# Changes in Metabolism and Lipid Composition with Nitrogen Starvation and Recovery in a New Productive Strain of Neochlorella semenenkoi Using N15-Isotopic Labeling and HRMS

## TL;DR

This study explores how a productive green algae strain adapts metabolically and in lipid composition during nitrogen starvation and recovery using isotopic labeling and mass spectrometry.

## Contribution

The study introduces a new productive strain of Neochlorella semenenkoi and applies 15N isotopic labeling to reveal metabolic and lipid changes under nitrogen stress.

## Key findings

- The algae strain utilizes ammonium acetate as a nitrogen source, primarily consuming nitrogen in the ammonium form.
- Isotopic labeling revealed significant divergence between isotopic labeling and compound concentrations, suggesting isotopic analysis offers advantages over quantitative methods.
- In vivo isotopic labeling helps identify compounds not found in standard mass spectrometry databases.

## Abstract

Microscopic green algae are active producers of beneficial compounds, particularly those containing nitrogen. However, the metabolism of nitrogen-containing compounds is diverse and depends on the conditions of the nitrogen source. As a result, the approach to studying the metabolism of nitrogen-containing compounds becomes more complicated. This work demonstrates the metabolic changes in the high-productive green algae Neochlorella semenenkoi IPPAS C-1210 under conditions of nitrogen starvation and subsequent reintake, using high-performance liquid chromatography–mass spectrometry (HPLC–MS) with 15N isotopic labeling. The presented results include semi-quantitative chromatography–mass spectrometric analysis for 17 amino acids, a metabolomic profile of over 40 isotopically labeled compounds, an assessment of metabolic flux via isotopic incorporation, and an analysis of cellular lipid composition under varying growth conditions. The findings indicate that this strain can utilize ammonium acetate as a nitrogen source, consuming nitrogen in the ammonium form. The degree of isotopic labeling in compounds often diverged significantly from their quantitative changes (concentrations and chromatographic peak areas), suggesting that isotopic analysis may offer advantages over purely quantitative analysis for biological systems. Furthermore, in vivo biological isotopic labeling is shown to assist in identifying compounds absent from standard mass spectrometric databases.