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The effects of different ferrous sulfate (FeSO4) concentrations on the fatty acid profile of the blue-green alga Arthrospira platensis cultured in Zarrouk’s medium to produce biodiesel were studied. Different ferrous sulfate concentrations (0, 0.005, 0.01, 0.05, and 0.1 g/L) and 0.01 g/L were examined on the biochemical composition of the alga and fatty acids profile of A. platensis. Findings revealed that the highest and lowest yields of fatty acid were 0 and 0.1 g/L FeSO4, respectively. It was also noticed an increase of palmitic acid, oleic acid, linoleic acid, γ-linolenic acid, and docosahexaenoic acid when ferrous sulfate was between 0.05 and 0.1 g/L, while these fatty acids decreased at low concentration. Ferrous sulfate at a concentration of 0.1 g/L exhibited an increase and best yields in the following: growth rate and the shortest doubling time, chlorophyll-a, phycocyanin, allophycocyanin, phycobiliproteins, and carotenoids. Thus, increasing the FeSO4 concentration to 0.1 g/L has led to the increase in fatty acid individuals, which in turn, resulted in potential enhancement of the biodiesel production from A. platensis.