Anaerobic digestion of microalgal biomass is one option for sustainable energy production. Therefore, several microalgae i.e. Chlorella vulgaris, C. emersonii and Acutodesmus obliquus were produced in a sleevebag photobioreactor system (maximum volume 800 L) and investigated in so-called biochemical methane potential tests (BMP). Different pre-treatments were selected in order to enhance methane (CH4) yields. A. obliquus was selected to screen for effective pretreatment methods: a combined milling and enzyme approach (+57%), ultrasonication (+53%) and milling (+51%) resulted in significantly higher CH4 yields. In the follow-up experiments, milling and ultrasonication were applied to cooled biomass of C. emersonii and A. obliquus. In both cases, ultrasonication led again to significantly higher CH4 yields (+41%) for A. obliquus and (+35%) for C. emersonii. In experiments with frozen biomass, highest CH4 yields were reached with freeze-thawed C. vulgaris biomass i.e. 406 Nm³ t-1 VSharvest. Freeze-thawed A. obliquus biomass yielded 337 Nm³ t-1 VSharvest, an increase of 47% if compared to unfrozen biomass i.e. 229 Nm³ t-1 VSharvest. Ultrasonication of frozen A. obliquus biomass increased CH4 yields for only 20% indicating that freeze-thawing strongly affects CH4 yields and provokes cell disruption by ice crystals. In addition, our work proposes that pretreatments seem to act strain-specific. ; SeriesInformation ; Proceedings of the 23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria, pp. 612-615