Conventional wastewater treatment (WWT) is not able to recycle nutrients from the wastewater (WW) directly. Microalgae integrate the valuable nutrients nitrogen and phosphorus within their biomass very efficiently, making them predestined for an application in WWT. Nevertheless, microalgae-based processes are driven by natural sunlight as energy source, making a continuous process mode during day and night difficult. The aim of this study was therefore to investigate metabolic activities of the continuously cultivated microalgae Chlorella vulgaris at light and dark periods (16 h,8 h) with focus on nutrient uptake during night from a synthetic WW. Varying the dilution rate D (D = 0.0–1.0 d−1 in 0.1 d−1-steps) causes different limitations for algae growth. Nutrient limitations at low D's cause maximum accumulation of intracellular storage components (sum of carbohydrates and lipids) of ~70 % of dry biomass, starch is converted to lipids at the absence of light. From middle to high D's, the growth rate is determined by light limitation, reducing the intracellular storage components to ~20 % of dry biomass. Complete nutrient uptake is measurable up to D = 0.5 d−1, marking the maximum operating point for wastewater purification. At that point, cells are characterised by high protein (up to 57%DBM) and pigment (up to 6.9%DBM) quotas. During the night, the build-up of proteins at the degradation of intracellular storage components is furthermore visible. Applying the concept of active biomass (cells without storage components), a constant cellular protein (~68%ABM) and nitrogen quota (11.94%ABM) was revealed. A nitrogen spiking experiment clearly showed nitrogen uptake and proliferation during the night period. Based on the experimental data, a window of operation for a continuous WWT process was designed, allowing the hypothesis that continuous WWT using microalgae during day and night operation is possible without the supplementation of artificial light. This revealed the system's capacity to treat WW throughout 24 h applying cell recycling and storage of carbohydrate-rich biomass. At the end of the night, protein-rich biomass is available for further valorisation.