Metabolic Profiling of Microalgae under Oil Stress

The Dunaliella tertiolecta culture was exposed to Control (f/2 medium) and WAF at a cell density of 5 × 10⁴ cells · mL⁻¹ in batch culture for a period of 48 h in triplicates. The role of Kreb's cycle on metabolism during oil exposure was confirmed through determination of respiration rates after 48 h time point. Respiration rates were measured using a Clark‐type oxygen electrode (Hansatech, Norfolk, UK), as per methods described in Kamalanathan et al. (2015 , 2021a (link)). Further confirmation of the upregulation of Kreb's/tricarboxylic acid (TCA) cycle was confirmed by measuring the concentration of cycle metabolites and rates of respiration. Briefly, cells of D. tertiolecta were collected after 48 h incubation in the Control and WAF treatment. For measuring the concentration of metabolites in the cells, 400 mL of cultures were filtered through GF/F membrane (0.7 μm, Whatman, United States) and 0.5 ppm of ¹³C‐citrate was added to the tubes as an internal standard and the metabolites were extracted using chilled 100% methanol. The methanol extract was then analyzed on LC–MS/MS for organic acid analysis.