| Summary: | Microalgae have a high growth rate, high CO<sub>2</sub> absorption capacity, and high content of chlorophyll, proteins, vitamins, mineral salts, carbohydrates, antioxidant substances, and fatty acids. In recent years, <i>Chlorella vulgaris</i> has been widely used as a feedstock for producing third-generation biofuels, such as bioethanol. Thus, this work aims to develop a strategy to increase the production scale of the microalgae <i>Chlorella vulgaris</i> grown in distilled reused water, supplemented with a modified BG-11 medium, to use biomass in the production of bioethanol. The total cultivation of 72 L presented a concentration of 0.415 g·L<sup>−1</sup>·d<sup>−1</sup>, with 61.32 g of final biomass. To improve carbohydrate extraction, the biomass was pre-treated with sulfuric acid at different concentrations (1.5% and 3% <i>v</i>/<i>v</i>). The hydrolyzed solution was supplemented with YPD (yeast extract peptone dextrose) medium and inoculated with <i>Saccharomyces cerevisae</i> yeast, initiating fermentation. In each sample, the Brix degree, cell concentration, reducing sugar concentration, and alcohol content were analyzed. The sample pre-treated with sulfuric acid 1.5% <i>v/v</i> was the one that presented the best result, with alcohol content after distillation of 68 °GL (Gay-Lussac). It appears that the cultivation of the microalgae <i>Chlorella vulgaris</i> in scale-up, with reused water, has high potential in the production of third-generation biofuel.
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