The pre-treatment of microalgae cell walls is known to be a key factor to enhance methane (CH4) yields during anaerobic digestion. This study investigated the combined effects of two different biomass storage methods and physical pre-treatments on the anaerobic digestion for three different microalgae species. Acutodesmus obliquus, Chlorella vulgaris and Chlorella emersonii were cultivated in 80 L sleevebag photobioreactors (batch mode), and then subjected to different storage (cooling and freezing) and pretreatment methods prior to anaerobic digestion using the biochemical methane potential (BMP) test. A. obliquus was selected to evaluate pre-treatment methods for further experimentation. Significantly higher CH4 yields of cooled (4 degrees C) A. obliquus biomass were achieved through ultrasonication (+53% CH4) and wet-milling (+51% CH4). These methods were then applied in follow-up experiments to cooled (4 degrees C) biomass of C. emersonii and A. obliquus. Ultrasonication again led to significantly higher CH4 yields for A. obliquus biomass (323 dm(3) kg(-1) CH4 yield calculated at standard gas conditions of 273 K, and 101.5 kPa per unit volatile solids, +41% CH4), and C. emersonii biomass (308 dm(3) kg(-1); +35% CH4). In a third experiment series, frozen A. obliquus and C. vulgaris biomass were thawed prior to pre-treatment and BMP-testing. Among all BMP tests, the highest CH4 yields were achieved with untreated, freeze-thawed C. vulgaris biomass (406 dm(3) kg(-1)); pre-treatment did not enhance CH4 yields for C. vulgaris, but for A. obliquus (ultrasonication +20%). Pre-treatment was more effective for cooled than freeze-thawed microalgal biomass and combined effects acted strain dependently. (C) 2016 Elsevier Ltd. All rights reserved.