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 Chlorella sorokiniana 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 Chlorella 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 C. sorokiniana. According to the proposed model, the sludge extract allowed us to supplement iron values close to the growth activation concentration (KFe ~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 Chlorella and provide an alternative for the valorization of industrial anaerobic sludge.