Algae-driven processes, such as direct CO₂ fixation into glycerol, provide new routes for sustainable chemical production in synergy with greenhouse gas mitigation. The marine microalgae Dunaliella tertiolecta is reported to accumulate high amounts of intracellular glycerol upon exposure to high salt concentrations. We have conducted a comprehensive, time-resolved systems biology study to decipher the metabolic response of D. tertiolecta up to 24 h under continuous light conditions. Initially, due to a lack of reference sequences required for MS/MS-based protein identification, a high-quality draft genome of D. tertiolecta was generated. Subsequently, a database was designed by combining the genome with transcriptome data obtained before and after salt stress. This database allowed for detection of differentially expressed proteins and identification of phosphorylated proteins, which are involved in the short- and long-term adaptation to salt stress, respectively. Specifically, in the rapid salt adaptation response, proteins linked to the Ca²⁺ signaling pathway and ion channel proteins were significantly increased. While phosphorylation is key in maintaining ion homeostasis during the rapid adaptation to salt stress, phosphofructokinase is required for long-term adaption. Lacking β-carotene, synthesis under salt stress conditions might be substituted by the redox-sensitive protein CP12. Furthermore, salt stress induces upregulation of Calvin–Benson cycle-related proteins.

The salt-tolerant marine microalgae Dunaliella tertiolecta is reported to generate significant amounts of intracellular glycerol as an osmoprotectant under high salt conditions. This study highlights the phylogenetic distribution and comparative glycerol biosynthesis of seven new Dunaliella isolates compared to a D. tertiolecta reference strain. Phylogenetic analysis indicates that all Dunaliella isolates are newly discovered and do not relate to the D. tertiolecta reference. Several studies have identified light color and intensity and salt concentration alone as the most inducing factors impacting glycerol productivity. This study aims to optimize glycerol production by investigating these described factors singularly and in combination to improve the glycerol product titer. Glycerol production data indicate that cultivation with white light of an intensity between 500 and 2000 μmol m⁻² s⁻¹ as opposed to 100 μmol m⁻² s⁻¹ achieves higher biomass and thereby higher glycerol titers for all our tested Dunaliella strains. Moreover, applying higher light intensity in a cultivation of 1.5 M NaCl and an increase to 3 M NaCl resulted in hyperosmotic stress conditions, providing the highest glycerol titer. Under these optimal light intensity and salt conditions, the glycerol titer of D. tertiolecta could be doubled to 0.79 mg mL⁻¹ in comparison to 100 μmol m⁻² s⁻¹ and salt stress to 2 M NaCl, and was higher compared to singularly optimized conditions. Furthermore, under the same conditions, glycerol extracts from new Dunaliella isolates did provide up to 0.94 mg mL⁻¹. This highly pure algae-glycerol obtained under optimal production conditions can find widespread applications, e.g., in the pharmaceutical industry or the production of sustainable carbon fibers.