Some microalgae are able to grow not only on light but also in darkness, utilizing organic substrates as the sole source of carbon and energy. Such microalgae are called facultative heterotrophic. In nature, however, the use of both trophic modes, i.e. photoautotrophy and heterotrophy in parallel, by certain microalgal species is far more common (mixotrophy). Among ten thousands of known microalgal species, only few are known to be facultative heterotrophic or mixotrophic, some of them having a biotechnological potential (Chlorella spp., Galdieria sulphuraria, Crypthecodinium cohnii, Haematococcus pluvialis). Heterotrophic or mixotrophic cultivations of microalgae have recently attracted attention due to many advantages: cultures grown in bioreactors are independent of weather and location, their growth is continuous up to high biomass densities, and the products are very clean. Such cultivation is, however, sensitive to contamination by alien microorganisms and demands nutrient solution enriched with expensive organic substances, e.g. glucose or acetate. Therefore, by-product substrates such as glycerol, lactose and molasses, are prospective to reduce production costs. Another obstacle to a larger use of heterotrophic/mixotrophic cultivation is the low number of known facultative heterotrophic microalgal. Some microalgae are able to grow not only on light but also in darkness, utilizing organic substrates as the sole source of carbon and energy. Such microalgae are called facultativestrains and lack of detailed information about them. Hence, we screened about 40 less-studied strains for their heterotrophy in the BIORAF project during last years. Several facultative heterotrophic species were newly identified (Botryosphaerella sudetica, Bracteacoccus sp., Dictyosphaerium spp., Lemmermannia sp., Parachlorella kessleri). Mixotrophic and photoautotrophic production of specific metabolites, carotenoids, EPA and exopolysaccharides were compared in Scenedesmus sp., Tribonema aequale and Dictyosphaerium chlorelloides, respectively. T. aequale showed significantly higher production of EPA under the mixotrophic cultivation mode than photoautotrophically. The ease of harvest by filtration is an additional benefit of this filamentous species.