Polyhydroxyalkanoates (PHAs) are promising alternatives that accumulate as energy and carbon storage material in various microorganisms, including bacteria and microalgae, being biodegradable and suitable for a wide variety of applications. Among these compounds, the most prevalent and well-characterized biopolymer is polyhydroxybutyrate (PHB), which belongs to the short-chain PHAs.The present study was designed to evaluate algae-based PHB production in two Chlorophyta (Desmodesmus communis and Chlorella vulgaris) under a two-phase nutritional mode of cultivation, namely a phototrophic growth phase (PGP) and a mixotrophic stress phase (MSP) with N,P-depleted media and organic carbon supply (i. e., glucose or sodium acetate, NaOAc). The highest PHB productivity (0.11 g PHB/g biomass/d; 0.015 g PHB/L/ d), corresponding to 32.1 % w/w of intracellular PHB, was observed for D. communis after 3 days of cultivation under mixotrophic conditions in batch cultures (e.g., low light, phosphorus-free medium, 1 g/L of NaOAc). A scaled-up cultivation (10 L) was set up to evaluate for the first time PHB yields and biomass composition in a semi-continuous system. A PHB content of 34 % w/w was achieved on day 8, corresponding to a maximum PHB productivity of 0.10 g PHB/g biomass/d (or 0.011 g PHB/L/d), which increased up to 54 % w/w on day 15. The biomass was composed of about 30 % w/w proteins, 6 % w/w polysaccharides, and 11 % w/w lipids, which can be valorised from a biorefinery perspective. The scaled-up D. communis cultivation in 10 L PBRs confirmed the potential utilization of this algal species for PHB production with productivity up to 2-times higher than those reported for several cyanobacterial species and similar to the maximum value obtained with batch cultures in previous works performed with Scenedesmaceae.