In the present investigation, sodium hydroxide (NaOH) and sulfuric acid (H2SO4) pretreatment were conducted using response surface methodology (RSM) technique for maximum cellulose exposure and delignification of banana peel (BP) biomass. H2SO4 pretreatment revealed higher cellulose exposure (585.2 mg/g of biomass) as compared to NaOH pretreatment. H2SO4 pre-treated banana peel (PBP) was further subjected to separate enzymatic hydrolysis and co-fermentation (SHCF) using L16 Taguchi orthogonal array design. SHCF demonstrated a maximum glucose and xylose yield of 677.3 and 165.1 mg/ g of PBP respectively. Further, higher butanol titers of 7.4 g/L were achieved from 20 g/L of sugar loadings by co-culture of Saccharomyces cerevisiae and Pichia sp. at 72 h of incubation time which was 89.1% and 75.67% higher as compared to monocultures of S cerevisiae and Pichia sp. respectively. Scale-up exper-iments with higher sugar loadings (90 g/L) demonstrated lower butanol concentrations of 15.7 g/L with total butanol productivity of 0.2 g/L/h. The overall energy consumption for 1 g of fuel-grade butanol from 20 g/L (shake flask) and 90 g/L (fermenter) of reducing sugar loadings was 138.1 KJ and 85.7 KJ respectively. (c) 2020 Elsevier Ltd. All rights reserved.