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Abstract: Mixotrophy can be exploited to support algal growth over night or in dark-zones of a photobioreactor. In order to achieve the maximal productivity, however, it is fundamental also to provide CO2 in excess to maximize photosynthetic activity and phototropic biomass production. The aim of this paper is to verify the possibility of exploiting mixotrophy in combination with excess CO2. Two species with high biomass productivity were selected, Nannochloropsis salina and Chlorella protothecoides. Different organic substrates available at industrial scale were tested, and glycerol chosen for its ability to support growth of both species. In mixotrophic conditions, excess CO2 stimulated photosynthesis but blocked the metabolization of the organic substrate, thus canceling the advantages of mixotrophy. By cultivating microalgae under day–night cycle, organic substrate supported growth during the night, but only if CO2 supply was not provided. This represents thus a possible method to reconcile CO2 stimulation of photosynthesis with mixotrophy. [Copyright &y& Elsevier]