Effectiveness of microalgae and cyanobacteria cryopreservation (for the strains from the All-Russian Collection of Microorganisms)

封面

如何引用文章

全文:

开放存取 开放存取
受限制的访问 ##reader.subscriptionAccessGranted##
受限制的访问 订阅存取

详细

The efficiency of cryopreservation of cyanobacteria and eukaryotic microalgae of different taxonomic groups, including charophyte, chlorophyte and ochrophyte microalgae, was assessed using 24 strains of the algological part of the All-Russian Collection of Microorganisms (VKM) as an example. Microalgae cultures differing in cell size, morphological organization of the thallus, reproduction method, presence and size of mucous membranes, and ability to form dormant cells were selected for comparative study. Two types of cryoprotectors (methanol and dimethyl sulfoxide), two types of nutrient media (slant agar and liquid medium), and three methods for determining the survival of microalgae after cryopreservation were tested. It was shown that a two-step cryopreservation protocol using both cryoprotectants (methanol and dimethyl sulfoxide) was successfully applied to all 24 studied strains, regardless of their taxonomic affiliation and morphological features. Based on the results of the experiment, a standard operating procedure for cryopreservation of microalgae was developed, including a liquid culture medium with dimethyl sulfoxide, as well as two methods for determining the survival of microalgae after cryopreservation – growth in a liquid medium and streaks on agar. The proposed protocol not only ensures the preservation of cell viability and the possibility of further use of the strain as a morphologically and genetically representative sample, but also minimizes time and resource costs, as well as the risk of contamination of cultures.

全文:

受限制的访问

作者简介

V. Redkina

FRC PSCBI RAS

编辑信件的主要联系方式.
Email: kalmykova_v_v@mail.ru

All-Russian Collection of Microorganisms (VKM), G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences

俄罗斯联邦, Pushchino, 142290

A. Temraleeva

FRC PSCBI RAS

Email: kalmykova_v_v@mail.ru

All-Russian Collection of Microorganisms (VKM), G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences

俄罗斯联邦, Pushchino, 142290

参考

  1. Beaty M.H. Cryopreservation of eukaryote algae. Master of Science in Biology thesis. Blacksburg, Virginia: Virginia Polytechnic Institute and State University, 1991. 116 p.
  2. Cameron R. E. Species of Nostoc vaucher occurring in the Sonoran Desert in Arizona // Trans. Am. Microsc. Soc. 1962. V. 81. P. 379–384. https://doi.org/10.2307/3223790
  3. Day J. G., Priestley I. M., Codd G. A. Storage, recovery and photosynthetic activities of immobilised algae // Plant and animal cells: process possibilities / Eds. Webb C., Mavituna F. Chichester, West Sussex: Ellis Horwood Limited, 1987. P. 257–261.
  4. Ettl H., Gärtner G. Syllabus der boden-, luft-und flechtenalgen. 2., ergänzte Auflage. Berlin, Heidelberg: Springer Spektrum, 2014. 773 p. https://doi.org/10.1007/978-3-642-39462-1
  5. Fernandes M. S., Calsing L. C., Nascimento R. C., Santana H., Morais P. B., de Capdeville G., Brasil B. S. Customized cryopreservation protocols for chlorophytes based on cell morphology // Algal Res. 2019. V. 38. Art. 101402. https://doi.org/10.1016/j.algal.2018.101402
  6. Foo S. C., Mok C. Y., Ho S. Y., Khong N. M.H. Microalgal culture preservation: progress, trends and future developments // Algal Res. 2023. V. 71. Art. 103007. https://doi.org/10.1016/j.algal.2023.103007
  7. Friedl T., Lorenz M. The culture collection of algae at Göttingen University (SAG): a biological resource for biotechnological and biodiversity research // Procedia Environ. Sci. 2012. V. 15. P. 110–117. https://doi.org/10.1016/J.PROENV.2012.05.015
  8. Guiry M. D. How many species of algae are there? A reprise. Four kingdoms, 14 phyla, 63 classes and still growing // J. Phycol. 2024. V. 60. P. 214–228. https://doi.org/10.1111/jpy.13431
  9. Holm-Hansen O. Viability of blue-green and green algae after freezing // Physiol. Plant. 1963. V. 16. P. 530–540. https://doi.org/10.1111/j.1399-3054.1963.tb08330.x
  10. Kapoore R. V., Huete-Ortega M., Day J. G., Okurowska K., Slocombe S. P., Stanley M. S., Vaidyanathan S. Effects of cryopreservation on viability and functional stability of an industrially relevant alga // Sci. Rep. 2019. V. 9. Art. 2093. https://doi.org/10.1038/s41598-019-38588-6
  11. Leeson E. A., Cann J. P., Morris G. J. Maintenance of algae and protozoa // Maintenance of Microorganisms / Eds. Kirsop B. E., Snell J. J.S. London: Academic Press, 1984. P. 131–160.
  12. Lewis L. A., Trainor F. R. Survival of Protosiphon botryoides (Chlorophyceae, Chlorophyta) from a Connecticut soil dried for 43 years // Phycologia. 2012. V. 51. P. 662–665. https://doi.org/10.2216/11-108.1
  13. Leya T. The CCCryo Culture Collection of Cryophilic Algae as a valuable bioresource for algal biodiversity and for novel, industrially marketable metabolites // Appl. Phycol. 2022. V. 3. P. 167–188. https://doi.org/10.1080/26388081.2020.1753572
  14. Prieto-Guevara M., Alarcón-Furnieles J., Jiménez-Velásquez C., Hernández-Julio Y., Espinosa-Araujo J., Atencio-García V. Cryopreservation of the microalgae Scenedesmus sp. // Cells. 2023. V. 12. Art. 562. https://doi.org/10.3390/cells12040562
  15. Morris G. J. Cryopreservation of 250 strains of Chlorococcales by the method of two-step cooling // Br. Phycol. J. 1978. V. 13. P. 15–24. https://doi.org/10.1080/00071617800650031
  16. Müller J., Day J. G., Harding K., Hepperle D., Lorenz M., Friedl T. Assessing genetic stability of a range of terrestrial microalgae after cryopreservation using amplified fragment length polymorphism (AFLP) // Am. J. Bot. 2007. V. 94. P. 799–808. https://doi.org/10.3732/ajb.94.5.799
  17. Osório H. C., Laranjeiro C. N., Santos L. M., Santos M. F. First attempts to cryopreserve strains from the Coimbra Collection of Algae (ACOI) and the use of image analysis to assess viability // Nova Hedwigia. 2004. V. 79. P. 227‒235. https://doi.org/10.1127/0029-5035/2004/0079-0227
  18. Paredes E., Ward A., Probert I., Gouhier L., Campbell C. N. Cryopreservation of Algae // Cryopreservation and freeze-drying protocols. Methods in Molecular Biology / Eds. Wolkers W. F., Oldenhof H. NY: Humana New York, 2021. V. 2180. P. 607–621. https://doi.org/10.1007/978-1-0716-0783-1_32
  19. Puchkov E. O. Preservation of viable microorganisms in the laboratory: an overview of basics, methods and practical recommendations for beginners // Austin J. Biotechnol. Bioeng. 2023. V. 10. Art. 1119. https://doi.org/10.26420/austinjbiotechnolbioeng.2023.1119
  20. Rastoll M. J., Ouahid Y., Martín-Gordillo F., Ramos V., Vasconcelos V., Del Campo F. F. The development of a cryopreservation method suitable for a large cyanobacteria collection // J. Appl. Phycol. 2013. V. 25. № 5. P. 1483–1493. https://doi.org/10.1007/s10811-013-0001-z
  21. Urmeneta J., Navarrete A., Huete J., Guerrero R. Isolation and characterization of cyanobacteria from microbial mats of the Ebro Delta, Spain // Curr. Microbiol. 2003. V. 46. P. 0199‒0204. https://doi.org/10.1007/s00284-002-3856-9
  22. Wan M. C., Qin W., Lei C., Li Q. H., Meng M., Fang M., Song W., Chen J. H., Tay F., Niu L. N. Biomaterials from the sea: Future building blocks for biomedical applications // Bioact. Mater. 2021. V. 6. P. 4255‒4285.

补充文件

附件文件
动作
1. JATS XML
下载
2. Fig. 1. Microphotographs of strains of microalgae and cyanobacteria: a – Chlamydomonas applanata VKM Al-18; b – Spongiosarcinopsis terrestris VKM Al-23; c – Bracteacoccus bullatus VKM Al-97; d – Chlorosarcinopsis eremi VKM Al-132; e – Chlorococcum sphacosum VKM Al-188; f – Tetracystis pampae VKM Al-230; g – Deasonia granata VKM Al-250; h – Coelastrella striolata VKM Al-355; and – Edaphochloris andreevii VKM Al-104; j – Pseudostichococcus sp. VKM Al-293; l – Micractinium thermotolerans VKM Al-332; m – Neochlorella semenenkoi VKM Al-342; n – Chloroidium sacharophilum VKM Al-400; o – Pseudochlorella sp. VKM Al-474; n – Vischeria magna VKM Al-26; p – Vischeria sp. VKM Al-463; c – Botrydiopsis eriensis VKM Al-98; t – Xanthonema bristolianum VKM Al-290; y – Klebsormidium flaccidum VKM Al-9; f – Interfilum terricola VKM Al-274; x – Nostoc sp. VKM Al-391; c – Microcoleus sp. VKM Al-312; h – Anabaena cf. pirinica VKM Al-153; w – Leptolyngbya sp. VKM Al-382. Scale mark – 10 µm.

下载 (790KB)
索引源数据
3. Fig. 2. Scheme of the experiment to evaluate the effectiveness of cryopreservation of microalgae and cyanobacteria strains.

下载 (323KB)
索引源数据
4. Fig. 3. Evaluation of the survival rate of microalgae and cyanobacteria during cryopreservation: a, c – Spongiosarcinopsis terrestris VKM Al-23, cultivation period after thawing is 8 weeks; b – Vischeria magna VKM Al-26, cultivation period is 21 days; d – Leptolyngbya sp. VKM Al-382, cultivation period is 4 weeks. Legend in the captions on the dishes and test tubes: M – MetOH, D – DMSO.

下载 (325KB)
索引源数据

版权所有 © Russian Academy of Sciences, 2025