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The aim of this work was to evaluate biomethane production by anaerobic digestion using aqueous phase from Fast Pyrolysis of Douglas Fir Wood as substrate. The effect of biomass acid treatment and condensation temperature on the bio-oil chemical composition and aqueous phase separation with a fractional condensation system coupled to Auger reactor during pyrolysis at 500 °C was reported. As the first condenser temperature was augmented from 40 and 80 °C, the bio-oil yield obtained decreased from 30 to 16 wt% and the aqueous phase yield in the second condenser (kept at 25 °C) increased from 27 till 37 wt%. When the untreated biomass was pyrolyzed, the aqueous phase collected in the second condenser (at the first condenser operated at 80 °C) was anaerobically digested at 100 ppm for 400 h results in 31.3 NmL of CH4/batch. However, the best result was attained after washed biomass with acetic acid (10%) previously to fast pyrolysis, obtaining 86.8 NmL of CH4/batch using just 10 ppm, perhaps due to halved of formed hydroxyacetaldehyde and estimated levoglucosan content increases by more than three times at this condition. Thus, the attained results confirmed the viability of the adopted strategy to improve the anaerobic digestion of the aqueous phase obtained by fractional condensation systems for biomethane production. [ABSTRACT FROM AUTHOR]