The anaerobic digestion of three microalgae mixtures was evaluated at different substrate to inoculum (S/I) ratios (0.5, 1 and 3), biomass concentrations (3, 10 and 20 gTS/kg) and pretreatments (thermal hydrolysis, ultrasound and biological treatment). An S/I ratio of 0.5 and 10 gTS/kg resulted in the highest final methane productivities regardless of the microalgae tested (ranging from 188 to 395 mL CH4/gVS(added)). The biological pretreatment supported negligible enhancements on CH4 productivity, while the highest increase (46-62%) was achieved for the thermal hydrolysis. The optimum temperature of this pretreatment depended on the microalgae species. The ultrasound pretreatment brought about increases in CH4 productivity ranging from 6% to 24% at 10,000 kJ/kgTS, without further increases at higher energy inputs. The results here obtained confirmed the lack of correlation between the solubilization degree and the methane enhancement potential and pointed out that anaerobic digestion of algae after thermal pretreatment is a promising technology for renewable energy production. (C) 2012 Elsevier Ltd. All rights reserved.