Citation: Onu Olughu, O.; Tabil, L.G.; Dumonceaux, T.; Mupondwa, E.; Cree, D. Optimization of Solid-State Fermentation of Switchgrass Using White-Rot Fungi for Biofuel Production. Fuels 2022, 3, 730–752. https://doi.org/10.3390/ fuels3040043 Academic Editors: Gaetano Zuccaro and Neha Arora Received: 3 October 2022 Accepted: 11 November 2022 Published: 6 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Optimization of Solid-State Fermentation of Switchgrass Using White-Rot Fungi for Biofuel Production Onu Onu Olughu 1 , Lope G. Tabil 1, * , Tim Dumonceaux 2 , Edmund Mupondwa 1,2 and Duncan Cree 3 1 Department of Chemical and Biological Engineering, University of Saskatchewan, Saskatoon, SK S7N 5A9, Canada 2 Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre, 107 Science Place, Saskatoon, SK S7N 0X2, Canada 3 Department of Mechanical Engineering, University of Saskatchewan, Saskatoon, SK S7N 5A9, Canada * Correspondence: lope.tabil@usask.ca Abstract: Biological delignification using white-rot fungi is a possible approach in the pretreatment of lignocellulosic biomass. Despite the considerable promise of this low-input, environmentally-friendly pretreatment strategy, its large-scale application is still limited. Therefore, understanding the best combination of factors which affect biological pretreatment and its impact on enzymatic hydrolysis is essential for its commercialization. The present study was conducted to evaluate the impact of fungal pretreatment on the enzymatic digestibility of switchgrass under solid-state fermentation (SSF) using Phanerochaete chrysosporium (PC), Trametes versicolor 52J (Tv 52J), and a mutant strain of Trametes versicolor that is cellobiose dehydrogenase-deficient (Tv m4D). Response surface methodology and analysis of variance (ANOVA) were employed to ascertain the optimum pretreatment conditions and the effects of pretreatment factors on delignification, cellulose loss, and total available carbohydrate (TAC). Pretreatment with Tv m4D gave the highest TAC (73.4%), while the highest delignification (23.6%) was observed in the PC-treated sample. Fermentation temperature significantly affected the response variables for the wild-type fungal strains, while fermentation time was the main significant factor for Tv m4D. The result of enzymatic hydrolysis with fungus-treated switchgrass at optimum pretreatment conditions showed that pretreatment with the white-rot fungi enhanced enzymatic digestibility with wild-type T. versicolor (52J)-treated switchgrass, yielding approximately 64.9% and 74% more total reducing sugar before and after densification, respectively, than the untreated switchgrass sample. Pretreatment using PC and Tv 52J at low severity positively contributed to enzymatic digestibility but resulted in switchgrass pellets with low unit density and tensile strength compared to the pellets from the untreated switchgrass. Keywords: fungal pretreatment; enzymatic digestibility; delignification; white rot fungi; cellulose loss 1. Introduction The potential of dedicated energy crops as suitable feedstock for producing cellulosic ethanol has attracted increasing attention because of their high yield, low costs, decreased environmental impacts, and the lack of competition for fertile lands between energy crops and food crops [1]. Out of the many energy crops which have been studied, Wright [2] recommended further development of switchgrass as a high-potential dedicated energy crop, which has led to extensive research on its utilization and enhancement [3,4]. This development has made switchgrass a promising feedstock for cellulosic ethanol production. However, the enzymatic digestibility of lignocellulosic materials to produce sugars that can be fermented into ethanol and other platform chemicals is generally low. Studies have shown that only about 20% of the theoretical maximum yield of lignocellulose to fermentable sugar can be obtained via enzymatic hydrolysis without a pretreatment step [5, 6]. The structural integrity of lignocellulose, mainly due to the presence of lignin, limits Fuels 2022, 3, 730–752. https://doi.org/10.3390/fuels3040043 https://www.mdpi.com/journal/fuels