Second-generation ethanol process uses lignocellulose, but a pre-treatment is required to degrade lignin before saccharification. Biological pre-treatment, using phenoloxidases, represents a cheap and eco-friendly option. Mould contamination can be overcome by heat pre-conditioning of substrates. Pasteurisation can be a mild and sustainable option. A 5-level response-surface experimental design was performed to test the effect of different time and temperatures on lignocellulolytic activities and substrate dephenolisation. Substrate microbial communities were characterised via catabolic profiles to determine functional diversity changes after pasteurisation. Temperatures ranging from 70 to 75 degrees C and time from 5 to 10 h led to the highest laccase activities and 30% of substrate dephenolisation was achieved while avoiding mould contamination. The per cent of O-Alkyl (polysaccharides) did not vary significantly, meaning that polysaccharides were not extensively consumed. Microbial communities were less catabolically diversified (H' = 1.2) but more active (AWCD = 0.9) and could degrade polysaccharides. (C) 2020 Published by Elsevier Ltd.