Fe (III)-Mediated Antioxidant Response of the Acidotolerant Microalga Coccomyxa onubensis

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TL;DR

This study investigates how the acidotolerant microalga Coccomyxa onubensis responds to iron(III)-induced oxidative stress. Moderate Fe(III) supplementation (up to 2 mM) improved growth and photosynthetic efficiency while triggering antioxidant defenses, including increased superoxide dismutase activity (62% higher at 1 mM Fe) and flavonoid accumulation (8% higher at 2 mM Fe). These findings suggest that controlled Fe(III) stress can be used to produce microalgal biomass enriched with valuable antioxidant compounds for potential nutraceutical applications.

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**Background:** Coccomyxa onubensis is an acidotolerant microalga isolated from the Tinto River (Huelva, Spain), an environment characterized by low pH (0.8–3) and high concentrations of metal cations, particularly Fe(II) and Fe(III). Iron is more bioavailable at low pH due to increased solubility of both ionic forms. While iron is essential for redox processes in microalgae—serving as a component of Fe-S clusters in electron transport proteins—it can also trigger oxidative stress via the Fenton reaction, producing reactive oxygen species (ROS) that damage proteins, lipids, and nucleic acids. This study hypothesized that modulating Fe(III)-mediated stress in acid-tolerant microalgae could increase the productivity of commercially valuable antioxidant molecules (flavonoids, polyphenols, PUFAs, carotenoids) while maintaining culture viability.

**Methods:** C. onubensis was cultivated in optimized K9 liquid medium at pH 2.5, 25°C ± 2°C, under continuous light (150 µmol photon·m⁻²·s⁻¹) and aeration with 2.5% CO₂. Cultures were supplemented with Fe(III) at concentrations from 0 mM (control) to 2 mM. Growth was assessed by dry weight biomass productivity. Photosynthetic viability was determined through chlorophyll fluorescence measurements (Fv/Fm) and OJIP protocol analysis using a pulse amplitude-modulated fluorimeter. Chlorophyll and carotenoid content were measured spectrophotometrically and by HPLC. Antioxidant capacity was determined by the DPPH free radical method. Total polyphenols were measured using the Folin-Ciocalteu reagent, and flavonoids were determined spectrophotometrically. Superoxide dismutase (SOD) activity was assayed by monitoring nitroblue-tetrazolium (NBT) reduction inhibition. Lipid content was measured gravimetrically after chloroform:methanol extraction, and fatty acid profiles were analyzed by gas chromatography after transesterification to FAMEs.

**Key Results:** Biomass productivity increased with Fe(III) supplementation, reaching a maximum of 0.35 g·L⁻¹·d⁻¹ in the 2 mM Fe(III) culture—17% higher than the control. While intracellular chlorophyll concentration decreased by 19% in the 2 mM Fe(III) culture compared to control, photosynthetic efficiency (quantum yield) increased by 27%. OJIP analysis revealed that the net closing rate of reaction centers (Mo) decreased by 22% at 2 mM Fe(III), while electron transport flux per reaction center (ETo/RC) increased by 5.4%. SOD specific activity increased with Fe(III) concentration, reaching a maximum at 1 mM Fe(III)—approximately 62% higher than the control. Total cellular protein decreased by 18% at 1 mM Fe(III). Total antioxidant capacity (DPPH assay) decreased by 22% in the 2 mM Fe(III) culture on day 2. Polyphenol content decreased by approximately 30% at 2 mM Fe(III), while flavonoid content increased by 8% at 2 mM Fe(III). Carotenoid analysis showed lutein content remained relatively stable (4–5 mg·g⁻¹ dw), while neoxanthin and beta-carotene increased by 30–50% under long-term cultivation. Total lipid content increased by approximately 11% at 1 mM Fe(III), while total fatty acid content decreased by 23% at 2 mM Fe(III). The PUFA/SAFA ratio remained stable during early cultivation but increased from day 4 in control and 0.25 mM Fe(III) cultures. Notably, C18:2 (omega-6) content was 48% higher at 0.5 mM Fe(III) compared to control, and C18:3 (omega-3) was 36% higher at 0.25 mM Fe(III).

**Clinical Implications:** This study demonstrates that moderate Fe(III) stress can be used to enhance the production of antioxidant molecules in C. onubensis without compromising growth. The increased accumulation of flavonoids and essential polyunsaturated fatty acids (linoleic and linolenic acids) under controlled Fe(III) supplementation suggests that this microalga could be cultivated to produce biomass enriched with compounds having potential anti-inflammatory and antioxidant activities relevant to human health. The findings provide a foundation for designing large-scale biotechnological processes to obtain microalgal extracts with nutraceutical value. However, the study is preclinical and does not directly test effects in human or animal models; further research would be needed to validate the bioactivity of extracts in biological systems.