Harnessing spent mushroom substrate for bioethanol: Optimizing pretreatment, hydrolysis, and fermentation with mass–energy balance

Abstract

Spent mushroom substrate (SMS) contains substantial lignocellulosic carbohydrates and nutrients. This study developed an SMS-to-bioethanol route, combining mild alkaline pretreatment, enzymatic hydrolysis, and Saccharomyces cerevisiae fermentation without external nutrients, supported by mass–energy balance. Oyster mushroom (Pleurotus ostreatus) SMS (air-dried to ~10% moisture; milled to <5 mm) contained ~38–40% cellulose, 18–20% hemicellulose, 14–16% lignin, ~5% protein, and ~10% ash. Pretreatment with 1% (w/v) NaOH at 121°C for 30 min (10% solids), followed by washing to neutral pH, reduced lignin from ~15% to ~5% and hemicellulose from ~19% to ~10%, enriching cellulose to ~60%. Enzymatic hydrolysis (5% solids, 5 FPU g⁻¹ cellulase, 50°C, pH 4.8) achieved ~72% glucan-to-glucose conversion by 72 h, yielding ~25–30 g L⁻¹ glucose. Fermentation at 32°C produced ~30±1 g L⁻¹ ethanol within 72 h, with glucose depletion (<0.5 g L⁻¹), ~0.49 g g⁻¹ ethanol per glucose (~96% theoretical), indicating adequate nitrogen/minerals from fungal biomass. Per 100 kg dry SMS, the process yielded ~16 kg ethanol (~20 L) and ~15 kg CO₂, leaving ~64 kg residual solids. Energy analysis showed favorable net energy when residues supply process heat, with ethanol energy exceeding inputs by 20–30%. Coupling mushroom cultivation with ethanol production advances circular bioeconomy while enabling energy recovery from residues; future work should target scale-up and C5 sugar utilization.