Inclusion of Hybrid Pennisetum and Probiotics Enhanced Anaerobic Fermentation Quality and Bacterial Diversity of Alfalfa Silage

This study aims to assess the impact of Bacillus subtilis (BS) and Lactobacillus buchneri (LB) on the fermentation quality, microbial communities, and predicted metabolic pathways in mixed silage made from alfalfa and hybrid Pennisetum . We prepared mixed silage from fresh alfalfa and hybrid Pennisetum in a 1:1 ratio and inoculated it with BS, LB, or a combination of both (BSLB) or left it untreated as a control. The silage was fermented for 30 and 60 days. The results showed that inoculation with BS, LB, or their combination increased the lactic acid and crude-protein content while reducing the fiber content compared to the control. Additionally, BS and LB inoculation raised ( p < 0.05) the acetic acid content, and the combination of both strains increased ( p < 0.05) the ratio of lactic acid to acetic acid. LB alone and the combined inoculation also increased the relative abundance of Lactobacillus during the pre-silage period. Functional analysis through the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed considerable variations among the different probiotic treatments. The silage process reduced nucleotide metabolism but enhanced carbohydrate, amino acid, energy, cofactor, and vitamin nucleotide metabolism. High-throughput sequencing combined with KEGG functional prediction demonstrated significant differences in community composition and functional changes at 30 and 60 days of fermentation. These findings enhance our understanding of bacterial communities and functional changes in mixed silage of alfalfa and hybrid Pennisetum , offering valuable insights into the fermentation mechanisms of legume and grass silage and informing practices for producing high-quality mixed silage.