Microwave alkali pretreated wheat straw was used for in-house enzyme production by Aspergillus flavus and Trichoderma reesei. Produced enzymes were concentrated, pooled and assessed for the hydrolysis of pretreated wheat straw. Factors affecting hydrolysis were screened out by Placket-Burman design (PBD) and most significant factors were further optimized by Box-Behnken design (BBD). Under optimum conditions, 82% efficiency in hydrolysis yield was observed. After the optimization by response surface methodology (RSM), a model was proposed to predict the optimum value confirmed by the experimental results. The concentrated enzymatic hydrolyzate was fermented for ethanol production by Saccharomyces cerevisiae, Pichia stipitis and co-culture of both. The yield of ethanol was found to be 0.48 g(p)/g(s), 0.43 g(p)/g(s) and 0.40 g(p)/g(s) by S. cerevisiae, P. stipitis and by co-culture, respectively, using concentrated enzymatic hydrolyzate. During anaerobic fermentation 42.31 mu mol/mL, 36.69 mu mol/mL, 43.35 mu mol/mL CO2 was released by S. cerevisiae, P. stipitis and by co-culture, respectively. (c) 2011 Elsevier Ltd. All rights reserved.