Three ligno-cellulosic substrates representing varying levels of biodegradability (giant reed, GR; fibre sorghum, FS; barley straw, BS) were combined with mild alkaline pre-treatments (NaOH 0.05, 0.10 and 0.15 N at 25 degrees C for 24 h) plus untreated controls, to study pretreatment effects on physical-chemical structure, anaerobic digestibility and methane output of the three substrates. In a batch anaerobic digestion (AD) assay (58 days; 35 degrees C; 4 g VS l(-1)), the most recalcitrant substrate (GR) staged the highest increase in cumulative methane yield: +30% with NaOH 0.15 N over 190 ml CH4 g(-1) VS in untreated GR. Conversely, the least recalcitrant substrate (FS) exhibited the lowest gain (+10% over 248 ml CH4 g(-1) VS), while an intermediate behaviour was shown by BS (+15% over 232 ml CH4 g(-1) VS). Pre-treatments speeded AD kinetics and reduced technical digestion time (i.e., the time needed to achieve 80% methane potential), which are the premises for increased production capacity of full scale AD plants. Fibre components (cellulose, hemicellulose and acid insoluble lignin determined after acid hydrolysis) and substrate structure (Fourier transform infra-red spectroscopy and scanning electron microscopy) outlined reductions of the three fibre components after pre-treatments, supporting claims of loosened binding of lignin with cellulose and hemicellulose. Hence, mild alkaline pre-treatments were shown to improve the biodegradability of ligno-cellulosic substrates to an extent proportional to their recalcitrance. In turn, this contributes to mitigate the food vs. fuel controversy raised by the use of whole plant cereals (namely, maize) as feedstocks for biogas production. (C) 2014 Elsevier Ltd. All rights reserved.