Efficient biohydrogen production from lignocellulosic hydrolysate assumes considerable practical and academic importance. The impact of glucose to xylose ratio, medium pH, inoculum size and age on biohydrogen production indicated that glucose to xylose ratio is the critical parameter for effective H(2) production compared to either pure glucose or xylose as carbon source. Inoculum size and its age contributed more than 70% to overall H(2) production and revealed significance at individual as well as interactive level. Maximum interaction of 39% and 32% was noticed with inoculum size and its age vs. glucose to xylose ratio (2:3), respectively. The H(2) production yield enhanced from 140 to 357 ml/g substrate upon statistical optimization revealing >240% improvement. (C) 2010 Elsevier Ltd. All rights reserved.