Improving silage fermentation of distilled grain waste-based spent mushroom substrate using indigenous lactic acid bacteria: metagenomic insights.

Abstract

Background: Rising global mushroom production calls for recycling spent mushroom substrate (SMS). However, its high moisture content, susceptibility to spoilage, and low abundance of indigenous lactic acid bacteria (LAB) limit its use as silage. In this study, an effective silage inoculation strategy was developed to transform SMS derived from composted distilled grain waste (DGW) and colonized by Pleurotus ostreatus.

Results: Three indigenous lactic acid bacteria, including Limosilactobacillus fermentum L2, Weissella paramesenteroides W1, and Pediococcus acidilactici P1, were isolated and selected for their rapid growth and acidification capacity, and applied individually or in combination at different stages of fungal colonization. Compared with a commercial inoculant, all indigenous lactic acid bacteria (LAB) treatments, especially a combination of L. fermentum L2 and P. acidilactici P1, significantly enhanced silage fermentation, as evidenced by lower pH (4.04-4.73), 31-fold and 1.87-fold increases in lactic acid and acetic acid production, respectively, improved crude protein retention, and preserved dry matter contents. KEGG enrichment analysis demonstrated that carbon metabolic flux was markedly redirected toward lactic acid fermentation, while pathways related to protein degradation and biosafety were significantly suppressed in LAB-treated SMS silage. Weighted metabolic analysis revealed that LAB contributed most to fermentative processes, thereby enriching Pediococcus, Weissella, and Lactobacillus, whereas the contributions of non-LAB and potentially harmful microorganisms were substantially reduced.

Conclusion: These results indicate that indigenous LAB enhance the ensiling efficiency of SMS by driving functional reallocation and pathway-level dominance, thereby improving fermentation efficiency, stability, and nutritional quality, highlighting their potential for promoting SMS silage as a high-quality ruminant feed resource.