Effects of microbial inoculants, cellulase, and sodium diacetate on fermentation profiles, bacterial succession, and predicted functional profiles of alfalfa silage

Abstract

Alfalfa is a high-protein forage crop widely used in ruminant production, yet its ensiling is generally challenged by low water-soluble carbohydrate content and high buffering capacity. This study systematically evaluated the effects of microbial inoculants, cellulase, and sodium diacetate on fermentation quality, microbial succession, and predicted metabolic functions of alfalfa silage using fermentation analyses and 16S rRNA sequencing. Lactic acid was detected only in the microbial inoculant-treated silages on day 1, accompanied by a rapid decline in pH below 5.8 (p < 0.05). After 60 days, all microbial inoculants except BSM lowered pH below 4.5 and reduced ammonia nitrogen (NH₃-N) (p < 0.05), while promoting Lactiplantibacillus- or Pediococcus-dominated communities and suppressing Enterobacter and Serratia (p < 0.05). Predicted functional profiles suggested lower lysine degradation and enhanced lysine biosynthesis. Among inoculants, KL yielded the most favorable profile, with the highest Lactiplantibacillus abundance (78.23%), lower gas loss, Chao1, Shannon, and NH₃-N (4.35% total nitrogen (TN)). Cellulase improved fermentation quality compared with the control and displayed the lowest fiber content, but showed slower early acidification, resulting in higher NH₃-N (6.46% TN), gas loss, and Enterobacter abundance (2.94%) after 60 days (p < 0.05). Sodium diacetate followed a distinct pathway, producing acetic acid (1.32% dry matter (DM)) rather than lactic acid on day 1 and reducing pH to 5.58. After 60 days, sodium diacetate produced well-preserved silage with a lower pH (4.28), NH₃-N (3.72% TN), and gas loss, higher lactic acid concentration (6.93% DM) (p < 0.05), and Lactiplantibacillus-dominated bacterial community. These findings highlight that silage additives govern fermentation outcomes through distinct mechanisms, with the KL inoculant and sodium diacetate providing the most effective preservation strategies in this study.

Graphical abstract

Keywords

Microbial inoculants / Cellulase / Sodium diacetate / Microbial community / Predicted metabolic pathway

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Funding

Agriculture Science and Technology Development, Rural Development Administration, Republic of Korea.(RS-2021-RD010124)

RIGHTS & PERMISSIONS

The Author(s)