Bacterial Diversity, Chemical Composition, and Fermentation Quality of Alfalfa-Based Total Mixed Ration Silage Inoculated with Lactobacillus reuteri and Lentilactobacillus buchneri

This study examined the effects of lactic acid bacteria (LAB) from extremely cold environments on the bacterial diversity, chemical composition, and fermentation quality of alfalfa-based TMR silage. The TMR was treated with Lactobacillus reuteri A4-2 (Lr A4-2) and Lentilactobacillus buchneri 9-2 (Lb 9-2) at an application rate of 1.0 × 105 cfu/g fresh material, respectively, and the control received the same volume of distilled water. The TMR was ensiled for 7, 15, 30, 60, and 210 days. The Lr A4-2 treatment produced higher lactic acid (62.53 g/kg DM at 210 days) and maintained a diverse bacterial community throughout the ensiling compared with the control and Lb 9-2 treatment. The Lb 9-2 treatment increased acetic acid (51.42 g/kg DM at 210 days) and formed a distinct bacterial community profile. The 16S rRNA sequencing revealed a shift from initial Weissella dominance to Lactobacillus-dominated communities across the treatments by day 210. Alpha diversity indices decreased over time, with Lr A4-2 treatment maintaining higher diversity. Principal coordinate analysis demonstrated significant temporal shifts in bacterial community composition among treatments (p < 0.01). The results suggest that different heterofermentative LAB strains modulated the fermentation and microbial balance in alfalfa-based TMR silage in different ways.