The metabolism of heterotrophic and mixotrophic cultivation modes of Chlorella vulgaris, a potential source ofbiofuel and CO2 mitigation, was studied in immobilized cultures. The gas concentration (O2 and CO2) wasmeasured thanks to an original device manufactured using 3D printing. The biomass was monitored by 3Dimaging and image processing. Net O2 and CO2 sources were obtained by a balance equation considering acalibrated leakage and the dissolved gas. Combined experimental and theoretical gas yields (mass of gas permass of biomass), the photosynthesis proportion of mixotrophic colony was determined. Its increase with lightintensity is not linear. Therefore, the highest light intensity (104 μmol∙m−2∙s−1) revealed the limit of photosynthesispotential in the growth of mixotrophic colony. In the presence of light, the colony adopts a cylindricalshape instead of a spherical cap. This study proposed mechanisms of synergy inside the colony for heterotrophicand mixotrophic modes.