Nitrate Versus Ammonium Assimilation in Neochlorella semenenkoi: 15N-Tracking Study of Metabolomic Dynamics
- 1Center for Molecular and Cellular Biology, Bolshoy Boulevard 30, bld. 1, Moscow 121205, Russia
- 2K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow 121205, Russia
- 3Project Center of Omics Technologies and Advanced Mass Spectrometry, Bolshoy Boulevard 30, bld. 1, Moscow 121205, Russia
Abstract
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.
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BibTeX
@article{vishnevskaya2026nitrate,
title = {{Nitrate Versus Ammonium Assimilation in Neochlorella semenenkoi: 15N-Tracking Study of Metabolomic Dynamics}},
author = {Anna Vishnevskaya and Maria Sinetova and Nikita Malyshev and Anastasya Malyukova and Eugene Nikolaev and Yury Kostyukevich},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27219497},
url = {https://www.mdpi.com/1422-0067/27/21/9497},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Nitrate Versus Ammonium Assimilation in Neochlorella semenenkoi: 15N-Tracking Study of Metabolomic Dynamics
AU - Anna Vishnevskaya
AU - Maria Sinetova
AU - Nikita Malyshev
AU - Anastasya Malyukova
AU - Eugene Nikolaev
AU - Yury Kostyukevich
PY - 2026
DA - 2026/10/24
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27219497
UR - https://www.mdpi.com/1422-0067/27/21/9497
AB - 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.
SN - 1422-0067
ER -