Dry matter content and inoculant alter the metabolome and bacterial community of alfalfa ensiled at high temperature.

Alfalfa silage fermentation quality, metabolome, bacterial interactions, and successions as well as their predicted metabolic pathways were explored under different dry matter contents (DM) and lactic acid bacteria (LAB) inoculations. Silages were prepared from alfalfa with DM contents of 304 (LDM) and 433 (HDM) g/kg fresh weight and inoculated with Lactiplantibacillus plantarum (L. plantarum, LP), Pediococcus pentosaceus (P. pentosaceus, PP), or sterile water (control). The silages were stored at a simulated hot climate condition (35°C) and sampled at 0, 7, 14, 30, and 60 days of fermentation. The results revealed that HDM significantly improved the alfalfa silage quality and altered microbial community composition. The GC-TOF-MS analysis discovered 200 metabolites in both LDM and HDM alfalfa silage, mainly consisting of amino acids, carbohydrates, fatty acids, and alcohols. Compared with LP and control, PP-inoculated silages had increased concentrations of lactic acid (P < 0.05) and essential amino acids (threonine and tryptophan) as well as decreased pH, putrescine content, and amino acid metabolism. However, alfalfa silage inoculated with LP had higher proteolytic activities than control and PP-inoculated silage, as revealed by a higher concentration of ammonia nitrogen (NH-N), and also upregulated amino acid and energy metabolism. HDM content and P. pentosaceus inoculation significantly altered the composition of alfalfa silage microbiota from 7 to 60 days of ensiling. Conclusively, these results indicated that inoculation with PP exhibited great potential in enhancing the fermentation of silage with LDM and HDM via altering the microbiome and metabolome of the ensiled alfalfa, which could help in understanding and improving the ensiling practices under hot climate conditions. KEY POINTS: • HDM improved fermentation quality and declined putrescine content of alfalfa silage • P. pentosaceus inoculation enhanced the fermentation quality of alfalfa silage • P. pentosaceus is an ideal inoculant for alfalfa silage under high temperature.