Abstract: Carbohydrate content of microalgae biomass can be increased and modulated. The nutrient limitation strategy may be promising when employed in a two-stage process, to limit carbohydrate productivity compromising biomass production. In this study, the effects of pH, nitrogen, sulfur, and phosphorus concentration on Chlorella vulgaris growth and carbohydrate accumulation were evaluated. Carbohydrate content reached 58% on the 14th cultivation day, with a biomass concentration of 0.876?±?0.037 g/L, when microalga was cultured in modified BG-11 medium (35 mg/L K2HPO4, 80 MgSO4·7H2O mg/L, nitrogen-depleted and pH 9). Under these conditions, carbohydrate productivity was enhanced by 72.04%, in comparison to the standard medium. In addition, the sugar profile of the microalgae cultivated in the modified nitrogen-depleted medium was altered, displaying a shift in microalgae metabolism towards carbohydrate accumulation. The hydrolysis and fermentation performance of the carbohydrate-rich microalgae biomass was assessed through different methods for process endorsement, and resulted in high reducing sugars release (5.48 g/L of glucose from 10 g/L of biomass), and theoretical ethanol yield (69%), highlighting the impact and feasibility of bioethanol 3G production Ver menos