Xylose is a by-product of lignocellulosic biomass processing for production of second-generation biofuels and could be suitable for bioproduct manufacturing. This paper describes an innovative approach that enables the system to achieve high yielding for hydrogen production. The study compared 4 physico-chemical pre-treatments performed in an anaerobic mixed culture (acidic, thermal, acidic-thermal and thermal acidic) to achieve an inoculum with a high-efficiency xylose to hydrogen conversion under mesophilic conditions (30 degrees C). The acidic pre-treatment was the most efficient to select microorganisms able to produce hydrogen and volatile acid from xylose. Kinetics has shown that acidic pre-treatment had a hydrogen/xylose molar yielding factor of 1.57 (molar base) and a hydrogen maximum production rate of 253 mL H-2 h(-1). Mass balance considered all possible metabolic pathways using xylose as a substrate. Anaerobic degradation of ethanol was the most active pathway for hydrogen production in all experiments, except for the control. Each pre-treatment performed for the original inoculum resulted in different microbiological profiles, but the genus Clostridium was the most abundant in all assays. Acidic pre-treatment stimulated the growth of organisms from the genera Peptostreptococcaceae, Truepera and Kurthia, which could be related to the better results in hydrogen production found in this condition. (C) 2019 Elsevier Ltd. All rights reserved.