Fermentation Quality Divergence of Alfalfa Silage within the Same Batch: Insights from 16S rRNA Sequencing and Untargeted Metabolomics

Abstract Alfalfa silage produced from the same batch of raw material frequently exhibits divergent fermentation quality, yet the microbial and metabolic basis of this divergence remains poorly understood. This study aimed to characterize the differences in bacterial community and metabolite profiles between relatively well- and poorly fermented alfalfa silages within the same batch. A total of 100 laboratory-scale silos of wilted alfalfa were prepared and ensiled for 60 days. Based on the pH value and lactic acid (LA) concentration, 49 silos were classified as the relatively well-fermented group (LPGood), 51 silos as the poorly fermented group (LPBad), and six silos were randomly selected from each group for further analysis. The LPGood group showed significantly (p < 0.05) lower pH and higher LA and acetic acid (AA) concentrations than the LPBad group, whereas no significant (p > 0.05) differences were detected in dry matter content or ammonia nitrogen. No significant (p > 0.05) differences in bacterial α diversity were observed between the two groups; however, the genus Lactiplantibacillus was the predominant microorganism in both groups and was significantly enriched in the LPGood group. Metabolomic analysis identified 231 significantly upregulated and 133 significantly downregulated metabolites in the LPGood group relative to the LPBad group (VIP > 1.0, FDR-adjusted q < 0.05), with the LPGood group significantly (p < 0.05) enriched in palmitic acid, oleamide, linoleic acid, succinic acid, and phenylacetic acid. In conclusion, the fermentation quality divergence of alfalfa silage within the same batch was closely associated with the bacterial community composition and metabolic reprogramming, and Lactiplantibacillus, together with its associated metabolic pathways, may serve as potential indicators for monitoring and regulating the fermentation quality of alfalfa silage.