Production of natural pigment by Dunaliella salina: Key factors screening through Placket-Burman design
Subject Areas : food biotechnologyMaryam Araj-Shirvani 1 , Masoud Honarvar 2 , Mahshid Jahadi 3 , Maryam Mizani 4
1 - Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 - Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 - Department of Food Science and Technology, Faculty of Agriculture, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
4 - Department of Food Science and technology, Science and Research Branch, Islamic AzadUniversity, Tehran, Iran.
Keywords: Dunaliella salina, Placket-Burman, Carotenoid,
Abstract :
The distinctive biological and technical characteristics of Dunaliella, including the need for cheap culture medium, fast growth rate, simple genetic manipulation, and easy scale-up methods, have made this microorganism the prime candidate for molecular agriculture, and a suitable host for the production of antibodies, vaccines and valuable compounds such as carotenoids, glycerol, unsaturated fats, vitamins, proteins, and bioactive substances. Therefore, this alga may be one of the most appropriate models to investigate and utilize to produce useful compounds by optimizing its environment. This study investigated the feasibility of high biomass and pigment (chlorophyll a, chlorophyll b, and carotenoids) accumulation in a species of Dunaliella salina native to Iran by creating mixotrophic conditions using the Placket-Burman screening design. In this design, the effects of 10 variables, including pH, light intensity, carbon source (date waste), Nitrogen source, NaCl, Fe (ferrous sulfate), vitamin B1, vitamin B12, Incubation time and Inoculum concentration were investigated. The results showed the significant effects of carbon source, sodium chloride, pH, inoculum concentration, and incubation time on biomass accumulation the value of which varied from 1.90-8.54 g/100. All variables except vitamins had a significant effect on the accumulation of chlorophyll and increased its amount from 0.60-1.35 mg/l. While variables such as pH, incubation time, sodium chloride, light intensity, and iron effected the accumulation of chlorophyll b significantly. pH, carbon source, sodium chloride, nitrogen source, and light intensity affected the accumulation of carotenoids, and the highest amounts of chlorophyll b and carotenoids were obtained as 2.8 and 8.6 mg/l, respectively.
Abdel-Wahab, M. A., El-Samawaty, A. M. A., Elgorban, A. M., & Bahkali, A. H. (2022). Utilization of low-cost substrates for the production of high biomass, lipid and docosahexaenoic acid (DHA) using local native strain Aurantiochytrium sp. YB-05. Journal of King Saud University - Science, 34(7),102224. doi:10.1016/j.jksus.2022.102224
Abedin, R. M. A., & Taha, H. M. (2008). Antibacterial and antifungal activity of cyanobacteria and green microalgae. Evaluation of medium components by Plackett-Burman design for antimicrobial activity of Spirulina platensis. Global Journal of Biotechnology and Biochemistry, 8(3), 22-31.
Andrade, M. R., & Costa, J. A.V. (2007). Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture, 264(1-4), 130-4.
doi:10.1016/j.aquaculture.2006.11.021
Araj-Shirvani, M., Honarvar, M., Jahadi, M., & Mizani, M. (2024). Biochemical profile of Dunaliella isolates from different regions of Iran with a focus on pharmaceutical and nutraceutical potential applications. Food Science & Nutrition
doi:10.1002/fsn3.4137
Aziz, M. A., Kassim, K. A., Shokravi, Z., Jakarni, F. M., Liu, H. Y., Zaini, N., et al. (2020). Two-stage cultivation strategy for simultaneous increases in growth rate and lipid content of microalgae: A review. Renewable and Sustainable Energy Reviews, 119(4), 109621.
doi:10.1016/j.rser.2019.109621
Banayan, S., Jahadi, M., & Khosravi-Darani, K. (2022). Pigment Productions by Spirulina platensis as a Renewable Resource. J Appl Biotechnol Rep, 9(2),614-621.
https://doi.org/10.30491/jabr.2021.292076.1406
Baquerisse, D., Nouals, S., Isambert, A., dos Santos, P. F., & Durand, G. (1999). Modelling of a continuous pilot photobioreactor for microalgae production. Progress in Industrial Microbiology, 35, 335-42. doi:10.1016/S0079-6352(99)80125-8
Benavente-Valdés, J. R., Aguilar, C., Contreras-Esquivel, J. C., Méndez-Zavala, A., & Montañez, J. (2016). Strategies to enhance the production of photosynthetic pigments and lipids in chlorophycae species. Biotechnology Reports,10, 117-25.
doi:10.1016/j.btre.2016.04.001
Besbes, S., Drira, L., Blecker, C., Deroanne, C., & Attia, H. (2009). Adding value to hard date (Phoenix dactylifera L.): Compositional, functional and sensory characteristics of date jam. Food Chemistry, 112(2), 406-11. doi:10.1016/j.foodchem.2008.05.093
Borowitzka, M. A., & Siva, C. J. (2007). The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species. Journal of Applied Phycology, 19(5), 567-90. doi:10.1007/s10811-007-9171-x
Carvalho, A.P., Silva, S. O., Baptista, J. M., & Malcata, F.X. (2010). Light requirements in microalgal photobioreactors: an overview of biophotonic aspects. Appl Microbiol Biotechnol, 89(5), 1275-88. doi:10.1007/s00253-010-3047-8
Chavoshi, Z. Z., & Shariati, M. (2019). Lipid production in Dunaliella salina under autotrophic, heterotrophic, and mixotrophic conditions. Biologia, 74, 1579–90.
doi:10.1590/s1679-87592019024906709
Colusse, G. A., Mendes, C. R. B., Duarte, M. E. R., Carvalho, J. C. D., & Noseda, M. D. (2020). Effects of different culture media on physiological features and laboratory scale production cost of Dunaliella salina. Biotechnology Reports, 27, e00508.
https://doi.org/10.1016/j.btre.2020.e00508
Croft, M. T., Warren, M. J., & Smith, A.G. (2006). Algae need their vitamins. ASM Journals, 6(8), 1175–1183. https://doi.org/10.1128/ec.00097-06
El-Awady, R. M., El-Sayed, A. B., El-Zabalawy, K. M., & El-Mohandes, M. (2020). Bio-mass Production of Chlorella vulgaris grown on date wastes under different stress conditions. Al-Azhar Journal of Agricultural Research, 45(2), 62-75.
doi:10.21608/ajar.2020.149423
El-Sheekh, M. M., Khairy, H. M., Gheda, S. F., & El-Shenody, R. A. (2016). Application ofPlackett–Burman design for the high production of some valuable metabolites in marine alga Nannochloropsis oculata. The Egyptian Journal of Aquatic Research, 42(1), 57-64. doi:10.1016/j.ejar.2015.10.001
Espinosa-Gonzalez, I., Parashar, A., & Bressler, D. C. (2014). Heterotrophic growth and lipid accumulation of Chlorella protothecoides in whey permeate, a dairy by-product stream, for biofuel production. Bioresource Technology, 155, 170-176.
doi:10.1016/j.biortech.2013.12.028
Fawzy, M. A., & Gomaa, M. (2020). Pretreated fucoidan and alginate from a brown seaweed as a substantial carbon source for promoting biomass, lipid, biochemical constituents and biodiesel quality of Dunaliella salina. Renewable Energy, 157, 246-55.
doi:10.1016/j.renene.2020.05.065
Gantar. M., & Svirčev. Z. (2008). Microalgae and cyanobacteria: food for thought 1. J Phycol, 44(2), 260-8. doi:10.1111/j.1529-8817.2008.00469.x
García, F., Freile-Pelegrín, Y., & Robledo, D. (2007). Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico. Bioresource Technology, 98(7),1359–65. doi:10.1016/j.biortech.2006.05.051
Giordano, M. (2001). Interactions between C and N metabolism in Dunaliella salina cells cultured at elevated CO 2 and high N concentrations. Journal of Plant Physiology, 158(5), 577-81. doi:10.1078/0176-1617-00234
Goericke, R., & Montoya, J. P. (1998). Estimating the contribution of microalgal taxa to chlorophyll a in the field-variations of pigment ratios under nutrient- and light-limited growth. Marine Ecology Progress Series, 169, 97-112. doi:10.3354/meps169097
Kadkhodaei, S., Abbasiliasi, S., Shun, T.J., Fard Masoumi, H. R., Mohamed, M.S., Movahedi, A., et al. (2015). Enhancement of protein production by microalgae Dunaliella salina under mixotrophic condition using response surface methodology. RSC Advances, 5(48).
doi:10.1039/C5RA04546K
Koutra, E., Kopsahelis, A., Manolia, M., Grammatikopoulos, G., & Kornaros, M. (2019). Effect of organic carbon and nutrient supplementation on the digestategrown microalga, Parachlorella kessleri. Bioresource Technology, 294, 122-232.
doi:10.1016/j.biortech.2019.122232
Koyandea, A. K., Chew, K. W., Rambabub, K., Taoc, Y., & Chud, D.T. (2019). Show Pl. Microalgae: A potential alternative to health supplementation for humans. Food Science and Human Wellness, 8(1),16-24. doi: 10.1016/j.fshw.2019.03.001.
Kumar, Y., Kaur, S., Kheto, A., Munshi, M., Sarkar, A., Pandey, H.O., et al. (2022) Cultivation of microalgae on food waste: Recent advances and way forward. Bioresource Technology, 363, 127834. doi:10.1016/j.biortech.2022.127834
Lamers, P. P., Janssen, M., De Vos, R .C., Bino, R. J., & Wijffels, R. H. (2012). Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga. Biotechnology and Bioengineering, 162(1), 21-27.
doi: 10.1016/j.jbiotec.2012.04.018
Lichtenthaler, H. K., & Buschmann, C. (2001). Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy. Current Protocols in Food Analytical Chemistry, 1, F4(1). doi:10.1002/0471142913.faf0403s01
Liu, J., Sun, Z., Zhong, Y., Gerken, H., Huang, J., & Chen, F. (2013). Utilization of cane molasses towards cost-saving astaxanthin production by a Chlorella zofingiensis mutant. Journal of Applied Phycology, 25(5). 1447–56.
doi:10.1007/s10811-013-9974-x
Luo, S. W., Alimujiang, A., Cui, J., Chen, T. T., Balamurugan, S., Zheng, J. W., et al. (2020). Molybdenum disulfide nanoparticles concurrently stimulated biomass and β-carotene accumulation in Dunaliella salina. Bioresource Technology, 320, 124391.
doi:10.1016/j.biortech.2020.124391
Malakar, B., Das, D., & Mohanty, K. (2022). Utilization of waste peel extract for cultivation of microalgal isolates: a study of lipid productivity and growth kinetics. Biomass Conversion and Biorefinery, 13(18),17017-26.
https://link.springer.com/article/10.1007%2Fs13399-022-02313-7
McLachlan, J. (1964). Some considerations of the growth of marine algae in artificial media. Canadian Journal of Microbiology, 10(5), 769-82. doi:10.1139/m64-098
Mojaat, M., Pruvost, J., Foucault, A., & Legrand, J. (2008). Effect of organic carbon sources and Fe2+ ions on growth and β-carotene accumulation by Dunaliella salina. Biochemical Engineering Journal, 39(1),177–84. doi:10.1016/j.bej.2007.09.009
Morowvat, M. H., & Ghasemi, Y. (2016). Culture medium optimization for enhanced β-carotene and biomass production by Dunaliella salina in mixotrophic culture. Biocatalysis and Agricultural Biotechnology, 7, 217-23. doi:10.1016/j.bcab.2016.06.008
Najafi, G., Ghobadian, B., Tavakoli, T., & Yusaf, T. (2009). Potential of bioethanol production from agricultural wastes in Iran. Renewable and Sustainable Energy Reviews, 13(6-7), 1418-2 doi:10.1016/j.rser.2008.08.010
Pal, A., Kamthania, M. C., & Kumar, A. (2014). Bioactive Compounds and Properties of Seaweeds. Open Access Library Journal, 1(4). doi:10.4236/oalib.1100752
Pereira, S., & Otero, A. (2019). Effect of light quality on carotenogenic and non-carotenogenic species of the genus Dunaliella under nitrogen deficiency. Algal Research, 44, 101725. doi:10.1016/j.algal.2019.101725
Polle, J. E., Jin, E., & Ben-Amotz, A. (2020). The alga Dunaliella revisited: Looking back and moving forward with model and production organisms. Algal Research, 49, 101948. doi:10.1016/j.algal.2020.101948
Priyadharshini, S. D., & Bakthavatsalam, A. k. (2016). Optimization of phenol degradation by the microalga Chlorella pyrenoidosa using Plackett–Burman Design and Response Surface Methodology. Bioresource Technology, 207, 150-156.
doi:10.1016/j.biortech.2016.01.138
Priyadarshani, I., & Rath, B. (2012). Bioactive compounds from microalgae And cyanobacteria: utility and applications. IJPSR, 3 (11), 4123-30. doi: 10.13040/IJPSR.0975-8232.3(11).4123-30
Qin, R., Li, Y., Zhang, L., & Liu, J. (2021). The effect of salinity shock on the growth and rapid light curve of dunaliella salina. Aquaculture Research, 52(6), 2569-2579.
https://doi.org/10.1111/are.15105
Ra, C. H., Kang, C. H., Kim, N. K., Lee, C.G., & Kim, S. K. (2015). Cultivation of four microalgae for biomass and oil production using a two-stage culture strategy with salt stress. Renewable Energy, 80, 117-22. doi:10.1016/j.renene.2015.02.002
Reshma, R., Devi, K. C., Kumar, S. D., Santhanam, P., Perumal, P., Krishnaveni N., et al. (2021). Enhancement of pigments production in the green microalga Dunaliella salina (PSBDU05) under optimized culture condition. Bioresource Technology Reports, 14, 100672.
doi:10.1016/j.biteb.2021.100672
Rehman, M., Kesharvani, S., Dwivedi, G., & Suneja, K. G. (2022). Impact of cultivation conditions on microalgae biomass productivity and lipid content. Materials Today: Proceedings, 56(1), 282-290. https://doi.org/10.1016/j.matpr.2022.01.152
Schoefs, B. (2002). Chlorophyll and carotenoid analysis in food products. Properties of the pigments and methods of analysis. Trends in Food Science & Technology, 13(11), 361–71.
doi:10.1016/S0924-2244(02)00182-6
Spolaore, P., Joannis-Cassan, C., Duran, E., & Isambert, A. (2006) Commercial applications of microalgae. J Biosci Bioeng, 101(2), 87-96. doi:10.1263/jbb.101.87
Vanaja, K., & Shobha Rani, R. H. (2007). Design of Experiments: Concept and Applications of Plackett Burman Design. Clinical Research and Regulatory Affairs, 24(1), 1–23.
doi:10.1080/10601330701220520
Vassiliev, I. R., Kolber, Z., Wyman, K. D., Mauzerall, D., Shukla, V. K., & Falkowski, P.G. (1995). Effects of Iron Limitation on Photosystem II Composition and Light Utilization in Dunaliella tertiolecta. Plant Physiol, 109(3), 963–72. doi:10.1104/pp.109.3.963
Varsano, T., Kaftan, D., & Pick, U. (2003). Effects of Iron Deficiency on Thylakoid Membrane Structure and Composition in the Alga Dunaliella salina. Journal of Plant Nutrition, 26(10-11), 2197–210. doi:10.1081/PLN-120024275
Vo, T.H., Mai, T., Vu, H., Van, D., Dao, H., Tran, P., Nguyen, N. et al. Effect of Osmotic Stress and Nutrient Starvation on the Growth, Carotenoid and Lipid Accumulation in Dunaliella salina A9. Research in Plant Sciences, 5(1), 1-8.
doi:10.12691/plant-5-1-1
Wood, A.M. (1979). Chlorophill a:b ratios in marine planktonic algae1. Journal of Phycology, 15 (13), 330-2. doi:10.1111/j.0022-3646.1979.00330.x
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, 305-15. doi:10.1016/j.plaphy.2016.05.021
Xu, Y., Ibrahim, I. M., Wosu, C. I., Ben-Amotz, A., & Harvey, P. J. (2018). Potential of New Isolates of Dunaliella Salina for Natural β-Carotene Production. Biology, 7(1), 14.
doi:10.3390/biology7010014
Zhu, Z.H., & Jiang, J.G. (2008). Continuous cultivation of Dunaliella salina in photobioreactor for the production of β-carotene. European Food Research and Technology, 227(3), 953-9. doi:10.1007/s00217-007-0789-3