BackgroundSugarcane trash (SCT) represents up to 18% of the aboveground biomass of sugarcane, surpassing 28 million tons globally per year. The majority of SCT is burning in the fields. Hence, efficient use of SCT is necessary to reduce carbon dioxide emissions and global warming and establish agro-industrial biorefineries. Apart from its low costs, conversion of whole biomass with high production efficiency and titer yield is mandatory for effective biorefinery systems. Therefore, in this study, we developed a simple, integrated method involving a single step of glycerolysis pretreatment to produce antiviral glycerolysis lignin (AGL). Subsequently, we co-fermented glycerol with hydrolyzed glucose and xylose to yield high titers of bioethanol.ResultsSCT was subjected to pretreatment with microwave acidic glycerolysis with 50% aqueous (aq.) glycerol (MAG50); this pretreatment was optimized across different temperature ranges, acid concentrations, and reaction times. The optimized MAG50(opMAG50) of SCT at 1:15 (w/v) in 1% H2SO4,360 µM AlK(SO4)2at 140 °C for 30 min (opMAG50) recovered the highest amount of total sugars and the lowest amount of furfural byproducts. FollowingopMAG50, the soluble fraction, i.e., glycerol xylose-rich solution (GXRS), was separated by filtration. A residual pulp was then washed with acetone, recovering 7.9% of the dry weight (27% of lignin) as an AGL. AGL strongly inhibited the replication of encephalomyocarditis virus (EMCV) in L929 cells without cytotoxicity. The pulp was then saccharified in yeast peptone medium by cellulase to produce a glucose concentration similar to the theoretical yield. The total xylose and arabinose recoveries were 69% and 93%, respectively. GXRS and saccharified sugars were combined and co-fermented through mixed cultures of two metabolically engineeredSaccharomyces cerevisiaestrains: glycerol-fermenting yeast (SK-FGG4) and xylose-fermenting yeast (SK-N2). By co-fermenting glycerol and xylose with glucose, the ethanol titer yield increased to 78.7 g/L (10% v/v ethanol), with a 96% conversion efficiency.ConclusionThe integration of AGL production with the co-fermentation of glycerol, hydrolyzed glucose, and xylose to produce a high titer of bioethanol paves an avenue for the use of surplus glycerol from the biodiesel industry for the efficient utilization of SCT and other lignocellulosic biomasses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02333-z.