Bacillus coagulans inoculation generates an alternative microbial succession trajectory associated with nitrogen preservation and aerobic stability in alfalfa silage

Background Microbial inoculants can redirect community succession during plant biomass fermentation, but how functionally distinct inoculants shape stability across anaerobic storage and aerobic exposure remains unclear. This study compared an uninoculated control with Lactiplantibacillus plantarum MTD/1 (LP), Bacillus coagulans (BC), and their combination (BL) during alfalfa ensiling. Results LP rapidly lowered pH and established Lactiplantibacillus dominance, whereas BC generated a staged succession involving Bacillus -, Pediococcus -, and Lactiplantibacillus -related taxa and maintained higher acetic acid concentrations. After 60 d, BC-treated silage had the highest crude protein and the lowest non-protein nitrogen and ammonia nitrogen concentrations. BC and BL extended aerobic stability to 133.5 h, compared with 105 h for LP and 40.5 h for the control. Shotgun metagenomics associated the BC trajectory with greater acetate-related functional potential and retention of selected nitrogen- and sulphur-related modules during anaerobic fermentation, followed by localized rather than broad functional changes after aerobic exposure. Conclusions These findings distinguish rapid dominance and acidification by LP from a staged, community-level trajectory associated with BC, identifying microbial succession as a design principle for improving nitrogen preservation and cross-stage stability in high-protein biomass fermentation.