A novel sequential pretreatment strategy using biodiesel industry generated waste glycerol assisted transition metal and alkali pretreatment of sugarcane trash were developed for the production of bioethanol. Various process parameters affecting pretreatment as well as hydrolysis were optimized by adopting a Taguchi design. This novel method was found to be superior when compared to conventional pretreatment strategies like acid and alkali in removing hemicelluloses and lignin and the hydrolyzate is devoid of major fermentation inhibitors like organic acids and furfurals. Physico-chemical changes of the native and the pretreated biomass were evaluated by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) analysis. Under optimized hydrolysis conditions 0.796 g of reducing sugar (pentoses and hexoses) per g of dry biomass after saccharification was produced. Fermentation of the non-detoxified hydrolyzate using Saccharomyces cerevisiae produced 31.928 g of bioethanol per g of dry biomass with an efficiency of 78.89%. (C) 2015 Elsevier Ltd. All rights reserved.