Design dark fraction reflects the unlit part of a microalgal culture system, as for example a hydraulic loop used for temperature or pH regulation, or a circulating pump for mixing purposes. This study investigates the impact of design dark fraction on photosynthetic biomass productivity of the eukaryotic microalgae Chlorella vulgaris. The effect of the volume of the dark fraction and the residence time spent in this dark fraction was investigated with two different nitrogen sources (N–NH4+, N–NO3−). Results showed a decrease of biomass productivity when the volume of the dark fraction and the dark residence time increased. Up to 47% loss of biomass productivity could be reached for a design dark fraction fd = 30% of the total culture system volume. This loss was explained as a result of metabolic reactions related to an increase of respiration activity or a decrease of photosynthetic activity in the cells.