The production of organic deuterated compounds in microalgal systems represents a cheaper and more versatile alternative to more complicated chemical synthesis. In the present study, we investigate the autotrophic growth of two microalgae, Chlamydomonas reinhardtii and Desmodesmus quadricauda, in medium containing high doses of deuterated water, D2O. The growth of such cultures was evaluated in the context of the intensity of incident light, since light is a critical factor in the management of autotrophic algal cultures. Deuteration increases the light sensitivity of both model organisms, resulting in increased levels of singlet oxygen and poorer photosynthetic performance. Our results also show a slowdown in growth and cell division processes with increasing D2O concentrations. At the same time, impaired cell division leads to cell enlargement and accumulation of highly deuterated compounds, especially energy-storing molecules. Thus, considering the specifics of highly deuterated cultures and using the growth conditions proposed in this study, it is possible to obtain highly deuterated algal biomass, which could be a valuable source of deuterated organic compounds.

Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Prague Czechia

Faculty of Science University of South Bohemia České Budějovice Czechia

Institute of Physics Faculty of Mathematics and Physics Charles University Prague Czechia

Laboratory of Cell Cycles of Algae Centre Algatech Institute of Microbiology of the Czech Academy of Sciences Třeboň Czechia

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Amini Khoeyi Z., Seyfabadi J., Ramezanpour Z. (2011). Effect of light intensity and photoperiod on biomass and fatty acid composition of the microalgae, Chlorella vulgaris . Aquacult. Int. 20 (1), 41–49. 10.1007/s10499-011-9440-1 DOI

Annan J. N. (2014). Growth and photosynthesis response of the green alga, Picochlorum oklahomensis to iron limitation and salinity stress. Int. J. Plant Physiol. Biochem. 6 (1), 7–18. 10.5897/ijppb2013.0198 DOI

Becker E. W. (2007). Micro-algae as a source of protein. Biotechnol. Adv. 25 (2), 207–210. 10.1016/j.biotechadv.2006.11.002 PubMed DOI

Bhosale P., Serban B., Bernstein P. S. (2006). Production of deuterated lutein by Chlorella protothecoides and its detection by mass spectrometric methods. Biotechnol. Lett. 28, 1371–1375. 10.1007/s10529-006-9105-8 PubMed DOI

Bialevich V., Zachleder V., Bisova K. (2022). The effect of variable light source and light Intensity on the growth of three algal species. Cells 11 (8), 1293. 10.3390/cells11081293 PubMed DOI PMC

Bigeleisen J., Mayer M. G. (1947). Calculation of equilibrium constants for isotopic exchange reactions. J. Chem. Phys. 15 (5), 261–267. 10.1063/1.1746492 DOI

Bisova K., Zachleder V. (2014). Cell-cycle regulation in green algae dividing by multiple fission. J. Exp. Bot. 65 (10), 2585–2602. 10.1093/jxb/ert466 PubMed DOI

Brennan L., Owende P. (2010). Biofuels from microalgae - a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 14 (2), 557–577. 10.1016/j.rser.2009.10.009 DOI

Cargnin S., Serafini M., Pirali T. (2019). A primer of deuterium in drug design. Future Med. Chem. 11 (16), 2039–2042. 10.4155/fmc-2019-0183 PubMed DOI

Chavez-Fuentes P., Ruiz-Marin A., Canedo-Lopez Y. (2018). Biodiesel synthesis from Chlorella vulgaris under effect of nitrogen limitation, intensity and quality light: estimation on the based fatty acids profiles. Mol. Biol. Rep. 45 (5), 1145–1154. 10.1007/s11033-018-4266-9 PubMed DOI

Crespi H. L., Smith U., Katz J. J. (1968). Phycocyanobilin. Structure and exchange studies by nuclear magnetic resonance and its mode of attachment in phycocyanin. A model for phytochrome. Biochemistry 7 (6), 2232–2242. 10.1021/bi00846a028 PubMed DOI

de Carpentier F., Lemaire S. D., Danon A. (2019). When unity is strength: the strategies used by Chlamydomonas to survive environmental stresses. Cells 8 (11), 1307. 10.3390/cells8111307 PubMed DOI PMC

de Kouchkovsky Y., Haraux F., Sigalat C. (1982). Effect of hydrogen‐deuterium exchange on energy coupled processes in thylakoids. FEBS Lett. 139 (2), 245–249. 10.1016/0014-5793(82)80862-4 DOI

Deeba F., Kumar K. K., Gaur N. A. (2020). “Overview of microbial production of omega-3-polyunsaturated fatty acid,” in Nutraceutical fatty acids from oleaginous microalgae: a human health perspective. Editors Patel A. K., Matsakas L. (Hoboken: John Wiley & Sons; ).

Delente J. (1987). Perdeuterated chemicals from D2O-grown microalgae. Trends Biotechnol. 5, 159–160. 10.1016/0167-7799(87)90088-6 DOI

Difusa A., Talukdar J., Kalita M. C., Mohanty K., Goud V. V. (2015). Effect of light intensity and pH condition on the growth, biomass and lipid content of microalgae Scenedesmus species. Biofuels 6 (1-2), 37–44. 10.1080/17597269.2015.1045274 DOI

Erickson E., Wakao S., Niyogi K. K. (2015). Light stress and photoprotection in Chlamydomonas reinhardtii . Plant J. 82, 449–465. 10.1111/tpj.12825 PubMed DOI

Eriksen N. T. (2016). Research trends in the dominating microalgal pigments, β-carotene, astaxanthin, and phycocyanin used in feed, in foods, and in health applications. J. Nutr. Food Sci. 6 (3). 10.4172/2155-9600.1000507 DOI

Fischer B. B., Wiesendanger M., Eggen R. I. (2006). Growth condition-dependent sensitivity, photodamage and stress response of Chlamydomonas reinhardtii exposed to high light conditions. Plant Cell Physiol. 47 (8), 1135–1145. 10.1093/pcp/pcj085 PubMed DOI

Fu W., Guethmundsson O., Paglia G., Herjolfsson G., Andresson O. S., Palsson B. O., et al. (2013). Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution. Appl. Microbiol. Biotechnol. 97 (6), 2395–2403. 10.1007/s00253-012-4502-5 PubMed DOI PMC

Garg H., Loughlin P. C., Willows R. D., Chen M. (2017). The C2(1)-formyl group in chlorophyll f originates from molecular oxygen. J. Biol. Chem. 292 (47), 19279–19289. 10.1074/jbc.M117.814756 PubMed DOI PMC

Gireesh T., Jayadeep A., Rajasekharan K. N., Menon V. P., Vairamany M., Tang G., et al. (2001). Production of deuterated b-carotene by metabolic labelling of Spirulina platensis . Biotechnol. Lett. 23, 447–449. 10.1023/A:1010378401621 DOI

Harris E. H. (2001). Chlamydomonas as a model organism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 363–406. 10.1146/annurev.arplant.52.1.363 PubMed DOI

Hattori A., Crespi H. L., Katz J. J. (1965). Effect of side-chain deuteration on protein stability. Biochemistry 4 (7), 1213–1225. 10.1021/bi00883a002 PubMed DOI

Hlavová M., Vítová M., Bišová K. (2016). “Synchronization of green algae by light and dark regimes for cell cycle and cell division studies,” in Plant cell division. Editor Caillaud M.-C. (New York, Heilderberg, Dordrecht, London: Springer Science; ), 3–16. PubMed

Ivanov I. N., Zachleder V., Vítová M., Barbosa M. J., Bišová K. (2021). Starch production in Chlamydomonas reinhardtii through supraoptimal temperature in a pilot-scale photobioreactor. Cells 10 (5), 1084. 10.3390/cells10051084 PubMed DOI PMC

Jerez C. G., Malapascua J. R., Sergejevova M., Figueroa F. L., Masojidek J. (2015). Effect of nutrient starvation under high irradiance on lipid and starch accumulation in Chlorella fusca (Chlorophyta). Mar. Biotechnol. 18 (1), 24–36. 10.1007/s10126-015-9664-6 PubMed DOI

Kajiwara K., Kearns D. R. (1973). Direct spectroscopic evidence for a deuterium solvent effect on the lifetime of singlet oxygen in water. J. Am. Chem. Soc. 95 (18), 5886–5890. 10.1021/ja00799a009 DOI

Katsuda T., Lababpour A., Shimahara K., Katoh S. (2004). Astaxanthin production by Haematococcus pluvialis under illumination with LEDs. Enzyme Microb. Technol. 35 (1), 81–86. 10.1016/j.enzmictec.2004.03.016 DOI

Khona D. K., Shirolikar S. M., Gawde K. K., Hom E., Deodhar M. A., D'Souza J. S. (2016). Characterization of salt stress-induced palmelloids in the green alga, Chlamydomonas reinhardtii . Algal Res. 16, 434–448. 10.1016/j.algal.2016.03.035 DOI

Kollars B., Geraets R. (2008). Effects of deuterium on Chlamydomonas reinhardtii . J. Under. Res. 6 (1), 113–117.

Kotyk A., Dvořáková M., Koryta J. (1990). Deuterons cannot replace protons in active transport processes in yeast. FEBS Lett. 264 (2), 203–205. 10.1016/0014-5793(90)80248-h PubMed DOI

Krieger-Liszkay A. (2005). Singlet oxygen production in photosynthesis. J. Exp. Bot. 56 (411), 337–346. 10.1093/jxb/erh237 PubMed DOI

Kselíková V., Zachleder V., Bišová K. (2021). To divide or not to divide? How deuterium affects growth and division of Chlamydomonas reinhardtii . Biomolecules 11 (6), 861. 10.3390/biom11060861 PubMed DOI PMC

Kubásek J., Urban O., Šantrůček J. (2013). C4 plants use fluctuating light less efficiently than do C3 plants: a study of growth, photosynthesis and carbon isotope discrimination. Physiol. Plant. 149 (4), 528–539. 10.1111/ppl.12057 PubMed DOI

Kuroiwa T., Suzuki T. (1980). An improved method for the demonstration of the in situ chloroplast nuclei in higher plants. Cell Struct. Funct. 5, 195–197. 10.1247/csf.5.195 DOI

Kushner D. J., Baker A., Dunstall T. G. (1999). Pharmacological uses and perspectives of heavy water and deuterated compounds. Can. J. Physiol. Pharmacol. 77 (2), 79–88. 10.1139/y99-005 PubMed DOI

Lee E., Jalalizadeh M., Zhang Q. (2015). Growth kinetic models for microalgae cultivation: a review. Algal Res. 12, 497–512. 10.1016/j.algal.2015.10.004 DOI

Lee C., Ahn J. W., Kim J. B., Kim J. Y., Choi Y. E. (2018). Comparative transcriptome analysis of Haematococcus pluvialis on astaxanthin biosynthesis in response to irradiation with red or blue LED wavelength. World J. Microbiol. Biotechnol. 34 (7), 96. 10.1007/s11274-018-2459-y PubMed DOI

Lehmann W. D. (2016). A timeline of stable isotopes and mass spectrometry in the life sciences. Mass Spectrom. Rev. 36 (1), 58–85. 10.1002/mas.21497 PubMed DOI

León R., Galván F. (1999). Interaction between saline stress and photoinhibition of photosynthesis in the freshwater green algae Chlamydomonas reinhardtii. Implications for glycerol photoproduction. Plant Physiol. Biochem. 3, 623–628. 10.1016/S0981-9428(00)80115-1 DOI

Li X., Manuel J., Slavens S., Crunkleton D. W., Johannes T. W. (2021). Interactive effects of light quality and culturing temperature on algal cell size, biomass doubling time, protein content, and carbohydrate content. Appl. Microbiol. Biotechnol. 105, 587–597. 10.1007/s00253-020-11068-y PubMed DOI

Madadi R., Maljaee H., Serafim L. S., Ventura S. P. M. (2021). Microalgae as contributors to produce biopolymers. Mar. Drugs 19 (8), 466. 10.3390/md19080466 PubMed DOI PMC

Moudříková Š., Mojzeš P., Zachleder V., Pfaff C., Behrendt D., Nedbal L. (2016). Raman and fluorescence microscopy sensing energy-transducing and energy-storing structures in microalgae. Algal Res. 16, 224–232. 10.1016/j.algal.2016.03.016 DOI

Oldenhof H., Bisova K., van den Ende H., Zachleder V. (2004). Effect of red and blue light on the timing of cyclin-dependent kinase activity and the timing of cell division in Chlamydomonas reinhardtii . Plant Physiol. Biochem. 42 (4), 341–348. 10.1016/j.plaphy.2004.02.002 PubMed DOI

Olgun A. (2007). Biological effects of deuteronation: ATP synthase as an example. Theor. Biol. Med. Model. 4, 9. 10.1186/1742-4682-4-9 PubMed DOI PMC

Poppe F., Hanelt D., Wiencke C. (2002). Changes in ultrastructure, photosynthetic activity and pigments in the Antarctic red alga Palmaria decipiens during acclimation to UV radiation. Bot. Mar. 45 (3), 253–261. 10.1515/bot.2002.024 DOI

Possmayer M., Berardi G., Beall B. F., Trick C. G., Huner N. P., Maxwell D. P. (2011). Plasticity of the psychrophilic green alga Chlamydomonas Raudensis (Uwo 241) (Chlorophyta) to supraoptimal temperature stress. J. Phycol. 47 (5), 1098–1109. 10.1111/j.1529-8817.2011.01047.x PubMed DOI

Saha S. K., Hayes J., Moane S., Murray P. (2013). Tagging of biomolecules with deuterated water (D2O) in commercially important microalgae. Biotechnol. Lett. 35 (7), 1067–1072. 10.1007/s10529-013-1176-8 PubMed DOI

Salomonsson L., Branden G., Brzezinski P. (2008). Deuterium isotope effect of proton pumping in cytochrome c oxidase. Biochim. Biophys. Acta Bioenergetics 1777 (4), 343–350. 10.1016/j.bbabio.2007.09.009 PubMed DOI

Shu C. H., Tsai C. C., Liao W. H., Chen K. Y., Huang H. C. (2012). Effects of light quality on the accumulation of oil in a mixed culture of Chlorella sp. and Saccharomyces cerevisiae . J. Chem. Technol. Biotechnol. 87 (5), 601–607. 10.1002/jctb.2750 DOI

Sueoka N. (1960). Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii . Proc. Natl. Acad. Sci. U. S. A. 46, 83–91. 10.1073/pnas.46.1.83 PubMed DOI PMC

Torres-Romero I., Kong F., Legeret B., Beisson F., Peltier G., Li-Beisson Y. (2019). Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil. Biochimie 169, 54–61. 10.1016/j.biochi.2019.09.017 PubMed DOI

Unno K., Busujima H., Shimba S., Narita K., Okada S. (1987). Characteristics of growth and deuterium incorporation in Chlorella ellipsoidea grown in deuterium oxide. Chem. Pharm. Bull. 36, 1828–1833. 10.1248/cpb.36.1828 DOI

Unno K., Ando I., Hagima N., Yokogaki S., Koike C., Okada S. (1992). Growth delay and intracellular changes in Chlorella ellipsoidea C-27 as a result of deuteration. Plant Cell Physiol. 33 (7), 963–969. 10.1093/oxfordjournals.pcp.a078348 DOI

Vasilescu V., Katona E. (1986). Deuteration as a tool in investigating the role of water in the structure and function of excitable membranes. Methods Enzymol. 127, 662–678. 10.1016/0076-6879(86)27052-4 PubMed DOI

Vitova M., Bisova K., Kawano S., Zachleder V. (2015). Accumulation of energy reserves in algae: from cell cycles to biotechnological applications. Biotechnol. Adv. 33 (6), 1204–1218. 10.1016/j.biotechadv.2015.04.012 PubMed DOI

von Caemmerer S., Millgate A., Farquhar G. D., Furbank R. T. (1997). Reduction of ribulose-1, 5-bisphosphate carboxylase/oxygenase by antisense RNA in the C4 plant Flaveria bidentis leads to reduced assimilation rates and increased carbon isotope discrimination. Plant Physiol. 113 (2), 469–477. 10.1104/pp.113.2.469 PubMed DOI PMC

Wade D. (1999). Deuterium isotope effects on noncovalent interactions between molecules. Chem. Biol. Interact. 117 (3), 191–217. 10.1016/S0009-2797(98)00097-0 PubMed DOI

Woodson J. D. (2022). Control of chloroplast degradation and cell death in response to stress. Trends Biochem. Sci.. 10.1016/j.tibs.2022.03.010 PubMed DOI

Yang J. (2016). Deuterium: Discovery and applications in organic chemistry. Amsterdam, Netherlands: Elsevier.

Yao S. L., Brandt A., Egsgaard H., Gjermansen C. (2012). Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species. Plant Physiol. Biochem. 61, 71–79. 10.1016/j.plaphy.2012.09.007 PubMed DOI

Zachleder V., Brányiková I. (2014). “Starch overproduction by means of algae,” in Algal biorefineries. Editors Bajpai R. K., Prokop A., Zappi M. (Dordrecht, Heidelberg, London, New York: Springer; ), 217–240.

Zachleder V., Bišová K., Vítová M. (2016). “The cell cycle of microalgae,” in The physiology of microalgae. Editors Borowitzka M. A., Beardall J., Raven J. A. (Dordrecht: Springer; ), 3–46.

Zachleder V., Vítová M., Hlavová M., Moudříková Š., Mojzeš P., Heumann H., et al. (2018). Stable isotope compounds - production, detection, and application. Biotechnol. Adv. 36, 784–797. 10.1016/j.biotechadv.2018.01.010 PubMed DOI

Zachleder V., Ivanov I., Vítová M., Bišová K. (2019). Cell cycle arrest by supraoptimal temperature in the alga Chlamydomonas reinhardtii . Cells 8 (10), 1237–1257. 10.3390/cells8101237 PubMed DOI PMC

Zachleder V., Ivanov I. N., Kselíková V., Bialevich V., Vítová M., Ota S., et al. (2021a). Characterization of growth and cell cycle events as affected by light intensity in the green alga Parachlorella kessleri, as a new model for cell cycle research. Biomolecules 11, 891. 10.3390/biom11060891 PubMed DOI PMC

Zachleder V., Kselíková V., Ivanov I. N., Bialevich V., Vítová M., Ota S., et al. (2021b). Supra-optimal temperature: an efficient approach for overaccumulation of starch in the green alga Parachlorella kessleri . Cells 10, 1806. 10.3390/cells10071806 PubMed DOI PMC

Zuppini A., Andreoli C., Baldan B. (2007). Heat stress: an inducer of programmed cell death in Chlorella saccharophila . Plant Cell Physiol. 48 (7), 1000–1009. 10.1093/pcp/pcm070 PubMed DOI

Bio-removal of rare earth elements from hazardous industrial waste of CFL bulbs by the extremophile red alga Galdieria sulphuraria