (6.3 mM at G10) in xylan enrichments associated with Caproiciproducens and Megasphaera. Cryopreserved consortia retained core community structure and fermentation characteristics upon revival. These results demonstrate that substrate-driven enrichment can generate stable, functionally specialized rumen consortia and provide a framework for developing ecologically compatible microbial communities with potential applications in improving rumen fermentation efficiency.