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Livestock manure, traditionally used just as fertilizer, can be energetically valued to produce biogas as an attractive alternative, since nowadays, energy production and its cost stands for a pressing problem around the world. Nevertheless, the presence of lignin in manure hinders the production of methane. This could be improved by pre-treating the manure with ligninolytic fungi, able to break lignin and therefore facilitate the hydrolysis step for the hydrolytic bacteria, yielding higher volumes of biomethane. Three strategies of incubation with living fungi of genus Pleurotus were evaluated to enhance methane production from livestock manure mixed with bedding material: short term (two weeks 2L container) and long term (two months 400 L container) and 24 h (2 L container) with a crude water extraction of Pleurotus extracellular enzymes. The positive effect of the fungal treatment was observed in the three strategies obtaining an increase in methane production with respect to the control manure of 7% at short term, 111% at long term and 173% (crude enzymatic extract). Consequently, the strategy of using crude enzyme extracts from Pleurotus to improve hydrolysis step as pre-treatment of manure should be considered as a novel, easy, cheap and promising tool to optimize methane production. [Display omitted] • Short and long-term pre-treatments of manure with Pleurotus were assessed. • Manure storage in deep closed container enhanced biogas production. • Manure with crude water extracts from Pleurotus yielded significant higher biogas. • Crude enzyme extracts from Pleurotus, a novel, easy, cheap, promising pre-treatment. [ABSTRACT FROM AUTHOR]