Investigation of the biochemical content of microalgae using flow cytometry
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Abstract
Lipid content in microalgae cells belonging to different taxonomic groups and grown under different cultivation conditions was determined using spectrophotometric and flow cytometry methods in combination with fluorochrome Nile Red (NR), a fluorescent marker of neutral and polar lipids in algal cells. It was shown that all cultures used in the experiments stained well with Nile Red at different growth stages: orange fluorescence of FL2 channel (575 nm), was marked for lipid identification in the cells. The optimum staining time was 10 min, when NR working solution was added, 20 μl per 1 ml of culture medium. The results obtained showed that there was no significant difference between spectrophotometric and fluorescence methods for the determination of lipids in microalgae, R2 = 0.98. Combination of standard spectrophotometric method and fluorescence method realized by staining cells with Nile Red can serve as a reliable approach for estimation of lipids in microalgae cells.
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Железнова С. Н., Геворгиз Р. Г., Бобко Н. И., Лелеков А. С. Питательная среда для интенсивной культуры диатомовой водоросли Cylindrotheca closterium (Ehrenb.) Reimann et Lewin – перспективного объекта биотехнологий // Актуальная биотехнология. – 2015. – № 3. – C. 46–48.
Оценка плотности культуры фототрофных микроорганизмов методом йодатной окисляемости / Геворгиз Р. Г. [и др.] ; Рос. акад. наук, Ин-т мор. биол. исслед. им. А. О. Ковалевского. – Севастополь : ИМБИ, 2015. – 31 c.
Инструкция по массовому разведению морских одноклеточных водорослей и коловраток / Всесоюз. науч.-исслед. ин-т мор. рыб. хоз-ва и океанографии ; сост.: Л. В. Спекторова [и др.]. – Москва : ВНИРО, 1986. – 63 c.
Руководство по современным биохимическим методам исследования водных экосистем, перспективных для промысла и марикультуры / Всесоюз. науч.-исслед. ин-т мор. рыб. хоз-ва и океанографии ; под ред. А. И. Агатовой. – Москва : ВНИРО, 2004. – 123 c.
Соломонова Е. С., Муханов В. С. Оценка доли физиологически активных клеток в накопительных культурах Phaeodactylum tricornutum и Nitzschia sp с помощью проточной цитометрии // Морской экологический журнал. – 2011. – Т. 10, № 4. – С. 67–72.
Финенко З. З., Ланская Л. А. Рост и скорость деления водорослей в лимитированных объемах воды // Экологическая физиология морских планктонных водорослей (в условиях культур) / Акад. наук УССР, Ин-т биологии юж. морей им. А. О. Ковалевского ; под общ. ред. К. М. Хайлова. – Киев : Наук. думка, 1971. – С. 22–51.
Bligh E. G., Dyer W. J. A rapid method of total lipid extraction and purification // Canadian Journal of Biochemistry and Physiology. – 1959. – Vol. 37, nr 8. – P. 911–917. – https://doi.org/10.1139/O59-099
Brett M., Müller-Navarra D. The role of highly unsaturated fatty acids in aquatic foodweb processes // Freshwater Biology. – 1997. – Vol. 38, iss. 3. – P. 483–499. – https://doi.org/10.1046/j.13652427.1997.00220.x
Chen W., Zhang C., Song L., Sommerfeld M., Hu Q. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae // Journal of Microbiological Methods. – 2009. – Vol. 77, iss. 1. – P. 41–47. – https://doi.org/10.1016/j.mimet.2009.01.001
Cole T. A., Fok A. K., Ueno M. S., Allen R. D. Use of nile red as a rapid measure of lipid content in ciliates // European Journal of Protistology. – 1990. – Vol. 25, iss. 4. – P. 361–368. – https://doi.org/10.1016/S0932-4739(11)80129-X
Cooksey K. E., Guckert J. B., Williams S. A., Callis P. R. Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red // Journal of Microbiological Methods. – 1987. – Vol. 6, iss. 6. – P. 333–345. – https://doi.org/10.1016/0167-7012(87)90019-4
Davey H. M., Kell D. B. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses // Microbiological Reviews. – 1996. – Vol. 60, iss. 4. – P. 641–696. – https://doi.org/10.1128/mr.60.4.641-696.1996
Elsey D., Jameson D., Raleigh B., Cooney M. J. Fluorescent measurement of microalgal neutral lipids // Journal of Microbiological Methods. – 2007. – Vol. 68, iss. 3. – P. 639–642. – https://doi.org/10.1016/j.mimet.2006.11.008
Eltgroth M. L., Watwood R. L., Wolfe G. V. Production and cellular localization of neutral long-chain lipids in the Haptophyte algae Isochrysis galbana and Emiliania huxleyi // Journal of Phycology. – 2005. – Vol. 41, iss. 5. – P. 1000–1009. – https://doi.org/ 10.1111/j.1529-8817.2005.00128.x
Gao C., Xiong W., Zhang Y., Yuan W., Wu Q. Rapid quantitation of lipid in microalgae by timedomain nuclear magnetic resonance // Journal of Microbiological Methods. – 2008. – Vol. 75, iss. 3. – P. 437–440. – https://doi.org/10.1016/j.mimet.2008.07.019
Guillard R. R. L., Ryther J. H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran // Canadian Journal of Microbiology. – 1962. – Vol. 8, nr 2. – P. 229–239. – https://doi.org/10.1139/m62-029
Guzmán H. M., de la Jara Valido A., Presmanes K. F., Duarte L. C. Quick estimation of intraspecific variation of fatty acid composition in Dunaliella salina using flow cytometry and Nile Red // Journal of Applied Phycology. – 2012. – Vol. 24, iss. 5. – P. 1237–1243. – https://doi.org/10.1007/s10811011-9768-y
Gurr M. I., Harwood J. L., Frayn K. N. Lipid Biochemistry. – 5th ed. – Oxford : Blackwell Science, 2002. – 336 p.
Hu Q., Sommerfeld M., Jarvis E., Ghirardi M., Posewitz M., Seibert M., Darzins A. Microalgal Triacylglycerols as feedstocks for biofuel production: perspectives and advances // The Plant Journal. – 2008. – Vol. 54, iss. 4. – P. 621–639. – https://doi.org/10.1111/j.1365313X.2008.03492.x
Huang G. H., Chen G., Chen F. Rapid screening method for lipid production in alga based on Nile Red fluorescence // Biomass and Bioenergy. – 2009. – Vol. 33, iss. 10. – P. 1386–1392. – https://doi.org/10.1016/j.biombioe.2009.05.022
de la Jara A., Mendoza H., Martel A., Molina C., Nordströn L., de la Rosa V., Díaz R. Flow cytometric determination of lipid content in a marine dinoflagellate, Crypthecodinium cohnii // Journal of Applied Phycology. – 2003. – Vol. 15, iss. 5. – P. 433–438. – https://doi.org/10.1023/A:1026007902078
Mendoza H., de la Jara A., Freijanes K., Carmona L., Ramos A. A., de Sousa Duarte V., Varela J. C. S. Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains // Electronic Journal of Biotechnology. – 2008. – Vol. 11, iss. 4. – P. 2–13. – https://doi.org/10.2225/vol11-issue4-fulltext-2
Montero M. F., Aristizábal M., Reina G. G. Isolation of high-lipid content strains of the marine microalga Tetraselmis suecica for biodiesel production by flow cytometry and single-cell sorting // Journal of Applied Phycology. – 2011. – Vol. 23, iss. 6. – P. 1053–1057. – https://doi.org/10.1007/s10811-010-9623-6
Pereira H., Barreira L., Figueiredo F., Custódio L., Vizetto-Duarte C., Polo C., Rešek E., Engelen A., Varela J. Polyunsaturated fatty acids of marine macroalgae: potential for nutritional and pharmaceutical applications // Marine Drugs. – 2012. – Vol. 10, iss. 9. – P. 1920–1935. – https://doi.org/10.3390/md10091920
Satpati G. G., Pal R. Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile Red staining – an improved technique // Annals of Microbiology. – 2015. – Vol. 65, no 2. – P. 937–949. – https://doi.org/10.1007/s13213-014-0937-5
Shahidi F., Ambigaipalan P. Omega-3 polyunsaturated fatty acids and their health benefits // Annual Review of Food Science and Technology. – 2018. – Vol. 9. – P. 345–381. – https://doi.org/10.1146/annurev-food-111317-095850
Wawrik B., Harriman B. H. Rapid, colorimetric quantification of lipid from algal cultures // Journal of Microbiological Methods. – 2010. – Vol. 80, iss. 3. – P. 262–266. – https://doi.org/10.1016/j.mimet.2010.01.016
