Nitrate Versus Ammonium Assimilation in Neochlorella semenenkoi: 15N-Tracking Study of Metabolomic Dynamics

Microalgae of the Chlorellaceae family are promising producers of valuable metabolites, yet the choice of nitrogen source significantly modulates their physiology and biochemistry. In this study, we investigated the effect of two inorganic nitrogen sources—ammonium acetate (AA) and sodium nitrate—on the metabolic recovery of Neochlorella semenenkoi IPPAS C-1210 after nitrogen starvation. Using a combination of targeted amino acid quantification (HPLC-MS/MS) and untargeted high-resolution metabolomics, we tracked both concentration changes and de novo biosynthesis of nitrogen-containing metabolites via 15N isotopic labeling. Our results show that ammonium acetate and sodium nitrate enable equally rapid growth resumption and comparable high final biomass yields. Targeted analysis revealed that ammonium-fed cells restored their amino acid pools substantially within 1 day, with a pronounced accumulation of glutamate and glutamine, whereas nitrate-fed cells exhibited a slower and only partial recovery of their free amino acid pool, with several amino acids remaining undetectable even after 3 days. Untargeted metabolomics identified 308 unique metabolites, and the heatmaps of the most variable compounds highlighted a marked early upregulation of TCA cycle intermediates, stress-signaling molecules, and nucleotide precursors in AA cultures, in contrast to a delayed, lipid-centric response in nitrate cultures. The 15N tracing confirmed near-complete (>96%) enrichment of amino acids in both sources, demonstrating that recovery is driven by de novo synthesis rather than recycling, and that isotopic enrichment extends to nucleotides and the NAD+ precursor nicotinamide. These findings reveal the metabolic advantages of ammonium over nitrate for rapid metabolic recovery and restoration of nitrogen-rich compound pools, and provide a foundation for optimizing cultivation strategies for this biotechnologically relevant alga.