Carbon dioxide concentrations (pCO₂) in the atmosphere play an integral role in Earth system dynamics, especially climate. Past climates help us understand future ones, but reconstructing pCO₂ over the geologic record remains a challenge. This research demonstrates new approaches for exploring past pCO₂ via the carbon isotope fractionation in general algal lipids, which we test over a high CO₂ gradient from a naturally occurring CO₂ seep.