Metagenomics analysis reveals the performance of homo- and heterofermentative lactic acid bacteria in alfalfa silage fermentation, bacterial community, and functional profiles.

Alfalfa (Medicago sativa L.) is a kind of roughage frequently utilized as an animal feed but challenging to be ensiled due to its low water-soluble carbohydrate (WSC), high water content, and elevated buffering capacity, thus requiring the application of lactic acid bacteria (LAB) to improve its fermentation. This study employed high-throughput metagenomic sequence technology to reveal the effects of homofermentative LAB, Lactobacillus plantarum (Lp), or Pediococcus pentosaceus (Pp), and heterofermentative LAB, L. buchneri (Lb), or their combinations (LbLp or LbPp) (applied at 1.0 × 109 colony forming units (cfu) per kilogram of alfalfa biomass fresh material) on the fermentation, microbial community, and functional profiles of alfalfa silage after 7, 14, 30, and 60 ensiling days. The results indicated a reduction (P < 0.05) in glucose and pH and higher (P < 0.05) beneficial organic acid contents, xylose, crude protein, ammonia nitrogen, and aerobic stability in Lb-, LbPp-, and LbLp-inoculated alfalfa silages after 30 and 60 d. Also, higher (P < 0.05) WSC contents were recorded in LbLp-inoculated alfalfa silages after 30 d (10.84 g/kg dry matter [DM]) and 60 d (10.92 g/kg DM). Besides, LbLp-inoculated alfalfa silages recorded higher (P < 0.05) LAB count (9.92 log10 cfu/g) after 60 d. Furthermore, a positive correlation was found between the combined LAB inoculants in LbLp-inoculated alfalfa silages and dominant LAB genera, Lactobacillus and Pediococcus, with fermentation properties after 30 and 60 d. In addition, the 16S rRNA gene-predicted functional analyses further showed that the L. buchneri PC-C1 and L. plantarum YC1-1-4B combination improved carbohydrate metabolism and facilitated further degradation of polysaccharides in alfalfa after 60 d of ensiling. These findings reveal the significant performance of L. buchneri and L. plantarum in combination with dominant LAB species in suppressing the growth of Clostridia, molds, and yeasts and improving the fermentation characteristics and functional carbohydrate metabolism of alfalfa after 60 d ensiling, thus suggesting the need for further studies to uncover the diverse performance of the LAB combination and their consortium with other natural and artificial inoculants in various kinds of silages.