Using PICRUSt2 to explore the functional potential of bacterial community in alfalfa silage harvested at different growth stages

Abstract

Abstract Background This study evaluated the effects of growth stage and storage time on fermentation characteristics, bacterial communities and their functionality in alfalfa (Medicago sativa L.) silage. Alfalfa was harvested at initial flowering (10–20% bloom, AL1) and full flowering (> 80% bloom, AL2) stages, respectively. The harvested alfalfa was ensiled in 15 L plastic silos. Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of ensiling, respectively. Fermentation products were analyzed on each sampling day. The bacterial communities and their functional potential after 3 and 60 days were analyzed by high-throughput sequencing technique and PICRUSt2 method. Results AL2 had better fermentation quality than AL1 with lower pH, ammonia nitrogen and butyric acid concentrations and higher lactic acid concentrations on day 60. AL2 had higher abundances of Weissella and Lactobacillus after 3 days, and lower abundances of Enterobacter and Enterobacteriaceae on day 60 compared to AL1. In metabolic pathway analysis, ensiling promoted the carbohydrate and amino acid metabolism, and inhibited the signal transduction and membrane transport. In enzyme analysis, AL2 had lower abundances of nitrite reductase (NADH) and ornithine decarboxylase than AL1 on day 60. In phenotype analysis, AL2 had higher proportions of facultatively anaerobic and lower proportions of anaerobic, potentially pathogenic and gram negative than AL1 on day 60. Conclusions High throughput sequencing technique combined with PICRUSt2 can be successfully used to describe the changes of bacterial communities and their functionality in silage. This approach can improve our understanding of the silage microbiology to further regulate the fermentation products. Graphical Abstract