Euglena gracilis is a metabolically versatile microalga capable of thriving under photoautotrophic (light, no ethanol), mixotrophic (light with 1% v/v ethanol), and heterotrophic (dark with 1% v/v ethanol) conditions. Here, we applied untargeted LC-MS metabolomics (Agilent 1290 LC, 6545XT QTOF-MS; Agilent Technologies, Santa Clara, California, USA) to investigate its trophic-mode-dependent metabolic adaptations and assess its biotechnological potential. Metabolites were separated on a C18 column and analyzed in both positive and negative ion modes. Multivariate analyses (PCA and sPLS-DA) revealed clear and reproducible metabolic separations among growth modes (p < 0.001). Photoautotrophic cultures were enriched in phenolic acids, flavonoids, and lipid classes associated with oxidative stress protection. Mixotrophy induced a broader spectrum of upregulated metabolite classes, including saccharolipids, macrolactams, and triterpenoids, reflecting a hybrid metabolism combining photosynthesis and ethanol utilization. Heterotrophic cultures showed elevated levels of polyamines and amino acids (e.g., putrescine, proline), indicative of redox regulation and stress adaptation in dark, ethanol-rich conditions. Class-level comparisons identified distinct and shared metabolite categories, with photoautotrophy favoring antioxidant biosynthesis and mixotrophy supporting metabolic diversity. These findings provide metabolite-level insights into the extraordinary plasticity of E. gracilis and offer a framework for optimizing cultivation strategies to enhance the targeted production of high-value bioproducts.

Short-wavelength ultraviolet radiation can impair biological systems by causing DNA damage, oxidative stress, and disruption of photosynthetic processes. Although ultraviolet C (UVC) at 254 nm is widely used as a controlled laboratory stressor, the extent to which trophic condition influences repeated UVC tolerance in phototrophic protists remains unclear. Here, we examined the response of Euglena gracilis grown under photoautotrophic or ethanol-supported mixotrophic conditions and exposed to daily UVC pulses for five days. Cell growth, photosynthetic pigments, intracellular oxidative stress measured by 2′,7′ dichlorodihydrofluorescein diacetate fluorescence, and lipid peroxidation estimated as thiobarbituric acid reactive substances equivalent malondialdehyde were assessed, together with qualitative fluorescence microscopy. Repeated UVC exposure reduced cell density in both trophic conditions, with stronger inhibition under photoautotrophy. Photoautotrophic UVC-treated cultures showed the highest oxidative stress signal, whereas malondialdehyde displayed only a non-significant directional increase. Mixotrophic cultures maintained higher cell density under UVC and showed lower oxidative stress signals than photoautotrophic UVC-treated cultures. Pigment responses also differed between trophic conditions, with increased chlorophyll a and carotenoids per cell under photoautotrophic UVC treatment, while mixotrophic pigment levels remained comparatively stable. These findings show that trophic condition shapes repeated UVC stress responses in E. gracilis and that ethanol-supported mixotrophy is associated with improved physiological robustness under the present experimental conditions.