More efficient iron enrichment in heterotrophic Auxenochlorella protothecoides by nitrogen source manipulation

• Nitrogen starvation and organic matter removal boost iron uptake in A. protothecoides. • Yeast extract limits iron bioavailability due to organic complexation effects. • Acid µMRR validated as fast, low-cost alternative for intracellular iron quantification. • Iron enrichment strategy preserves cell viability and biomass under all tested conditions. Microalgae are increasingly explored as sustainable carriers for micronutrient delivery in food, feed, and agriculture, particularly for iron enrichment. Among them, Auxenochlorella protothecoides is a promising strain owing to its ability to grow heterotrophically to high cell densities using food industry side streams and organic carbon and nitrogen sources. However, under such conditions, direct iron supplementation in the cultivation medium is largely ineffective because ferric ions are poorly soluble at near-neutral pH and readily form complexes with organic matter, limiting cellular uptake. To overcome this limitation, this study investigated how nitrogen source and availability affect iron accumulation under heterotrophic conditions. Four cultivation strategies were compared, revealing that transfer to a nitrogen- and organic-free medium and supplementation of iron with ferric ammonium citrate at 200 μg mL −1 resulted in efficient iron accumulation with the highest intracellular iron content (3170 ± 254 mg kg DW –1 ), representing a 25-fold increase over direct supplementation in a normal medium and exceeding previously reported values for other microalgae by up to one order of magnitude. Intracellular iron was quantified using acid micromagnetic resonance relaxometry (acid μMRR), which showed strong agreement with conventional inductively coupled plasma mass spectrometry (ICP-MS) while enabling faster, simpler, and more accessible analysis. Overall, the study shows that eliminating dissolved organic matter, combined with controlled nitrogen deprivation, enhances iron bioaccumulation and introduces acid μMRR as a useful tool for analyzing nutrient-enriched microalgae for sustainable food and feed applications.