This study focused on evaluating whether the injection of CO2, which is associated with the use of thermoelectric fly ashes and a reduced supply of nitrogen, affects the production of intracellular carbohydrates from Spirulina. For this purpose, the addition of 0.25 g L-1 of NaNO3, along with a 10% (v v-1) of CO2 injection, a flow rate of 0.3 vvm for 1 or 5 min, as well as 0, 120 and 160 ppm of fly ashes, was studied. The assays with 120 ppm of fly ashes presented the best kinetic parameters and CO2 biofixation rate, regardless of the CO2 injection time. Meanwhile, the experiments with 120 and 160 ppm of fly ash and CO2 injection for 1 min presented 63.3 and 61.0% (w w-1) of carbohydrates, respectively. Thus, this study represents an important strategy to increase the accumulation of carbohydrates in Spirulina, with potential application in the production of bioethanol.