Bioactive metabolites in Hypsizygus marmoreus spent substrate enhanced Pleurotus ostreatus growth

The rapid accumulation of spent mushroom substrate (SMS) presents a critical environmental challenge globally, but efficient and cost-effective recycling approaches remain limited. Here, to advance valorization of SMS, a proof-of-concept study was conducted in which Hypsizygus marmoreus SMS ( Hm SMS) was recycled in the cultivation of Pleurotus ostreatus . Impacts of the Hm SMS-derived bioactive metabolites were further examined systematically. Results showed that integration of 10 %–30 % Hm SMS shortened the harvest time by 2–6 days, significantly improved fruiting body differentiation and productivity of P. ostreatus . The Hm SMS-derived water-soluble and volatile metabolites significantly enhanced P. ostreatus growth, albeit through different metabolic pathways: the former act by reprogramming of the amino acid-related metabolisms, whereas the latter act by upregulating purine metabolism, nucleotide metabolism, and the biosynthesis of phenylalanine, tyrosine and tryptophan. Chemical profiles of SMS were comprehensively investigated using a combined multi-omics approach. The Water-soluble metabolites within Hm SMS comprised primarily lipids and lipid-like molecules (26.8 %; LC–MS) and organic acids and derivatives (31.9 %; GC–MS). Volatile metabolites in Hm SMS were primarily lipids and lipid-like molecules (33.6 %; GC–MS). Five of the examined water-soluble metabolites (fulvic acid, dl-arabinose, erythritol, l-valine and l-isoleucine) significantly promoted P. ostreatus productivity. These findings highlight the untapped potential of SMS-derived bioactive metabolites and imply their potential high-value valorization in forestry and agricultural practices.