Multi-omics insights into the regulatory mechanism of citric acid in silage fermentation

A meta-analysis was conducted to assess the effects of citric acid (CA) on silage fermentation, and then used whole-plant cassava silage as a model to explore the underlying microbiological mechanisms with metagenomic and metabolomic data. The meta-analysis revealed that CA supplementation increased the dry matter, crude protein, water-soluble carbohydrate, and lactic acid contents in silage, but decreased the pH, dry matter loss, and the contents of fiber, NH₃-N, and acetic acid, all of which meet the expectations for an ideal silage additive. The fermentation parameter responses of whole-plant cassava silage to CA were consistent with those in the meta-analysis. Metabolomic analysis revealed that CA increased the level of antimicrobial metabolites and decreased the level of amino acids and their derivatives in cassava silage. By constructing microbial genome and gene catalogs, we found that CA supplementation increased the abundance of lactic acid-rods (Levilactobacillus, Lentilactobacillus, and Companillactobacillus) and inhibited the abundance of lactic acid cocci (Leuconostoc, Pediococcus, and Weissella) and undesirable bacteria (Acinetobacter, Serratia, Klebsiella, and Pantoea), which resulted in an increased abundance of genes involved in structural carbohydrate hydrolysis (cellulase and pectinase), lactic acid production (ldh), and amino acid synthesis (CKase and CPS1) and a decreased abundance of genes involved in acetate (porA, acs, pdhC, and pct) and NH₃ production (glsA). Additionally, CA reduced the abundance of antibiotic resistance genes in silage by inhibiting the bacteria that hosted more resistance genes. Accordingly, CA supplementation could improve the nutritional value, preservation, and biosafety of silage by regulating its microbial composition and function.