Influence of Photoperiod on Lipid, Protein and Astaxanthin Content of Cystic Haematococcus pluvialis Cultivated in High-Concentration Molasses Medium

Year 2025, Volume: 9 Issue: 2, 127 - 141, 30.12.2025

Gökçe Kendirlioğlu Şimşek , A. Kadri Çetin

Abstract

This study investigates the response of Haematococcus pluvialis to controlled induction of the cyst stage using a high-carbon molasses medium and evaluates how distinct photoperiod regimes (16L/8D, 12L/12D, 8L/16D, 4L/20D, and 24D) regulate cell development and metabolite accumulation. The applying of molasses at 8 g/L effectively reduced light penetration and generated a carbon-rich stress environment, accelerating encystment and promoting the activation of metabolic pathways associated with astaxanthin, protein and lipid enrichment. Growth analyses indicated that the 16L/8D treatment supported the highest cell density even under cyst-inducing conditions, highlighting the stimulatory role of extended illumination. Protein formation was maximized under this regime, reaching 2.409 µg/mL on day 10, whereas the 12L/12D cycle provided the strongest stimulation for astaxanthin synthesis, with a peak value of 2.997 µg/mL. Lipid levels followed a similar pattern, with the most highest accumulation (65.9%) observed under long-light conditions, while continuous darkness markedly suppressed all metabolic outputs. Correlation mapping revealed that the photoperiod exerted a coordinated regulatory influence on growth, astaxanthin, protein, and lipid production, indicating an integrated metabolic adjustment to light–dark cycling during the cyst phase. Overall, the findings demonstrate that molasses is a low-cost and efficient substrate for inducing the red/cyst stage and that optimizing of photoperiod conditions can substantially enhance the biosynthesis of high-value metabolites. These results provide a promising framework for cost-effective and environmentally sustainable production strategies in astaxanthin biotechnology, functional food development, nutraceutical formulations, and microalgae-based biofuel applications.

Keywords

Haematococcus pluvialis , Molasses , Astaxanthin

Thanks

The authors would like to express their sincere gratitude to the Algal Biotechnology Laboratory at Fırat University for providing the research infrastructure, laboratory facilities, and technical assistance that made this study possible.

References

• Allen, M. M., & Stanier, R. Y. (1968). Growth and Division of Some Unicellular Blue-green Algae. Journal of General Microbiology, 51 (2), 199–202.
• Alzahrani, M. A. J., Perera, C. O., Sabaragamuwa, R., & Hemar, Y. (2019). Assessment of bioactive potential of aqueous protein extracts from diatoms nitzschia laevis, spirulina platensis, and Chlorella vulgaris. Journal of Aquatic Food Product Technology, 28 (2), 177–193. https://doi.org/10.1080/10498850.2019.1571551
• Al-Zuhair, S., Ashraf, S., Hisaindee, S., Darmaki, N. A., Battah, S., Svistunenko, D., et al. (2017). Enzymatic pre-treatment of microalgae cells for enhanced extraction of proteins. Engineering in Life Sciences, 17 (2), 175–185. https://doi.org/10.1002/elsc.201600127
• Batan, L., Quinn, J., Willson, B., & Bradley, T. (2010). Net energy and greenhouse gas emission evaluation of biodiesel derived from microalgae. Environmental Science & Technology, 44 (20), 7975–7980. https://doi.org/10.1021/es102052y
• Bligh, E., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917.
• Boussiba, S. (2000). Carotenogenesis in the green alga Haematococcus pluvialis: Cellular physiology and stress response. Physiologia Plantarum, 108 (2), 111–117.
• Boussiba, S., & Vonshak, A. (1991). Astaxanthin accumulation in the green alga Haematococcus pluvialis. Plant Cell Physiology, 32, 1077–1082.
• Davies, B. H. (1976). Carotenoids. In: Goodwin, T. W. (Ed.), Chemistry and Biochemistry of Plant Pigments, Vol. 2, Academic Press., pp. 38-166.
• García-Malea, M. C., Acién, F. G., Fernández-Sevilla, J. M., Rodrigo, M. A., Molina, E., & Christenson, L. (2006). Continuous light as a strategy to promote astaxanthin accumulation in Haematococcus pluvialis. Enzyme and Microbial Technology, 38 (7), 981–989. https://doi.org/10.1016/j.enzmictec.2005.08.001
• Grossmann, L., Hinrichs, J., & Weiss, J. (2019). Solubility and aggregation behavior of protein fractions from the heterotrophically cultivated microalga Chlorella protothecoides. Food Research International, 116, 283–290. https://doi.org/10.1016/j.foodres.2018.08.037
• Gupta, S., Sengupta, A., Singh, P., & Banerjee, S. (2025). Stress-induced enhancement of microalgal biochemical composition under variable light regimes. SN Applied Sciences, 7 (2), 254. https://doi.org/10.1007/s42452-025-07545-6
• Henchion, M., Hayes, M., Mullen, A. M., Fenelon, M., & Tiwari, B. (2017). Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium. Foods, 6 (7), 53. https://doi.org/10.3390/foods6070053
• Hildebrand, G., Poojary, M. M., O’Donnell, C., Lund, M. N., Garcia-Vaquero, M., & Tiwari, B. K. (2020). Ultrasound-assisted processing of Chlorella vulgaris for enhanced protein extraction. Journal of Applied Phycology, 32 (3), 1709–1718. https://doi.org/10.1007/s10811-020-02105-4
• Ho, S.-H., Kondo, A., Hasunuma, T., & Chang, J.-S. (2013). Engineering strategies for improving the CO₂ fixation and carbohydrate productivity of Scenedesmus obliquus CNW-N used for bioethanol fermentation. Bioresource Technology, 143 (6), 163–171. https://doi.org/10.1016/j.biortech.2013.05.043
• Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., & Darzins, A. (2008). Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant Journal, 54, 621–639. https://doi.org/10.1111/j.1365-313X.2008.03492.x
• Karan, H., Funk, C., Grabert, M., Oey, M., & Hankamer, B. (2019). Green bioplastics as part of a circular bioeconomy. Trends in Plant Science, 24, 237–249.
• Katiyar, R., Gurjar, B. R., Biswas, S., Pruthi, V., Kumar, N., & Kumar, P. (2017). Microalgae: An emerging source of energy-based bioproducts and a solution for environmental issues. Renewable and Sustainable Energy Reviews, 72, 1083–1093. https://doi.org/10.1016/j.rser.2016.10.028
• Köse, A., Gökpınar, Ş., & Açıkel, Y. (2020). Effect of continuous light on biomass production and biochemical composition of Scenedesmus acutus. Turkish Journal of Botany, 44 (5), 521–531. https://doi.org/10.3906/bot-2003-12
• Kumar, V., Kumar, R., Rawat, D., & Nanda, M. (2018). Synergistic dynamics of light, photoperiod and chemical stimulants influencing biomass and lipid productivity in Chlorella singularis. Applied Biological Chemistry, 61 (1), 43–52. https://doi.org/10.1007/s13765-017-0332-6
• Li, J., Zhu, D., Niu, J., Shen, S., & Wang, G. (2011). An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnology Advances, 29, 568–574. https://doi.org/10.1016/j.biotechadv.2011.04.001
• Lim, Y. A., Chong, M. N., Foo, S. C., & Ilankoon, I. M. S. K. (2021). Analysis of direct and indirect quantification methods of CO₂ fixation via microalgae cultivation in photobioreactors: A critical review. Renewable and Sustainable Energy Reviews, 137, 110579. https://doi.org/10.1016/j.rser.2020.110579
• Lio, E., Esposito, C., Paini, J., Gandolfi, S., Secundo, F., & Ottolina, G. (2025). Valorizing agro-industrial by-products for sustainable cultivation of Chlorella sorokiniana. Metabolites, 15 (3), 212. https://doi.org/10.3390/metabo15030212
• Liu, J., Sun, Z., Zhong, Y., Huang, J., & Chen, F. (2021). The regulation of carotenoid accumulation in microalgae by environmental stresses: A review. Marine Drugs, 19 (9), 518. https://doi.org/10.3390/md19090518
• Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. https://doi.org/10.1016/S0021-9258(19)52451-6
• Lorenz, R. T., & Cysewski, G. R. (2000). Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends in Biotechnology, 18 (4), 160–167.
• Maltsev, Y., Maltseva, K., Kulikovskiy, M., & Maltseva, S. (2021). Influence of light conditions on microalgae growth and content of lipids, carotenoids, and fatty acid composition. Biology, 10 (10), 1060. https://doi.org/10.3390/biology10101060
• Mata, T. M., Martins, A. A., & Caetano, N. S. (2010). Microalgae for biodiesel production and other applications: A review. Renewable and Sustainable Energy Reviews, 14, 217–232.
• Melo, R. G., de Andrade, A. F., Bezerra, R. P., Viana Marques, D. d. A., da Silva, V. A., Paz, S. T., et al. (2019). Hydrogel-based Chlorella vulgaris extracts: A new topical formulation for wound healing treatment. Journal of Applied Phycology, 31 (6), 3653–3663. https://doi.org/10.1007/s10811-019-01837-2
• Meng, Y., Jiang, J., Wang, H., Cao, X., Xue, S., Yang, Q., & Wang, W. (2015). The characteristics of TAG and EPA accumulation in Nannochloropsis oceanica IMET under different nitrogen supply regimes. Bioresource Technology, 179, 483–489. https://doi.org/10.1016/j.biortech.2014.12.012
• Mustafa, H. M., Hassan, M. S., & Abd El-Baky, H. H. (2013). Utilization of sugarcane molasses as an efficient organic carbon source for enhancing growth and biochemical composition of microalgae. Journal of Applied Phycology, 25 (6), 1869–1877. https://doi.org/10.1007/s10811-013-0014-z
• Patel, A. K., Tambat, V. S., Chen, C. W., Chauhan, A. S., Kumar, P., Vadrale, A. P., Huang, C. Y., Dong, C. D., & Singhania, R. R. (2022). Recent advancements in astaxanthin production from microalgae: A review. Bioresource Technology, 128030. https://doi.org/10.1016/j.biortech.2022.128030
• Qiao, T., Zhao, Y., Zhong, D.-b., & Yu, X. (2021). Hydrogen peroxide and salinity stress act synergistically to enhance lipids production in microalga. Algal Research, 53. https://doi.org/10.1016/j.algal.2020.102017
• Richmond, A. (2004). Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Science.
• Safi, C., Zebib, B., Merah, O., Pontalier, P.-Y., & Vaca-Garcia, C. (2014). Morphology, composition, production, processing and applications of Chlorella vulgaris: A review. Renewable and Sustainable Energy Reviews, 35, 265–278. https://doi.org/10.1016/j.rser.2014.04.007
• Sanchez-Saavedra, M. P., Jimenez, C., & Figueroa, F. L. (1996). Far-red light inhibits growth but promotes carotenoid accumulation in Dunaliella bardawil. Physiologia Plantarum, 98 (2), 419–423.
• Smith, K., Watson, A. W., Lonnie, M., Peeters, W. M., Oonincx, D., Tsoutsoura, N., … & Corfe, B. M. (2024). Meeting the global protein supply requirements of a growing and ageing population. European Journal of Nutrition, 63, 1425–1433. https://doi.org/10.1007/s00394-024-03358-2
• Ulianova, Y. V., Popov, A. M., Babichenko, N. P., Gorin, K. V., & Sergeeva, Y. E. (2020). Production of O/W emulsions containing astaxanthin by microfluidic devices. Nanotechnology in Russia, 15 (1), 63–68. https://doi.org/10.1134/S1995078020010103
• Wahidin, S., Idris, A., & Shaleh, S. R. M. (2013). The influence of light–dark cycles and photoperiod on lipid productivity of Nannochloropsis sp. in outdoor cultivation. Bioresource Technology, 148, 373–378. https://doi.org/10.1016/j.biortech.2013.09.035
• Wang, S. B., Chen, J., & Sommerfeld, M. (2013). Photoperiod-driven astaxanthin accumulation and cyst formation in Haematococcus pluvialis. Bioresource Technology, 143, 164–169. https://doi.org/10.1016/j.biortech.2013.05.106
• Wei, T., Gu, W., Li, J., Zhang, B., Pan, G., Zhu, D., & Wang, G. (2013). Effect of photoperiod on microalgae Haematococcus pluvialis. Chinese Bulletin of Botany, 48 (1), 70–78.
• Xu, Y., Ibrahim, I. M., & Harvey, P. J. (2016). The influence of photoperiod and light intensity on the growth and photosynthesis of Dunaliella salina (Chlorophyta) CCAP 19/30. Plant Physiology and Biochemistry, 106, 305–315. https://doi.org/10.1016/j.plaphy.2016.05.021
• Zhang, S., Zhang, L., Xu, G., Li, F., & Li, X. (2022). A review on biodiesel production from microalgae: Influencing parameters and recent advanced technologies. Frontiers in Microbiology, 13, 970028. https://doi.org/10.3389/fmicb.2022.970028
• Zhou, J., Wang, M., Bäuerl, C., Cortés-Macías, E., Calvo-Lerma, J., Carmen Collado, M., et al. (2023). The impact of liquid-pressurized extracts of Spirulina, Chlorella and Phaedactylum tricornutum on antioxidant, anti-inflammatory, bacterial growth effects and gut microbiota modulation. Food Chemistry, 401, 134083. https://doi.org/10.1016/j.foodchem.2022.134083