AbstractBioethanol production from lignocellulosic biomass is a viable option for improving energy security and reducing green house emissions. In the present studyProsopis juliflora, an invasive tree found through out India, with total carbohydrate content of 67.4 +/- 6% was selected as lignocellulosic feedstock for bioethanol production. The hydrolysate obtained after biphasic dilute acid hydrolysis contained initial sugar concentration of 18.70 +/- 0.16 g/L and hence to increase the ethanol yield it was concentrated to 33.59 +/- 0.52 g/L (about two-folds) by vacuum distillation. The concentration of sugars, phenols and furans was analyzed before and after concentration process. The concentrated hydrolysate was further detoxified by over liming, neutralization and charcoal treatment and later used for ethanol fermentation by mono- and co culture method. Monoculture ofSaccharomyces cerevisiae(VS3) andPichia stipitis(NCIM 3498) produced 8.52 +/- 0.43 and 4.52 +/- 0.23 g/L of ethanol with 66.21 +/- 3.26% and 60.46 +/- 3.02% of fermentation efficiency, 0.33 +/- 0.02 and 0.31 +/- 0.02 g/g of ethanol yield and 0.24 +/- 0.01 and 0.13 +/- 0.01 g/L/h of productivity, respectively. The co-culture ofS. cerevisiae(VS3) andP. stipitis(NCIM 3498) helped to improve all parameters i.e. 10.94 +/- 0.53 g/L of ethanol with fermentation efficiency, ethanol yield and productivity of 83.11 +/- 0.42%, 0.420 +/- 0.01 g/g and 0.30 +/- 0.01 g/L/h, respectively.