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Nitrogen deficiency can increase the lipid content in certain microalgae species, including diatoms. However, the molecular and metabolic basis of such changes remains rather unclear. We analyzed strains of freshwater Nitzschia palea collected from a eutrophic pond and from an artificial rock. The habitats, differing in light and nutrient availability, lead to two metabolically distinct strains, BR006 and BR022. Differential accumulation of primary compounds, membrane lipid composition and fatty acid saturation were observed. Metabolic and biophysical analysis demonstrated differential sensitivity to N regimes: depleted, replete and saturated. Whereas N depletion leads to typical stress-related responses in both strains, including reduction of protein and photosynthesis, the response observed in BR006 is far more severe. Our results demonstrated that these strains developed distinct metabolic responses to N …