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Microalgae provides an alternative for the valorization of industrial by-products, in which the nutritional content varies substantially and directly affects microalgae system performance. Herein, the heterotrophic cultivation of <i>Chlorella sorokiniana</i> was systematically studied, allowing us to detect a nutritional deficiency other than the carbon source through assessing the oxygen transfer rate for glucose or acetate fermentation. Consequently, a mathematical model of the iron co-limiting effect on heterotrophic microalgae was developed by exploring its ability to regulate the specific growth rate and yield. For instance, higher values of the specific growth rate (0.17 h⁻¹) compared with those reported for the heterotrophic culture of <i>Chlorella</i> were obtained due to iron supplementation. Therefore, anaerobic sludge from an industrial wastewater treatment plant (a baker’s yeast company) was pretreated to obtain an extract as a media supplement for <i>C. sorokiniana</i>. According to the proposed model, the sludge extract allowed us to supplement iron values close to the growth activation concentration (K_Fe ~12 mg L⁻¹). Therefore, a fed-batch strategy was evaluated on nitrogen-deprived cultures supplemented with the sludge extract to promote biomass formation and fatty acid synthesis. Our findings reveal that nitrogen and iron in sludge extract can supplement heterotrophic cultures of <i>Chlorella</i> and provide an alternative for the valorization of industrial anaerobic sludge.