The greatest limitation to utilization of maize stover by ruminants as a feed is the high concentration of lignin, which limits fibre digestibility. However, ruminants can effectively utilize maize stover if its nutritive value is improved using white rot fungal species. This study was designed to determine optimal bio-physical conditions for mycelial growth and select the most ideal fungal species and pre-treatment options for improving nutritive value of maize stover. Four popular edible Pleurotus fungal species (viz. Pleurotus florida, Pleurotus ostreatus, Pleurotus sajor caju and Pleurotus pulmonarius) were subjected to varying temperatures, pH levels, hydrogen peroxide (H₂O₂) concentration and illumination to establish the extent of mycelial growth rate. Inclusion of H₂O₂ was used to determine optimal levels for preservation and prevention of contamination from other indigenous microbiota. Effects of pre-treatment options on chemical composition and nutritive value of maize stover were also examined. Mycelial growth rate of Pleurotus species on potato dextrose agar (PDA) varied (P < 0.05) with temperature, pH level and H₂O₂ concentration following a quadratic trend. Optimal temperature, pH and H₂O₂ concentration for mycelial growth on PDA were 25 °C, 5 and 0.01 mL/L, respectively. Under the different bio-physical conditions, P. sajor caju had the highest mycelia density and growth rate. Chemical composition of solid-state fermented maize stover differed (P < 0.05) among the Pleurotus species. Maize stover fermented with P. sajor caju had the highest crude protein (CP) of 86.6 g/kg DM, in-vitro dry matter digestibility (IVDMD) of 731 g/kg DM, in-vitro organic matter digestibility (IVOMD) of 670.4 g/kg DM and metabolizable energy (ME) of 10.0 MJ/kg DM but with the lowest lignin (sa) of 50 g/kg DM. At 25 °C, P. sajor caju had the highest mycelial growth rate on PDA and highest lignin (sa) breakdown in the maize stover substrate. It was, therefore, selected as the most ideal fungal species for improving nutritive value of maize stover. Pre-treatment of maize stover with Lactobacillus plantarum and molasses under anaerobic condition for 7 days before inoculation with P. sajor caju resulted into a substrate with the highest (P < 0.05) CP (96.6 g/kg DM), IVDMD (752.3 g/kg DM), IVOMD (687.2 g/kg DM) and ME (10.2 MJ/kg DM). However, neutral detergent fiber exclusive of residual ash (NDFom) and lignin (sa) fractions decreased (P < 0.05) as a result of subjecting maize stover to pre-treatment with L. plantarum and molasses prior to fermentation with P. sajor caju. Therefore, pre-treatment of maize stover with L. plantarum and molasses for 7 days prior to fermentation with P. sajor caju for 14 days in darkness at 25 °C offered the greatest potential for breaking the lignin barrier.

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