Populous deltoides wood is a suitable raw material for ethanol production, since it has high cellulose content. Efficient degradation and subsequent conversion of lignocellulosic biomass to bio-ethanol, requires physico-chemical pretreatments followed by enzymatic hydrolysis to obtain high overall yields of sugars. Microbial hydrolytic enzymes are actively utilized for biomass degradation and bio-ethanol production. Four glycosyl hydrolases (GHs) namely; three subunits of cellulase, endoglucanase (ThWF5-endoglucanase), exoglucanase (ThWF5-exoglucanase) and b-glucosidase (ThWF5-glucosidase), and xylanase (ThWF5-xylanase), from fungal strain Trichoderma harzianum WF5-isolated from decayed wood, were pulled out and subsequently characterized. Analysis of full length sequences and prediction of secondary and tertiary structure showed that these enzymes shared homology with the respective enzymes of other Trichoderma species and have comparatively evolved structure to express catalytic activity under broad range of environmental conditions. Respective GHs were partially purified and used for the enzymatic hydrolysis of P. deltoids wood after pretreatments. The pretreatment of P. deltoides wood with sulfuric acid and hydrogen peroxide (H2SO4_H2O2) at 121 degrees C for 60 min was determined as the best condition to high recovery of reducing sugars after enzymatic hydrolysis. The overall high ethanol yield of 15.6 g/l and fermentation efficiency of 61.1% was obtained when fermentation was performed on pretreated poplar wood slurry followed by detoxification with calcium hydroxide. High bio-ethanol production from P. deltoides wood and molecular characterization of glycosyl hydrolases used, provide insights that this wood offers a potential source for biofuel production while T. harzianum WF5 is a treasure house of robust degrading enzymes. (C) 2015 Elsevier Ltd. All rights reserved.