Abstract
Production characteristics of the microalgal Dunaliella salina were studied. The maximum rate of carotenoid synthesis was observed at high light intensity and nitrogen deficiency. The applicability of cytometric and fluorescent indicators to assess the degree of β-carotene accumulation and to estimate the D. salina functional state was studied. The shift in growth conditions, which stimulated carotenoid accumulation, affected the algae photosynthetic apparatus and weakly affected cell enzyme activity and cell viability. Fluorescein diacetate (FDA) fluorescence and the ratio of live cells can be used to assess the degree of lethal effect of an external factor on the physiological state of microalgal. The measurement of cell autofluorescence at a wavelength of 575 nm was a highly effective approach to the rapid assessment of β-carotene content in D. salina and to the prediction of β-carotene formation. The correlations were obtained between cell autofluorescence ratios in the orange (575 nm) and red (680 nm) spectrum areas and the ratio content of carotenoid and chlorophyll in algae cells. A linear relationship was established between the Fm/D750 ratio (the ratio of the maximum chlorophyll fluorescence at closed PS II reaction centers to the suspension optical density at 750 nm) and intracellular chlorophyll content in microalgal. These relationships allow assessing pigment content in the cells as well as predicting β-carotene formation in D. salina.
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The datasets generated and/or analyzed during the present study are available from the corresponding author on reasonable request.
References
Alvarez E, Nogueira E, Lopez-Urrutia A (2017) In vivo single-cell fluorescence and size scaling of phytoplankton chlorophyll content. Appl Environ Microbiol 83:e03317-e3416. https://doi.org/10.1128/AEM.03317-16
Andersen RA (2005) Algal culturing techniques. Elsevier Academic Press, New York
Antal TK, Venediktov PS, Matorin DN, Ostrowska M, Wozniak B, Rubin AB (2001) Measurement of phytoplankton photosynthesis rate using a pump-and-probe fluorometer. Oceanologia 43(3):291–313
Ben-Amotz A (1995) New mode of Dunaliella biotechnology: two-phase growth for β-carotene production. J Appl Phycol 7:65–68. https://doi.org/10.1007/BF00003552
Ben-Amotz A, Avron M (1983) On the factors which determine massive β-carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol 72:593–597. https://doi.org/10.1104/pp.72.3.593
Ben-Amotz A, Shaish A, Avron M (1989) Mode of action of the massively accumulated β-carotene of Dunaliella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol 91(3):1040–1043. https://doi.org/10.1104/pp.91.3.1040
Borovkov AB, Gudvilovich IN, Avsiyan AL (2020) Scale-up of Dunaliella salina cultivation: from strain selection to open ponds. J Appl Phycol 32(3):1545–1558. https://doi.org/10.1007/s10811-020-02104-5
Borowitzka MA (1990) The mass culture of Dunaliella salina. In: Technical resource papers regional workshop on the culture and utilization of Seaweads, vol. 2. http://www.fao.org/3/ab728e/. Accessed 16 Feb 2020
Borowitzka MA (2018) The ‘stress’ concept in microalgal biology—homeostasis, acclimation and adaptation. J Appl Phycol 30:2815–2825. https://doi.org/10.1007/s10811-018-1399-0
Brookes JD, Geary SM, Ganf GG, Burch MD (2000) Use of FDA and flow cytometry to assess metabolic activity as an indicator of nutrient status in phytoplankton. Mar Freshw Res 51:817–823. https://doi.org/10.1071/MF00048
Chen J, Wei D, Pohnert G (2017) Rapid estimation of astaxanthin and the carotenoid-to-chlorophyll ratio in the green microalga Chromochloris zofingiensis using flow cytometry. Mar Drugs 15(7):231–254. https://doi.org/10.3390/md15070231
d’Alessandro S, Havaux M (2019) Sensing β-carotene oxidation in photosystem II to master plant stress tolerance. New Phytol 223(4):1776–1783. https://doi.org/10.1111/nph.15924
Dhanam DS, Dhandayuthapani K (2013) Optimization of β-carotene production by marine microalga - Dunaliella salina. Int J Curr Microbiol Appl Sci 2:37–43
Fachet M, Hermsdorf D, Rihko-Struckmann L, Sundmacher K (2016) Flow cytometry enables dynamic tracking of algal stress response: a case study using carotenogenesis in Dunaliella salina. Algal Res 13:227–234. https://doi.org/10.1016/j.algal.2015.11.014
Falkowski PG, Kolber Z (1995) Variations in chlorophyll fluorescence yields in phytoplankton in the world oceans. Funct Plant Biol 22(2):341–355. https://doi.org/10.1071/PP9950341
García-González M, Moreno J, Cañavate JP, Anguis V, Prieto A, Manzano C, Florencio FJ, Guerrero MG (2003) Conditions for open-air outdoor culture of Dunaliella salina in southern Spain. J Appl Phycol 15:177–184. https://doi.org/10.1023/A:1023892520443
Han P, Virtanen M, Koponen J, Straskraba M (2000) Effect of photoinhibition on algal photosynthesis: a dynamic model. J Plankton Res 22(5):865–885. https://doi.org/10.1093/PLANKT/22.5.865
Hyka P, Lickova S, Přibyl P, Melzoch K, Kovar K (2013) Flow cytometry for the development of biotechnological processes with microalgal. Biotechnol Adv 31(1):2–16. https://doi.org/10.1016/j.biotechadv.2012.04.007
Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanz 167:191–194. https://doi.org/10.1016/S0015-3796(17)30778-3
Jimenez C, Pick U (1994) Differential stereoisomer compositions of β, β-carotene in thylakoids and in pigment globules in Dunaliella. J Plant Physiol 143(3):257–263. https://doi.org/10.1016/S0176-1617(11)81628-7
Kleinegris DM, van Es MA, Janssen M, Brandenburg WA, Wijffels RH (2010) Carotenoid fluorescence in Dunaliella salina. J Appl Phycol 22(5):645–649. https://doi.org/10.1007/s10811-010-9505-y
Lamers PP, Van de Laak CCW, Kaasenbrood PS, Lorier J, Janssen M, De Vos RC, Bino RJ, Wijffels RH (2010) Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol Bioeng 106:638–648. https://doi.org/10.1002/bit.22725
Lee CS, Yeo YSW, Sin TM (2012) Bleaching response of Symbiodinium (zooxanthellae): determination by flow cytometry. Cytom Part A 81(10):888–895. https://doi.org/10.1002/cyto.a.22111
Lers A, Biener Y, Zamir A (1990) Photoinduction of massive β-carotene accumulation by the alga Dunaliella bardawil: kinetics and dependence on gene activation. Plant Physiol 93(2):389–395. https://doi.org/10.1104/pp.93.2.389
Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. Curr Protoc Food Anal Chem 1(1):F4.3.1-F4.3.8. https://doi.org/10.1002/0471142913.faf0403s01
Mendoza H, De la Jara A, Freijanes K, Carmona L, Ramos AA, de Sousa DV, Serafim Varela JC (2008) Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains. Electron J Biotechnol 11:1–6. https://doi.org/10.4067/S0717-34582008000400005
Olson RJ, Zettler ER, DuRand MD (2018) Phytoplankton analysis using flow cytometry. Handbook of methods in aquatic microbial ecology, 1st edn. CRC Press, pp 175–186
Onji M, Sawabe T, Ezura Y (2000) An evaluation of viable staining dyes suitable for marine phytoplankton. Bull Fac Fish Mie Univ 51:151–158
Peperzak L, Brussaard CPD (2011) Flow cytometric applicability of fluorescent vitality probes on phytoplankton. J Phycol 47:692–702. https://doi.org/10.1111/j.1529-8817.2011.00991.x
Pisal DS, Lele SS (2005) Carotenoid production from microalga, Dunaliella salina. Indian J Biotechnol 4:476–483
Pogosyan SI, Matorin DN (2005) Variability in the condition of the photosynthetic system of the Black Sea phytoplankton. Oceanology 45:139–148
Polle JE, Jin E, Ben-Amotz A (2020) The alga Dunaliella revisited: looking back and moving forward with model and production organisms. Algal Res 49:1–5. https://doi.org/10.1016/j.algal.2020.101948
Prieto A, Cañavatea JP, García-González M (2011) Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes. J Biotechnol 151:180–185
Richmond A (2000) Microalgal biotechnology at the turn of the millennium: a personal view. J Appl Phycol 12:441–451. https://doi.org/10.1023/A:1008123131307
Rioboo C, González-Barreiro Ó, Abalde J, Cid Á (2011) Flow cytometric analysis of the encystment process induced by paraquat exposure in Haematococcus pluvialis (Chlorophyceae). Eur J Phycol 46:89–97. https://doi.org/10.1080/09670262.2011.561775
Shoman NY (2015) The dynamics of the intracellular contents of carbon, nitrogen, and chlorophyll a under conditions of batch growth of the diatom Phaeodactylum tricornutum (Bohlin, 1897) at different light intensities. Russ J Mar Biol 41:356–362. https://doi.org/10.1134/S1063074015050132
Shoman NYU, Akimov AI (2013) Effect of irradiance and temperature on specific growth rate of diatoms Phaeodactulum tricornutum and Nitzschia sp. No 3. Morskoj Ehkologicheskij Zhurnal 12:85–91 (In Russian)
Shoman N, Solomonova E, Akimov A (2021) Application of structural, functional, fluorescent, and cytometric indicators for assessing physiological state of marine diatoms under different light growth conditions. Turk J Bot 45:511–521. https://doi.org/10.3906/bot-2102-39
Solovchenko AE (2013) Physiology and adaptive significance of secondary carotenogenesis in green microalgae. Russ J Plant Physiol 60:1–13. https://doi.org/10.1134/S1021443713010081
Solomonova ES, Akimov AI (2013) The assessment of functional status of Chlorella vulgaris suboblonga by flow cytometry and variable fluorescence. Morskoj Ehkologicheskij Zhurnal 1:73–81 (In Russian)
Solomonova ES, Akimov AI (2021) Assessing the physiological state of microalgal using cytometric and fluorescent indicators. Russ J Plant Physiol 68:981–987. https://doi.org/10.1134/S1021443721050204
Solomonova ES, Mykhanov VS (2011) Flow cytometry for the assessment of physiological active cells in batch cultures of Phaeodactylum tricornutum and Nitzschia specia. Morskoj Ehkologicheskij Zhurnal 10:67–72 (In Russian)
Solomonova ES, Akimov AI, Shoman NYu (2018) Investigation of applicability of relative variable chlorophyll fluorescence and diacetate fluorescein staining for estimation and control of the state of algae culture on the example Phaeodactylum tricornutum (Phaeodactylaceae). Botanicheskij Zhurnal 103:1177–1191. https://doi.org/10.7868/S0006813618090089. (In Russian)
Sosik HM, Chisholm SW, Olson RJ (1989) Chlorophyll fluorescence from single cells: interpretation of flow cytometric signals. Limnol Oceanogr 8:1749–1761. https://doi.org/10.4319/lo.1989.34.8.1749
Sui Y, Muys M, Van de Waal DB, D’Adamo S, Vermeir P, Fernandes TV, Vlaeminck SE (2019) Enhancement of co-production of nutritional protein and carotenoids in Dunaliella salina using a two-phase cultivation assisted by nitrogen level and light intensity. Bioresour Technol 287:121398. https://doi.org/10.1016/j.biortech.2019.121398
Ukibe K, Katsuragi T, Tani Y, Takagi H (2008) Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry. FEMS Microbiol Lett 286:241–248. https://doi.org/10.1111/j.1574-6968.2008.01278.x
Zhu C, Zhai X, Jia J, Wang J, Han D, Li Y, Yajie T, Chi Z (2018) Seawater desalination concentrate for cultivation of Dunaliella salina with floating photobioreactor to produce β-carotene. Algal Res 35:319–324. https://doi.org/10.1016/j.algal.2018.08.035
Funding
This work was supported by the Russian Foundation for Basic Research (grant number 16–34-00388) and A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (state project № 121041400077–1 “Functional, metabolic and toxicological aspects of aquatic organisms existence and their populations in habitats with different physical and chemical regime”).
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AA, formal analysis, investigation, and writing—review and editing. ES, methodology, investigation, formal analysis, and writing—original draft. NS, investigation, formal analysis, and writing—review and editing.
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Akimov, A.I., Solomonova, E.S. & Shoman, N.Y. Estimation physiological state and carotenoid content of Dunaliella salina (Teod.) using flow cytometry and variable fluorescence methods. Aquacult Int 32, 161–174 (2024). https://doi.org/10.1007/s10499-023-01153-0
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DOI: https://doi.org/10.1007/s10499-023-01153-0