| Summary: | The study aimed to determine the effectiveness of docosahexaenoic acid (DHA) production by <i>Schizochytrium</i> sp. biomass fed with waste glycerol depending on the concentration of extracellular polymeric substances (EPS) in the culture medium and medium aeration effectiveness. The microalgae from the genus <i>Schizochytrium</i> sp. were proved to be capable of producing EPS composed of glucose, galactose, mannose, fucose, and xylose. The highest EPS concentration, reaching 8.73 ± 0.09 g/dm<sup>3</sup>, was determined at the stationary growth phase. A high EPS concentration caused culture medium viscosity to increase, contributing to diminished oxygen availability for cells, lower culture effectiveness, and reduced waste glycerol conversion to DHA. The <i>Schizochytrium</i> sp. culture variant found optimal in terms of the obtained technological effects and operating costs was performed at the volumetric oxygen mass transfer coefficient of k<sub>L</sub>a = 600 1/h, which enabled obtaining dry cell weight (DCW) of 147.89 ± 4.77 g/dm<sup>3</sup>, lipid concentration of 69.44 ± 0.76 g/dm<sup>3</sup>, and DHA concentration in the biomass reaching 29.44 ± 0.36 g/dm<sup>3</sup>. The effectiveness of waste glycerol consumption in this variant reached 3.76 ± 0.31 g/dm<sup>3</sup>·h and 3.16 ± 0.22 g/gDCW.
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