Spent mushroom substrate substitutes silkworm pupae powder in the industrial cultivation of Cordyceps militaris: Agronomic validation, mechanism, and cost implications

The industrial cultivation of the medicinal mushroom Cordyceps militaris relies heavily on expensive animal-derived supplements such as silkworm pupae powder, creating economic and sustainability challenges. This study evaluated spent mushroom substrates (SMSs) from seven saprophytic species as alternative cultivation inputs. Among them, Hypsizygus marmoreus -SMS proved the most effective, increasing fresh fruiting body weight by 35.33% and matching the yield-promoting performance of silkworm pupae powder. To identify the key growth-enhancing factor, we integrated targeted metabolomic profiling and exogenous validation assays. Malic acid, highly enriched in H. marmoreus -SMS, was identified as a growth-associated organic acid; exogenous malic acid supplementation increased fresh weight by 20.97%. Spatiotemporal transcriptomic analysis further revealed that malic acid induced a stage-specific transcriptional response, prominently at the primordium stage, marked by coordinated remodeling of central carbon metabolism, including the TCA cycle, gluconeogenesis, the pentose phosphate pathway, and one-carbon metabolism. These metabolic shifts suggest an enhanced supply of precursors for fruiting body development. Our findings establish H. marmoreus -SMS not merely as an agro-industrial by-product repurposed as a nutrient source, but as a dual-function input that combines basal nutrition with malic acid-mediated developmental reprogramming. This work provides a mechanistic basis for replacing animal-derived supplements with valorized SMS in circular mushroom production.