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Consumption of mushrooms has recently increased due to their health benefits; consequently, increased mushroom cultivation generates large volumes of spent mushroom substrate (SMS) and effective methods for SMS valorization are thus required. Anaerobic digestion (AD) processes SMS with minimal energy and reduces the amount of waste generated; moreover, it contributes to energy generation through biogas production. To improve the energy efficiency of AD and promote sufficient biomass pretreatment, thermal pretreatment conditions require further investigation. Here, we evaluated the pretreatment efficiency and biogas production of the SMS thermal pretreatment process, studying different pretreatment temperatures to understand the formation of SMS degradation products and the changes in AD efficiency. Particularly, mechanical and hydrothermal pretreatment (HTP) methods were employed to improve SMS biodegradability. By increasing the substrate solubilization efficiency, HTP increased the biogas yield more effectively than mechanical pretreatment. Additionally, HTP improved the substrate's physicochemical properties and increased the reactive surface area for microorganisms by changing the substrate morphology. Further, the biodegradability of the hydrothermally pretreated SMS was higher (87.46%) than that of the mechanically pretreated SMS (61.37%). Thus, SMS could be employed in biogas production and HTP play a key role in improving the biogas yield during SMS processing. [ABSTRACT FROM AUTHOR]