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Heterotrophic cultivation of Euglena gracilis was carried out on synthetic (Hutner medium) and complex cultivation media in order to optimize production of β-1,3-glucan (paramylon). For preparation of complex media, various industrial by-products (e.g., molasses, corn steep solid, yeast extract, and beef extract) were used with or without addition of pure compounds [glucose, galactose, fructose, lactose, maltose, sucrose, and (NH)HPO]. Heterotrophic cultivation of E. gracilis was performed in Erlenmeyer flasks and additionally confirmed during research in the stirred tank bioreactor. The results clearly show that E. gracilis can easily metabolize glucose and fructose as carbon sources and corn steep solid as complex nitrogen and growth factors source for biomass growth and paramylon synthesis. Furthermore, it was also proved that addition of (NH)HPO, beef extract, or gibberellic acid did not have positive effect on the biomass growth and paramylon synthesis. After optimization of complex medium composition and verification in the stirred tank bioreactor, it was concluded that medium composed of glucose (20 g/L) and corn steep solid (30 g/L) is the most suitable complex medium for industrial cultivation of E. gracilis and paramylon production. [ABSTRACT FROM AUTHOR]