Impact of inoculants on alfalfa silage: a multi-omics analysis reveals microbial community and metabolic shifts despite undesirable fermentation quality

Lactic acid bacteria are extensively utilized in silage production to enhance fermentation quality, yet their effects remain variable. This study investigated the potential role and underlying mechanisms of inoculants in silage fermentation through microbiome and metabolome analyses, despite suboptimal fermentation outcomes. Alfalfa was wilted to 300 g/kg (LDM) and 430 g/kg (HDM) fresh weight and ensiled either without inoculation (CON) or with Lactiplantibacillus plantarum (LP) or Pediococcus pentosaceus (PP). After 60 days of fermentation at 20°C, samples were assessed using integrated microbiome sequencing and metabolome profiling alongside conventional fermentation parameters. All treatments exhibited elevated pH values (> 5.00), particularly under LDM condition, indicating inadequate fermentation. Although LP and PP inoculation resulted in lower pH values, they had no impact on proteolysis or nutritional preservation. Microbiome analysis revealed distinct bacterial community structures, with CON and PP-treated silages dominated by Weissella and Pediococcus, whereas LP-inoculated silage was characterized by high levels of Lactiplantibacillus abundance and substantial proportions of Hafnia-Obesumbacterium and Enterobacter, irrespective of DM content. LP inoculation enhanced amino acid and energy metabolism within bacterial communities while suppressing carbohydrate and nucleotide metabolism. Metabolome analysis confirmed significant metabolic shifts between DM contents; at LDM, LP-inoculated silage exhibited reduced lactulose and lyxose concentrations, while at LDM, LP and PP treatments increased tyrosine levels but decreased N-Methyl-DL-alanine and N(alpha), N(alpha)-dimethyl-L-histidine concentrations. These findings demonstrate that bacterial inoculants profoundly influence microbial composition and metabolic pathways in silage, even in the absence of substantial improvements in conventional fermentation metrics. The integration of microbiome and metabolome analyses provides novel insights into the complex mechanisms of inoculant-mediated silage fermentation.