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The feasibility of utilising spent mushroom substrates (SMSs) as a growing medium component for Pleurotus ostreatus cultivation was investigated. P. ostreatus was cultivated on traditional wheat straw (control) and wheat straw substrate supplemented with SMSs from Pholiota nameko (N-SMS), Hypsizygus marmoreus (M-SMS), and Hericium erinaceus (E-SMS) in varying supplementation rates (10%, 20%, and 30%). The yield, biological efficiency (BE), dry matter, and protein content of P. ostreatus fruiting bodies grown on ten substrates were evaluated. Significant differences in yield, BE, protein content, and dry matter of P. ostreatus were found among the studied substrates. The highest yield was recorded in 20% E-SMS (254.33 g), 20% N-SMS (253.43 g), and 10% E-SMS (251.67 g). The biological efficiency ranged from 66.48% (30% M-SMS) to 72.67% (20% E-SMS) and followed a similar trend to yield. The highest protein content was recorded in 30% M-SMS (29.93 g∙100 g dry weight−1). The highest dry matter of P. ostreatus was noticed in 30% of M-SMS (23.74 g) and 10% of M-SMS (23.06 g). Therefore, the spent mushroom substrates of Ph. nameko, H. marmoreus, and H. erinaceus could be used as a potential, low-cost sustainable alternative (10–30%) and as a renewable component of traditional growing media for P. ostreatus cultivation.

The Utilisation of Pholiota nameko, Hypsizygus marmoreus, and Hericium erinaceus Spent Mushroom Substrates in Pleurotus ostreatus Cultivation