Chlamydomonas is one of the most well-studied photosynthetic organisms that had important biotechnological potential for future bioproductions of biofuels. However, an energy balance from incident photons to the energy stored in the new biomass is still lacking. In this study, we applied a recently developed system to measure the energy balance for steady state growth of Chlamydomonas reinhardtii grown at pH 6.5, and C. acidophila that was grown at pH 6.5 and 2.6. Energy use efficiency was quantified on the basis of light absorption, photosynthetic quantum yield, photosynthetic and respiratory quotient, and electron partitioning into proteins, carbohydrates and lipids. The results showed that lower growth rates of C. acidophila under both pH conditions were not caused by the differences in the photosynthetic quantum yield or in alternative electron cycling, but rather by differences in the efficiency of light absorption and increased dark respiration. Analysis of the macromolecular composition of the cells during the light phase showed that C. acidophila uses biosynthetic electrons preferentially for carbohydrate synthesis but not for synthesis of lipids. This led to a strong diurnal cycle of the C/N ratio and could explain the higher dark respiration of C. acidophila compared with C. reinhardtii.