In recent years, researchers have highlighted the role of low cost-efficient agro-industrial by-products used as supplements in algal culture media. The aim of the study was to identify and characterize the basic metabolic pathways in Tetradesmus obliquus cells induced by supplementation with beet molasses in photoheterotrophic and mixotrophic culture conditions. to assess the impact of the nutritional strategy in unicellular algae, growth curves were plotted and lipid, carbohydrate, and protein levels were determined. Fourier Transform Infrared Spectroscopy was applied to measure the Tetradesmus obliquus cell composition. Additionally, the C16-C18 fatty acid profile of Tetradesmus obliquus was determined by gas chromatograph/mass spectrometry. The switch from autotrophy to photoheterotrophy and mixotrophy contributes to shortening of the adaptation growth phase. The highest protein content was obtained in the mixotrophic growth. This study has demonstrated high content of 18:1, cisΔ 9 , 18:2, cisΔ 9,12 , ω6, and 18:3, cisΔ 9,12,15 , ω3 in photoheterotrophic and mixotrophic culture conditions. High levels of proteins and essential fatty acids make Tetradesmus obliquus cell biomass important for human and animals health. Microalgae are not only a source of many valuable bio-products e.g. proteins, carbohydrates, lipids, ω3 and ω6 fatty acids or pigments applied in many different commercial sectors but can be a simple and economical solution to wastewater treatment and waste management 1. Algal cells utilize effectively agricultural, industrial and municipal wastewaters, waste raw materials, and by-products from many branches of industry. Algae are capable of assimilating organic compounds and other ingredients for biomass production and synthesis of both basic and specific metabolites. Additionally, the use of wastewaters and waste materials make microalgal biomass and bio-product production environmentally friendly 2. A number of studies have described supplementation of the algal culture medium with agricultural waste such as dairy manure 3 , liquid waste produced in piggeries 4 , post-fermentation effluents and wastewater from fruit, vegetable or cultivated plant processing, i.e. residues of cane bagasse and pineapple peel 5 , cassava 6 , sweet sorghum 7 , or hydrolysate of Jerusalem artichoke tubers 8. Most algae are autotrophs; however, some species such as Auxenochlorella protothecoides 9 Parachlorella kessleri 10 and Tetradesmus obliquus can assimilate organic compounds. Cultivation modes based on organic carbon sources include heterotrophy, photoheterotrophy, and mixotrophy 11. Compared to the heterotrophic mode, the photoheterotrophic and mixotrophic cultivation systems have several advantages e.g. intensification of algal growth and synthesis of valuable metabolites, such as fatty acids 12. Green algae growing in photoheterotrophic and mixotrophic culture conditions are natural producers of polyunsaturated fatty acids 10,13. In terms of human nutrition, one of the most important PUFAs are essential fatty...