Metabolic Adaptation of Chlorella vulgaris InaCCM49 to Cadmium-Salinity Stress: UPLC-MS/MS-Based Identification of Antioxidant Metabolites
Abstract
Chlorella vulgaris is a microalga species studied for its characteristics and potential applications since 1960. This study investigates the effect of combined salinity and cadmium stress on compound diversity, antioxidant activity, and pigment concentrations in C. vulgaris InaCCM49. Microalgae were cultivated in control and Cd-saline treatments (0.4 M NaCl and 95 μM CdCl₂), followed by biomass harvesting, pigment determination, IC₅₀ measurement, and metabolomic analysis. Cd-salinity stress enhanced antioxidant activity, showing 51.8% lower IC₅₀ values (79.47 ppm) compared to controls (164.99 ppm), and increased carotenoid content to 1.919 mg g⁻¹. Meanwhile, chlorophyll a and b concentrations were notably higher in control treatment during the half-logarithmic phase. Partial Least Squares Discriminant Analysis revealed clear metabolomic differences between treatments. Stigmatellin Y, maltose, glycolipid 1-hexadecanoyl-3-(6'-sulfo-α-D-quinovosyl)-sn-glycerol, and phenolic derivative (2-phenoxy-3-pyridinyl)[3-(2-thienyl)-1H-pyrazol-1-yl]methanone exhibited VIP scores >15. Stigmatellin Y and glycolipids were highly synthesized in controls, whereas maltose and phenolic derivatives were elevated in Cd-saline treatment. Pheophorbide a and 3-oxo-nonadecanoic acid showed the strongest negative correlations with IC₅₀ (regression coefficients of -14.6047 and -13.9555), indicating key roles in antioxidant activity. This study represents the first comprehensive metabolomic analysis of C. vulgaris InaCCM49 under combined cadmium-salinity stress, revealing metabolic adaptation through enhanced synthesis of phenolic derivatives and pheophorbide a-mediated antioxidant responses.
References
Abdi, H. & Williams, L.J., 2010. Tukey's honestly significant difference (HSD) test. Encyclopedia of Research Design, 1, pp.1–5.
Atabayeva, S.D. et al., 2022. Rice Plants (Oryza sativa L.) under Cd Stress in Fe Deficiency Conditions. BioMed Research International, 2022(1), 7425085 . doi: 10.1155/2022/7425085.
Bacova, R. et al., 2019. The effects of 5 azacytidine and cadmium on global 5 methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. Journal of Phycology, 55, pp.329–342. doi: 10.1111/jpy.12824
Barten, R. et al., 2022. Expanding the upper-temperature boundary for the microalga Picochlorum sp. (BPE23) by adaptive laboratory evolution. Biotechnology Journal, 17, 2100659. doi: 10.1002/biot.202100659
Blazenovic, I. et al., 2018. Comprehensive comparison of annotation strategies for untargeted metabolomics. Analytical Chemistry, 90(12), pp.732–742. doi: 10.1021/acs.analchem.8b00800
Chen, J. et al., 2020. Hydrothermal Carbonization of Microalgae-Fungal Pellets: Removal of Nutrients from the Aqueous Phase Fungi and Microalgae Cultivation. ACS Sustainable Chemistry & Engineering, 8(45), pp.16823–16832. doi: 10.1021/acssuschemeng.0c05441
Cheng, J. et al., 2016. The effect of cadmium on the growth and antioxidant response for freshwater algae Chlorella vulgaris. SpringerPlus, 5, 1290. doi: 10.1186/s40064-016-2961-4
Chia, M.A. et al., 2013. Effects of cadmium and nitrogen on lipid composition of Chlorella vulgaris (Trebouxiophyceae, Chlorophyta). European Journal of Phycology, 48(1), pp.1–11. doi: 10.1080/09670262.2012.750687
Chong, J., Wishart, D.S. & Xia, J., 2019. Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis. Current Protocols in Bioinformatics, 68(1), e86. doi: 10.1002/cpbi.86
Coronado-Reyes, J.A. et al., 2022. Chlorella vulgaris, a microalgae important to be used in Biotechnology: a review. Food Science and Technology, 42, e37320. doi: 10.1590/fst.37320
Coulombier, N. et al., 2020. Impact of Light Intensity on Antioxidant Activity of Tropical Microalgae. Marine Drugs, 18(2), 122. doi: 10.3390/md18020122
Creek, D.J. et al., 2024. Best practices and confidence levels in metabolite annotation. Metabolites, 14(1), 11. doi: 10.3390/metabo14010011
El-fayoumy, E.A. et al., 2021. Evaluation of antioxidant and anticancer activity of crude extract and different fractions of Chlorella vulgaris axenic culture grown under various concentrations of copper ions. BMC Complementary Medicine and Therapies, 21(1), 51. doi: 10.1186/s12906-020-03194-x
González-Domínguez, Á. et al., 2024. QComics: Recommendations and Guidelines for Robust, Easily Implementable and Reportable Quality Control of Metabolomics Data. Analytical Chemistry, 96(3), pp.1064–1072. doi: 10.1021/acs.analchem.3c03660
Gonzalez-Esquer, C.R. et al., 2019. Demonstration of the potential of Picochlorum soloecismus as a microalgal platform for the production of renewable fuels. Algal Research, 43, 101658. doi: 10.1016/j.algal.2019.101658
Gorrochategui, E. et al., 2021. Recent advances in high-resolution mass spectrometry for environmental metabolomics. Environmental Science & Technology, 55(7), pp.4139–4152. doi: 10.1021/acs.est.0c07979
Hameed, A. et al., 2011. Differential activation of the enzymatic antioxidant system of Abelmoschus esculentus L. under CdCl₂ and HgCl₂ exposure. Brazilian Journal of Plant Physiology, 23(1), pp.46–54. doi: 10.1590/S1677-04202011000100007
Hawrył, A. et al., 2020. HPLC Fingerprint Analysis with the Antioxidant and Cytotoxic Activities of Selected Lichens Combined with the Chemometric Calculations. Molecules, 25(18), 4301. doi: 10.3390/molecules25184301
Hiremath, S. & Mathad, P., 2022. Secondary Metabolites of Chlorella Vulgaris Under Saline Stress. International Journal of Scientific Research in Science and Technology, pp.424–429. doi: 10.32628/IJSRST229650
Jun, M. et al., 2003. Comparison of Antioxidant Activities of Isoflavones from Kudzu Root (Pueraria lobata Ohwi). Journal of Food Science, 68(6), pp.2117–2122. doi: 10.1111/j.1365-2621.2003.tb07029.x
Lemoine, Y. & Schoefs, B., 2010. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynthesis Research, 106(1–2), pp.155–177. doi: 10.1007/s11120-010-9573-2
Lichtenthaler, H.K. & Buschmann, C., 2001. Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy. Current Protocols in Food Analytical Chemistry, 1(1), pp.F4.3.1-F4.3.8. doi: 10.1002/0471142913.faf0403s01
Liu, Y. et al., 2021. Biofuels for a sustainable future. Cell, 184(6), pp.1636–1647. doi: 10.1016/j.cell.2021.01.052
Mendes, A.R. et al., 2024. Chemical Compounds, Bioactivities, and Applications of Chlorella vulgaris in Food, Feed and Medicine. Applied Sciences, 14(23), 10810. doi: 10.3390/app142310810
Metting, F.B., 1996. Biodiversity and application of microalgae. Journal of Industrial Microbiology, 17(5–6), pp.477–489. doi: 10.1007/BF01574796
Minhas, A.K. et al., 2016. A review on the assessment of stress conditions for simultaneous production of microalgal lipids and carotenoids. Frontiers in Microbiology, 7, 546. doi: 10.3389/fmicb.2016.00546
Nowicka, B., 2022. Heavy metal–induced stress in eukaryotic algae—mechanisms of heavy metal toxicity and tolerance with particular emphasis on oxidative stress in exposed cells and the role of antioxidant response. Environmental Science and Pollution Research, 29(12), pp.16860–16911. doi: 10.1007/s11356-021-18419-w
Pantami, H.A. et al., 2020. Comprehensive GCMS and LC-MS/MS Metabolite Profiling of Chlorella vulgaris. Marine Drugs, 18(7), 367. doi: 10.3390/md18070367
Pirouz, D.M., 2006. An Overview of Partial Least Squares. SSRN Electronic Journal, pp.1–16.
Reignier, O. et al., 2024. Effects of salinity and nutrient stress on a toxic freshwater cyanobacterial community and its associated microbiome: An experimental study. Environmental Microbiology Reports, 16, e70029. doi: 10.1111/1758-2229.13029
Saide, A., Lauritano, C. & Ianora, A., 2020. Pheophorbide a: State of the Art. Marine Drugs, 18(5), 257. doi: 10.3390/md18050257
Saide, M., Abdullah, M. & Hossain, M.A., 2020. Pheophorbide a as a chlorophyll derivative with multifunctional biological activities: A review. Journal of Photochemistry and Photobiology B: Biology, 204, 111801. doi: 10.1016/j.jphotobiol.2020.111801
Salem, M.A. et al., 2020. An improved UPLC–MS-based method for untargeted metabolomics analysis of plant tissues. Metabolomics, 16(3), 13. doi: 10.1007/s11306-020-1654-1
Sarkar, A. et al., 2025. Untargeted metabolomics for systems biology and environmental studies: Methods and applications. Trends in Analytical Chemistry, in press.
Sathasivam, R. et al., 2019. Microalgae metabolic engineering to improve the energy balance for biodiesel production. Applied Energy, 254, 113702. doi: 10.1016/j.apenergy.2019.113702
Schymanski, E.L. et al., 2014. Identifying small molecules via high resolution mass spectrometry: Communicating confidence. Environmental Science & Technology, 48(4), pp.2097–2098. doi: 10.1021/es5002105
Shi, T.-Q. et al., 2020. Stresses as First-Line Tools for Enhancing Lipid and Carotenoid Production in Microalgae. Frontiers in Bioengineering and Biotechnology, 8, 610. doi: 10.3389/fbioe.2020.00610
Smith, J. et al., 2023. SP3-based host cell protein monitoring in AAV-based gene therapy products using LC-MS/MS. European Journal of Pharmaceutics and Biopharmaceutics, 189, pp.276–280. doi: 10.1016/j.ejpb.2023.06.019
Sumner, L.W. et al., 2007. Proposed minimum reporting standards for chemical analysis. Metabolomics, 3(3), pp.211–221. doi: 10.1007/s11306-007-0082-2
Tsugawa, H. et al., 2015. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature Methods, 12(6), pp.523–526. doi: 10.1038/nmeth.3393
Umar, A.H. et al., 2021. Untargeted Metabolomics Analysis Using FTIR and UHPLC-Q-Orbitrap HRMS of Two Curculigo Species and Evaluation of Their Antioxidant and α-Glucosidase Inhibitory Activities. Metabolites, 11(1), 42. doi: 10.3390/metabo11010042
von Jagow, G. & Ohnishi, T., 1985. The chromone inhibitor stigmatellin ‐ binding to the ubiquinol oxidation center at the C‐side of the mitochondrial membrane. FEBS Letters, 185(2), pp.311–315. doi: 10.1016/0014-5793(85)80929-7
Walne, P.R.,1970. Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria and Mytilus, London: Her Majesty's Stationery Office.
Wang, J. et al., 2022. Application of Microalgal Stress Responses in Industrial Microalgal Production Systems. Marine Drugs, 20(1), 30. doi: 10.3390/md20010030
Want, E.J. et al., 2012. Global metabolic profiling of animal and human tissues via UPLC–MS. Nature Protocols, 7(5), pp.813–838. doi: 10.1038/nprot.2012.024
Westerhuis, J. et al., 2008. Assessment of PLSDA cross validation. Metabolomics, 4, pp.81–89. doi: 10.1007/s11306-007-0099-6
Xu, P. et al., 2024. Cadmium-Induced Physiological Responses, Biosorption and Bioaccumulation in Scenedesmus obliquus. Toxics, 12(4), 262. doi: 10.3390/toxics12040262
Zhang, L. et al., 2023. The differential modulation of secondary metabolism induced by a protein hydrolysate and a seaweed extract in tomato plants under salinity. Frontiers in Plant Science, 13, 1072782. doi: 10.3389/fpls.2022.1072782
