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The effectiveness of the niche of freshwater algae as a remover of carbon dioxide (CO₂) from the ecosystem, and its potential to perpetuate carbon cycle are to be systematically investigated. In this study, using unsupplemented natural freshwater medium, we investigated the freshwater microalgal sequestration of atmospheric CO₂ in the laboratory. The microalgal fixation of carbon, calculated by mass balance, showed that the freshwater microalgal CO₂ uptake rate could reach 175 mg/L/day, with a CO₂ consumption efficiency over 100%. We found that the natural freshwater medium system achieved much higher carbon fixation efficiency than artificial media which used CO₂ as the sole carbon source. This study demonstrated that in natural freshwater medium, the extent of microalgal carbon fixation transcended the substrate limitation by directly absorbing CO₂ from air and thus the carbon in the microalgal …