ELEVATED CO2 SHIFTS THE n-6/n-3 FATTY ACID BALANCE IN Nannochloropsis oceanica DURING THE LATE EXPONENTIAL TO STATIONARY PHASE TRANSITION UNDER DIFFERENT PHOTOPERIODS

Introduction

Mitigating elevated CO2 levels in the atmosphere due to anthropogenic sources is at the forefront of this study. The culturing of microalgae can be one of many tools for mitigating these elevated CO2 levels and its biomass is used in zooplankton culture and early fish and shellfish larval nutrition. Cultures are harvested between the late exponential and early stationary phases when biomass is at its highest, however, lipid and fatty acid profile remodeling also occurs during this period. Studies claim that differing photoperiods may cause changes in the cellular contents of proteins, lipids, and carbohydrates (Khoeyi et al., 2012). Increasing the CO2 levels in the culture media has shown an enhancement of the lipid content (Jiang et al., 2016; Dubey et al., 2024). Omega-3 polyunsaturated fatty acids (n-3 PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 PUFA arachidonic acid (ARA), are essential in the early development and survival of marine species and the cultured microalgae may supply these necessary biochemical compounds (Matsunari et al., 2013). Therefore, we tested the effects of varying CO2 levels and photoperiod to determine how CO₂ enrichment and photoperiod influence biomass production and fatty acid composition of the eustigmatophyte Nannochloropsis oceanica during the late exponential to stationary phase transition.

Materials and Methods

N. oceanica was cultured using a two-phase semi-continuous system in a 20 L photobioreactor with Walne's medium. Upon reaching late exponential phase, cultures were distributed into 2 L plastic bag photobioreactors. Three independent trials comprising of three photoperiod regimes (24:0, 18:6, and 12:12 Light:Dark) and three CO₂ concentrations (atmospheric 0.04%, 5%, and 10%), each in biological triplicate. Biomass and cellular density were recorded daily. After five days, at the transition to early stationary phase, biomass was harvested for total lipid and fatty acid analysis.

Results

Biomass increased significantly with CO₂ enrichment in the 24:0 h (F₂,₆ = 8.85, p = 0.016) and 18:6 h (F₂,₆ = 11.04, p = 0.010) trials, with 5% and 10% CO₂ both yielding significantly more biomass than atmospheric CO₂ in each; the 12:12 h trial showed a similar but non-significant trend (F₂,₆ = 5.17, p = 0.050). Total fatty acid content followed a comparable pattern across trials. Across all three independent photoperiod trials, elevated CO₂ produced a consistent, dose-dependent increase in the n-6 pathway fatty acid ARA, with the ARA/EPA ratio rising significantly from 0.24-0.28 at atmospheric CO₂ to 0.38-0.54 at 10% CO₂ (p < 0.05 in all trials). Furthermore, the entire n-6 pathway was upregulated under higher CO2 exposure across all photoperiods. This was accompanied by a significant reduction in EPA content in the 24:0 h and 18:6 h trials, evident in both relative and absolute terms. The n-6/n-3 ratio increased 2.0- to 2.2-fold across the CO₂ range in every photoperiod trial tested.

Conclusion

Although elevated CO₂ enhanced biomass production, it consistently shifted fatty acid composition toward higher ARA and lower EPA, increasing the n-6/n-3 ratio regardless of photoperiod. These findings highlight a trade-off between biomass productivity and nutritional quality when culturing N. oceanica under high CO2 in the late exponential and early stationary phases.

References

Dubey, S., Chen, C. W., Patel, A. K., Bhatia, S. K., Singhania, R. R., & Dong, C. di. (2024). Development in health-promoting essential polyunsaturated fatty acids production by microalgae: a review. In Journal of Food Science and Technology (Vol. 61, Issue 5, pp. 847–860). Springer. https://doi.org/10.1007/s13197-023-05785-1

Jiang, X., Han, Q., Gao, X., & Gao, G. (2016). Conditions optimising on the yield of biomass, total lipid, and valuable fatty acids in two strains of Skeletonema menzelii. Food Chemistry, 194, 723–732. https://doi.org/10.1016/j.foodchem.2015.08.073

Khoeyi, Z. A., Seyfabadi, J., & Ramezanpour, Z. (2012). Effect of light intensity and photoperiod on biomass and fatty acid composition of the microalgae, Chlorella vulgaris. Aquaculture International, 20(1), 41–49. https://doi.org/10.1007/s10499-011-9440-1

Matsunari, H., Hashimoto, H., Oda, K., Masuda, Y., Imaizumi, H., Teruya, K., Furuita, H., Yamamoto, T., Hamada, K., & Mushiake, K. (2013). Effects of docosahexaenoic acid on growth, survival and swim bladder inflation of larval amberjack (Seriola dumerili, Risso). Aquaculture Research, 44(11), 1696–1705. https://doi.org/10.1111/j.1365-2109.2012.03174.x