Effects of Bacillus coagulans and Lactobacillus plantarum on the Fermentation Characteristics, Microbial Community, and Functional Shifts during Alfalfa Silage Fermentation

SIMPLE SUMMARY: Dry matter loss (DM loss) occurs during silage production, with the potential to reach up to 4–20% due to the aerobic respiration. Lower water-soluble carbohydrates (WSC) and a higher buffering capacity can contribute to this; however, the presence of Bacillus can help create an anaerobic environment, which is more conducive to the growth of anaerobic bacteria. The objective of this study was to evaluate the potential of Bacillus coagulans (BC) as an inoculant for silage fermentation. The results showed that adding BC increased the fermentation quality of alfalfa silage, especially when applied together with Lactobacillus plantarum (LP). This was evidenced by a reduction in the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents, as well as an increase in Lactobacillus abundance and a decrease in Enterococcus abundance after 60 d of fermentation. Additionally, the application of LP, BC, and their combination stimulated cofactor and vitamin metabolism abundance, whilst suppressing drug resistance: antimicrobial pathway abundance. Thus, BC could be considered a viable bioresource for improving fermentation quality. ABSTRACT: This study aimed to investigate the potential of Bacillus coagulans (BC) as an inoculant in alfalfa silage fermentation. Fresh alfalfa was harvested at a dry matter (DM) content of 329.60 g/kg fresh weight (FW), and inoculated without (CON) or with BC (1 × 10(6) CFU/g FW), Lactobacillus plantarum (LP, 1 × 10(6) CFU/g FW), and their combinations (LP+BC, 1 × 10(6) CFU/g FW, respectively). Samples were taken at 3, 7, 14, 30, and 60 d, with three replicates for each. The prolonged ensiling period resulted in a decrease in pH values and an increase in lactic acid (LA) concentrations in alfalfa silages. After 60 d of fermentation, the application of BC and LP decreased the pH values and increased LA concentrations in treated silages, especially when their combination was applied. Application of BC preserved more water-soluble carbohydrates (WSC), and further application of BC increased WSC in LP+BC-treated silage compared to LP-treated silage. There was no significant difference in the crude protein (CP) content between the CON and treated silages, however, the BC and LP treatments reduced the ammonia nitrogen (NH(3)-N) concentration, especially when their combination was applied. Additionally, the BC and LP-treated silages had lower neutral detergent fiber (NDF) and acid detergent fiber (ADF) when compared to the CON silage (p < 0.001). Inoculants also increased Lactobacillus abundance and decreased Enterococcus abundance after 60 d of fermentation. Spearman’s rank correlation analysis revealed a positive correlation between LA concentration and Lactobacillus abundance. It was noteworthy that LP, BC, and their combination increased the relative abundances of carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism, decreasing the relative abundances of amino acid metabolism and drug resistance: antimicrobial. Therefore, the inclusion of BC increased the fermentation quality of alfalfa silage, with the optimal combination being LP+BC. According to the findings, BC could be considered a viable bioresource for improving fermentation quality.