Microalgae have received increasing attention as one of the most promising feedstocks in the development of new healthier food products and different strategies have been at-tempted to improve their growth. However, the high production costs and low pro-ductivities, commonly associated with photoautotrophic growths, are still a big challenge. In this study, a two-step optimization strategy was carried out in order to maximize the biomass production of a Chlorella vulgaris strain used at industrial scale under hetero-trophic conditions. From a total of 24 independent variables, which were studied si-multaneously, 10 have presented a positive effect over Xmax, while the remaining have shown to be negative. The amount of (NH4)2SO4 (6.3 g L-1), MgSO4middot7H2O (0.7 g L-1), and C6H12O6 (50% w/v) in the culture medium has revealed to be the only factors with a sig-nificant impact on biomass concentration, with optimum values of 25.5, 64.6, and 75 ml.L-1, respectively. The optimized medium resulted in an improvement of the Xmax by 99.6% when compared to the growth medium applied at industrial scale, proving the success of this strategy. Additionally, the carbohydrates production was enhanced by 48.0%.(c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Institution of Chemical Engineers. This is an open access article under the CC BY-NC-ND license (http://creati-vecommons.org/licenses/by-nc-nd/4.0/).