The storage of anhydrobiotic cultures of microalgae and cyanobacteria of A. O. Kovalevsky Institute of Biology of the Southern Seas of RAS

Main Article Content

I. A. Kharchuk

senior researcher, PhD

http://orcid.org/0000-0002-5771-2143

https://elibrary.ru/author_items.asp?id=789761

Abstract

Reliable preservation of microalgae cultures and creation of genetic banks of strains is one of the important tasks of modern biology. To date, 792 collections of various cultivated organisms from 76 countries are registered in the catalog of the World Federation for Culture Collections in the WDCM CCINFO database. This is the most extensive consolidated database of culture collections, which includes both well-known large collections and small repositories of research and educational institutions from all over the world. The database contains 47 algological collections and 80 collections of various microorganisms, which also include microalgae and cyanobacteria cultures. Only 30 biological collections are registered in Russia, from which only 13 contain algae strains. The most common technique of microalgae cultures storage is the method of their periodic re-sowing onto liquid media or agar. It is used in 127 collections (99 % of the total number in the catalog). Other methods used are: cryopreservation – in 33 collections (27 %), lyophilization – in 13 (11 %), L-drying – in 5 (4 %), freezing – in 19 (16 %),  and immobilization in alginate beads – in 1 (0.8 %). However, when using these methods, there is a change in morphological and functional features of cells of the cultures stored, as well as their shredding. In addition, cultures maintaining in a viable state is time-consuming and requires expensive equipment. Preservation of microalgae, transferred to the state of anhydrobiosis by dehydration, is simple and cost-effective. Anhydrobiosis is a deep and long-term inhibition of metabolism, reversible under favorable conditions; it is a quite common phenomenon in nature. The only collection in the WDCM CCINFO database that applies the method of transferring cells to a resting state (for soil algae) is the collection of algae cultures of the National University of Kyiv (ACKU WDCM 994). Many years of experiments on the transfer of microalgae to the state of anhydrobiosis allowed us to develop a method of long-term preservation of microalgae without the use of nutrient media. This technique includes cells transfer to the state of anhydrobiosis, their preservation in a dehydrated state, and subsequent removal to an active culture. In order to preserve algological biodiversity, IBSS RAS created a repository of microalgae transferred to the state of anhydrobiosis, which can be converted to active cultures if necessary. The objects of the repository were marine unicellular algae, as well as freshwater and halobic species of lower phototrophs which are perspective for biotechnology and aquaculture. The cultures were obtained as an inoculum from IBSS RAS collection of live cultures of planktonic microalgae. The algae were grown in an accumulative mode under constant lighting. The biomass was collected during cultivation of algologically pure microalgae cultures at the growth retardation or at the stationary stage. Cells were separated from the culture medium by centrifugation or by filtering them on a plankton sieve. Then the algae were dehydrated and maintained in hermetic zipper bags placed in plastic containers of 100 to 500 ml, at a temperature of +18…+21 °C in the dark in a specially equipped room. The main part of the collection is represented by strains from the phyla Chlorophyta, Cyanophyta, Bacillariophyta, and Rodophyta. The list of species, the number of isolates stored, and the information on preservation forms are provided in this article. The technological regulations for maintenance and replenishment of the storage of anhydrobiotic cultures are described. The repository is at the stage of formation. Its future lies in the fund expansion to include marine, freshwater, and halobic species. Optimization of the dehydration method will allow the transfer of microalgae belonging to different systematic phyla to the state of anhydrobiosis.

Article Details

Kharchuk I. A. The storage of anhydrobiotic cultures of microalgae and cyanobacteria of A. O. Kovalevsky Institute of Biology of the Southern Seas of RAS. Marine Biological Journal, 2020, vol. 5, no. 1, pp. 90-98. doi: 10.21072/mbj.2020.05.1.09
Keywords:
microalgae, anhydrobiosis, viability, dehydration, storage of microalgae and cyanobacteria
Section
Scientific communications

References

Айздайчер Н. А. Коллекция культур морских микроводорослей Института биологии моря им. А. В. Жирмунского ДВО РАН // Биология моря. 2008. Т. 34, № 2. С. 152–155. [Aizdaicher N. A. Collection of marine microalgae at the A. V. Zhirmunsky Institute of Marine Biology. Biologiya morya, 2008, vol. 34, no. 2, pp. 152–155. (in Russ.)]

Владимирова М. Г., Игнатьевская М. И. Изучение влияния условий хранения культур Chlorella в коллекции на последующую их продуктивность // Микробиология. 1966. Т. 35, № 3. С. 539–548. [Vladimirova M. G., Ignat’evskaya M. I. Izuchenie vliyaniya uslovii khraneniya kul’tur Chlorella v kollektsii na posleduyushchuyu ikh produktivnost’. Mikrobiologiya, 1966, vol. 35, no. 3, pp. 539–548. (in Russ.)]

Костиков И. Ю., Демченко Э. Н., Березовская М. А. Коллекция культур водорослей Киевского национального университета имени Тараса Шевченко. Каталог штаммов (2008 г.) // Чорноморський ботанічний журнал. 2009. Т. 5, № 1. С. 37–79. [Kostikov I. Yu., Demchenko E. N., Berezovskaya M. A. Microalgae culture collection at the Taras Shevchenko National University, Kyiv. Catalogue of strains (2008). Chornomorskyi botanichnyi zhurnal, 2009, vol. 5, no. 1, pp. 37–79. (in Russ.)]

Новаковская И. В., Патова Е. Н. Коллекция живых штаммов микроводорослей Института биологии Коми НЦ УрО РАН и перспективы ее использования // Известия Коми научного центра УрО РАН. 2012. Вып. 2, № 10. С. 36–41. [Novakovskaya I. V., Patova E. N. Collection of living microalgae strains of the Institute of Biology, Komi Science Centre, Ural Branch, RAS, and its perspective using. Izvestiya Komi nauchnogo tsentra UrO RAN, 2012, iss. 2, no. 10, pp. 36–41. (in Russ.)]

Одинцова Н. А., Борода А. В. Криосохранение клеток и личинок морских гидробионтов // Биология моря. 2012. Т. 38, № 2. С. 93–103. [Odintsova N. A., Boroda A. V. Cryopreservation of the cells and larvae of marine organisms. Biologiya morya, 2012, vol. 38, no. 2, pp. 93–103. (in Russ.)]

Пат. 2541452 Российская Федерация. МПК6 С 12 N 1/04. Способ длительного хранения микроводорослей / Харчук И. А. ; заявитель и патентообладатель Федеральное государственное бюджетное учреждение науки «Институт морских биологических исследований имени А. О. Ковалевского РАН». № 2014149881/93 ; заявл. 26.09.2014 ; приор. 17.03.2008 ; опубл. 10.02.2015 , Бюл. № 4. [Pat. 2541452 Rossiiskaya Federatsiya. MPK6 S 12 N 1/04. Sposob dlitel’nogo khraneniya mikrovodoroslei / Kharchuk I. A. ; zayavitel’ i patentoobladatel’ Federal’noe gosudarstvennoe byudzhetnoe uchrezhdenie nauki “Institut morskikh biologicheskikh issledovanii imeni A. O. Kovalevskogo RAN”. No. 2014149881/93; zayavl. 26.09.2014; prior. 17.03.2008; opubl. 10.02.2015, Byul. no. 4. (in Russ.)]

Харчук И. А. Хранение микроводорослей в состоянии ангидробиоза // Микроводоросли Чёрного моря: проблемы сохранения биоразнообразия и биотехнологического использования / под ред. Ю. Н. Токарева, З. З. Финенко, Н. В. Шадрина Севастополь : ЭКОСИ-Гидрофизика, 2008. Гл. 9. С. 237–267. [Kharchuk I. A. Khranenie mikrovodoroslei v sostoyanii angidrobioza. In: Mikrovodorosli Chernogo morya: problemy sokhraneniya bioraznoobraziya i biotekhnologicheskogo ispol’zovaniya / Yu. N. Tokarev, Z. Z. Finenko, N. V. Shadrin (Eds). Sevastopol : EKOSI-Gidrofizika, 2008, chap. 9, pp. 237–267. (in Russ.)]

Харчук И. А. Динамика жизнеспособности и компонентов биохимического состава Arthrospira (Spirulina) platensis (Nords) Gomont в зависимости от температуры дегидратации при переводе в состояние ангидробиоза // Вопросы современной альгологии. 2018. № 1 (16). [Kharchuk I. A. Dynamics of viability and components of biochemical composition Arthrospira (Spirulina) platensis (Nords) Gomont depending on the dehydration temperature transferring at anhydrobiosis state. Voprosy sovremennoi al’gologii, 2018, no. 1 (16). URL: http://algology.ru/1258 (accessed 23.07.2019). (in Russ.)]

Коллекция микроводорослей отдела экологической физиологии водорослей ФИЦ ИнБЮМ [Электронный ресурс]. : сайт. [Kollektsiya mikrovodoroslei otdela ekologicheskoi fiziologii vodoroslei FITs InBYuM [Electronic resource] : site. URL: http://ibss-ras.ru/?page_id=2828 (accessed 24.07.2019). (in Russ.)]

Cañavate J. P., Lubian L. M. Relationship between cooling rates, cryoprotectant concentrations and salinities in the cryopreservation of marine microalgae. Marine Biology, 1995, vol. 124, iss. 2, pp. 325–334. https://doi.org/10.1007/BF00347136

Chen Y. C. Immobilized microalgae Scenedesmus quadricanda (Chlorophyta, Chlorococcales) for long-term storage and for application for water quality control in fish culture. Aquaculture, 2001, vol. 195, no. 1–2, pp. 71–80. https://doi.org/10.1016/S0044-8486(00)00540-8

Chen Y. C. Immobilized Isochrysis galbana (Haptophyta) for long-term storage and applications for feed and water quality control in clam (Meretrix lusoria) cultures. Journal of Applied Phycology, 2003, vol. 15, no. 5, pp. 439–444. https://doi.org/10.1023/A:1026071714199

Crutchfield A., Diller K., Brand J. Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta). European Journal of Phycology, 1999, vol. 34, iss. 1, pp. 43–52. https://doi.org/10.1080/09670269910001736072

Day J. G., Watanable M. M., Morris G. H., Fleck R. A., McLellan M. R. Long-term viability of preserved eukaryotic algae. Journal of Applied Phycology, 1997, vol. 9, no. 2, pp. 121–127. https://doi.org/10.1023/A:1007991507314

Hur S. B., Bae J. H., Youn J., Jo M. J. KMMCC – Korea Marine Microalgae Culture Center: List of strains, 2nd edition. Algae, 2015, vol. 30, suppl., pp. S1–S188. http://dx.doi.org/10.4490/algae.2015.30.S.S1

Kumari N., Gupta M., Singh R. Open encapsulation-vitrification for cryopreservation of algae. Cryobiology, 2016, vol. 73, iss. 2, pp. 232–239. https://doi.org/10.1016/j.cryobiol.2016.07.005

Marsalek B., Rojickova-Padrtova R. Long-term maintenance of alga strains for use in biomassays and biotechnology. Archiv fur̈ Hydrobiologie. Supplementband: Algological Studies, 1988, vol. 124, pp. 121–136.

Meyer M. A. Cryopreservation of a Marine Diatom. PhD Thesis. Texas A & M University, College Station, 1985, 112 p.

Poncet J.-M., Véron B. Cryopreservation of the unicellular marine alga, Nannochloropsis oculata. Biotechnology Letters, 2003, vol. 25, no. 23, pp. 2017–2022. https://doi.org/10.1023/B:BILE.0000004395.04116.45

Richmond A., Lichtenberg E., Stahl B., Vonshak A. Quantitative assessment of the major limitations on productivity of Spirulina platensis in open raceways. Journal of Applied Phycology, 1990, vol. 2, no. 3, pp. 195–206. https://doi.org/10.1007/BF02179776

Sakane T. Preservation of microorganisms by L-drying. International Journal of Reefing, 1982, vol. 57, no. 6, pp. 767–775.

Scarbrough C., Wirschell M. Comparative analysis of cryopreservation methods in Chlamydomonas reinhardtii. Cryobiology, 2016, vol. 73, iss. 2, pp. 291–295. https://doi.org/10.1016/j.cryobiol.2016.07.011

von Schwartzenberg K., Bornfleth S., Lindner A., Hanelt D. The Microalgae and Zygnematophyceae Collection Hamburg (MZCH) – living cultures for research on rare streptophytic algae. Algological Studies, 2013, vol. 142, pp. 77–108. http://doi.org/10.1127/1864-1318/2013/0131

Surek B. Meeting report: International Symposium on the Cryopreservation of Algae (Austin, Texas, USA, 16–17 April, 1988). Protist, 1998, vol. 149, iss. 3. pp. 201–205. https://doi.org/10.1016/S1434-4610(98)70026-4

Tessarolli L. P., Day J. G., Vieira A. H. Establishment of a cryopreserved biobank for the Culture Collection of Freshwater Microalgae (CCMA-UFSCar), São Paulo, Brazil. Biota Neotropica, 2017, vol. 17, no. 2, e20160299. http://dx.doi.org/10.1590/1676-0611-bn-2016-0299

Tsuru S. Preservation of marine and fresh water algae by means of freezing and freeze-drying. Cryobiology, 1973, vol. 10, iss. 5, pp. 445–452. https://doi.org/10.1016/0011-2240(73)90074-6

WDCM CCINFO. World Data Centre for Microorganisms, Culture Collections Information Worldwide. 2014. [Electronic resource.] URL: http://www.wfcc.info/ccinfo/home (accessed 23.07.2019).

Zheng L., Lu Z., Zhang Q., Li T., Song L. A fluorescence ratio-based method to determine microalgal viability and its application to rapid optimization of cryopreservation. Cryobiology, 2018, vol. 81, pp. 27–33. https://doi.org/10.1016/j.cryobiol.2018.02.014