Designing more efficient mixtures of enzymes is necessary to produce molecules of interest from biomass lignocellulosic fractionation. The present study aims to investigate the strategies used by the thermophilic and hemicellulolytic bacterium Thermobacillus xylanilyticus to fractionate wheat bran and wheat straw during its growth. Results demonstrated ratios and levels of hemicellulases produced varied during growth on both biomasses. Xylanase activity was mainly produced during stationary stages of growth whereas esterase and arabinosidase activities were detected earlier. This enzymatic profile is correlated with the expression pattern of genes encoding four hemicellulases (two xylanases, one arabinosidase and one esterase) produced by T. xylanilyticus during growth. Based on identification of the bacterial strategy, the synergistic efficiency of the four hemicellulases during the hydrolysis of both substrates was evaluated. The four hemicellulases worked together with high degree of synergy and released high amounts of xylose, arabinose and phenolic acids from wheat bran and wheat straw. (C) 2014 Elsevier Ltd. All rights reserved.