Formulation of substrates based on three hardwood species combined with modulation of nitrogen content by whey addition (0–2%) was investigated in an experiment designed in D-optimal model for their effects on biological preproceesing of lignocellulosic feedstock by shiitake mushroom (Lentinula edodes) cultivation. Nitrogen loading was shown a more significant role than wood species for both mushroom production and lignocellulose degradation. The fastest mycelial colonisation occurred with no nitrogen supplementation, but the highest mushroom yields were achieved when 1% whey was added. Low nitrogen content resulted in increased delignification and minimal glucan consumption. Delignification was correlated with degradation of syringyl ligni...