BACKGROUND: Microalgae can synthesize starch with productivity higher than conventional terrestrial crops, without the need for arable land. However, little is known about processes to extract starch from microalgae. Here, a biorefinery process is described including microalgal cell disruption followed by extraction of starch and pigments with aqueous two-phase system (ATPS) using choline chloride and polypropylene glycol 400. Sonication and bead milling were compared for cell disruption rate and starch extraction efficiency.RESULTS: A first order kinetic model described well the cell disruption for both the methods, with a rate 2.6 times higher for bead milling than sonication. By applying ATPS on samples with comparable cell disruption (>93%), starch was separated better after sonication (67% recovery in the pellet) than after bead milling, for which it remained equally distributed between pellet (40%) and choline chloride phase. Pigments were extracted with 42-66% yield irrespective of the cell disruption method. Microalgal starch granules had a normal and narrow distribution for size (0.93 ± 0.14 ∼m) and a gelatinization temperature between 45-55 °C.CONCLUSION: For the same cell disruption yield, different starch separation efficiencies can be achieved, depending on the cell disruption method applied. Although bead milling was faster than sonication in disrupting cells, it gave worst starch separation efficiency. The properties of the extracted microalgal starch indicate potential technical advantages, with respect to conventional starch sources, for applications in the bioplastic and food sector.