Abstract
The improved aerobic stability of corn silage inoculated with heterofermentative lactic acid bacteria (LAB) has traditionally been attributed to acetic acid production, although the mechanisms involved likely extend beyond acetic acid production. The combined effects of delayed sealing on microbial community dynamics, volatile organic compound (VOC) production, and aerobic stability remain poorly understood. This study evaluated the effects of sealing time (immediate vs. 24-h delayed sealing) and two commercial mixture of Lentilactobacillus buchneri and L. hilgardii inocula on the fermentation characteristics, nutritive value, microbial communities, VOC profiles, and aerobic stability of whole-crop corn silage. Delayed sealing markedly affected silage preservation. Exposure to oxygen before sealing increased silage temperature by up to 14°C, increased dry matter losses, depleted water-soluble carbohydrates, and substantially modified the volatile profile through the accumulation of lipoxygenase-derived compounds, short-chain alcohols, organic acids, esters, and aromatic metabolites. Despite the reduced availability of fermentable substrates, lactic acid fermentation remained dominant, and delayed silages exhibited a numerical improvement of aerobic stability after silo opening. Amplicon-based microbiome analyses revealed that delayed sealing primarily altered fungal community structure, explaining 39% of the observed variance and promoting genera such as Candida and Nakaseomyces. In contrast, heterofermentative LAB inoculation mainly affected bacterial communities by enriching specific Lactobacillus amplicon sequence variants and reducing taxa, including Weissella, Leuconostoc, Serratia, and members of the Enterobacteriaceae. These microbial shifts were accompanied by lower yeast populations, improved fiber digestibility, and numerically greater aerobic stability. Notably, acetic acid concentration was not strictly associated with aerobic stability, whereas several VOCs showed significant correlations with stability, suggesting that volatile metabolites contribute to silage preservation beyond the effects traditionally attributed to acetic acid. These findings demonstrate that corn silage aerobic stability is jointly regulated by substrate availability, microbial succession, and volatile metabolite production. The integration of quantitative volatilomics and amplicon-based microbiome analyses provides new mechanistic insights into silage preservation and offers a valuable framework for optimizing sealing management and the application of commercial heterofermentative LAB inoculants.