Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Screening of cryoprotectants (CPAs) for cryopreservation in the Nitzschia sp. of marine microalgae

해양 규조류 Nitzschia sp.의 초저온동결보존을 위한 보존제의 영향 분석

  • 이인혜 (국립생물자원관 유용자원활용과 생물소재연구팀) ;
  • 전지영 (국립생물자원관 유용자원활용과 생물소재연구팀) ;
  • 김경미 (국립생물자원관 유용자원활용과 생물소재연구팀) ;
  • 강명석 (국립생물자원관 유용자원활용과 생물소재연구팀)
  • Received : 2018.09.21
  • Accepted : 2018.12.03
  • Published : 2018.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Biodiversity has continued to degrade in the $21^{st}$ century due to global warming occasioned by destruction of the environment around the world.. The Nagoya protocol places Korea in a unique position to effectively develop and protect its domestic genetic resources. Microalgae under study in this research contains large amount of antioxidant substances such as beta carotene and astaxanthin, that can be used as biological resource owing to the large amounts of biomass that can be secured through photosynthesis. However, it is difficult to preserve it since cryopreservation method used for long-term preservation is yet to be developed. A basic study for long term cryopreservation was carried out on Nizschia frustulum and Nitzschia amabilis which belong to marine diatoms. As cryoprotectants (CPAs), glycerol, DMSO, and methanol which penetrate into cells were prepared at 5%, 10%, and 15% concentrations each, in case of methanol, it was tested at concentrations of 5%, 10% and 12% by its nature. Two kinds of microalgae, N. frustulum and N. amabilis, were diluted with $10^2$, $10^3$ and $10^4cells\;ml^{-1}$, respectively. The highest survival rate was shown at12% concentration of methanol, and the figures were $6.94{\pm}0.31%$ in N. frustulum and $8.85{\pm}0.16%$ in N. amabilis. As a result of 3 weeks cultivation of thawed microalgae after freezing, the result is shows that N. frustulum increased about 10 times faster and N. amabilis increased about 12 times the original concentration.

21세기 들어 전 세계는 환경파괴에 따른 지구 온난화로 인하여 생물 다양성이 지속적으로 감소하였다. 생물다양성의 감소는 생태계의 자정 및 복원능력 등을 악화 시켜 환경오염이 더욱 심화 되었고 나아가 생물의 생존을 위협하고 있다. 우리나라는 현재 나고야 의정서로 인해 국내 유전자원을 효율적으로 발굴 보호해야 하는 중요한 시점에 있다. 본 연구에서 사용하는 미세조류는 유용 생물자원으로 활용도가 높으나 동결보존하는 방법이 개발되어 있지 않아 장기보존에 어려움이 있다. 대부분의 원핵생물이나 균류 등의 미생물은 종에 관계없이 glycerol 을 이용하여 저온동결보존이 가능하나 미세조류는 동결보존제의 종류와 농도, 유리화 과정 및 해동방법 등에 따라 세포 재생률에 차이가 있기 때문에 구체적인 연구가 필요하다. 미세조류 중 규조류는 전 세계적으로 발견되며 규산질로 구성된 cell wall 은 이미 산업적으로 많이 이용되고 있다. 본 연구에서는 해양 규조류 Nizschia frustulum과 Nitzschia amabilis를 이용하여 장기 동결보존을 위한 기초연구를 수행하였다. 동결보존제로는 세포막 침투성 보존제인 glycerol, DMSO, methanol을 각각 5, 10, 15% 농도로 제조하여 실험하였고 메탄올의 경우 시약 특성상 5, 10, 12% 농도로 실험하였으며 미세조류는 N. frustulum과 N. amabilis 두 종을 각각 $10^2$, $10^3$, $10^4cells\;ml^{-1}$로 희석하여 사용하였다. 실험에 사용한 미세조류 두 종 모두 메탄올 12%에서 가장 높은 생존율을 보였으며 N. frustulum은 $6.94{\pm}0.31%$, N. amabilis는 $8.85{\pm}0.16%$로 나타났다. 동결 후 해동한 미세조류를 3주간 재배양한 결과에서는 N. frustulum이 재배양 초기 농도보다 약10배 증가하였고 N. amabilis는 약12배 증가한 결과를 나타내었다. 본 연구에서는 유용생물자원인 미세조류 Nizschia sp.에 한하여 적합한 동결보존제를 탐색하였으며 이 자료를 바탕으로 더 많은 동결보존제에 대한 효과와 다양한 미세조류 종에 적합한 동결보존 기법 연구가 필요한 것으로 사료된다.

Keywords

References

  1. Andersen RA (2005) Algal culturing techniques. Elsevier Academic Press New York 578p
  2. Ben-Amotz A, Gilboa A (1980) Cryopreservation of marine unicellular algae. I. A survey of algae with regard to size, culture age, photosynthetic activity and chlorophyll-to-cell ratio. Mar. Ecol. Prog. Ser 2:157-161 https://doi.org/10.3354/meps002157
  3. Blanco JM, Long JA, Gee G, Donoghue AM, Wildt DE (2008) Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability. Cryobiology 56:8-14 https://doi.org/10.1016/j.cryobiol.2007.09.004
  4. Brand JJ, Diller KR (2004) Application and theory of algal cryopreservation. Nova Hedwigia 79:175-189 https://doi.org/10.1127/0029-5035/2004/0079-0175
  5. Buhmann MT, Day JG, Kroth PG (2013) Post-cryopreservation viability of the benthic freshwater diatom Planothidium frequentissimum depends on light levels. Cryobiology 67:23-29 https://doi.org/10.1016/j.cryobiol.2013.04.005
  6. Bui TV, Ross IL, Jakob G, Hankamer B (2013) Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae. PloS one 8:e78668 https://doi.org/10.1371/journal.pone.0078668
  7. Burnett KG (2001) Recent advances in marine biotechnology. Copeia 4:1165-1167
  8. Canavate J, Lubian L (1995) Relationship between cooling rates, cryoprotectant concentrations and salinities in the cryopreservation of marine microalgae. Marine Biology 124:325-334 https://doi.org/10.1007/BF00347136
  9. Canavate JP, Lubian LM (1997) Effects of slow and rapid warming on the cryopreservation of marine microalgae. Cryobiology 35:143-149 https://doi.org/10.1006/cryo.1997.2031
  10. Crutchfield A, Diller K, Brand J (1999) Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta). European Journal of Phycology 34:43-52 https://doi.org/10.1080/09670269910001736072
  11. Day J, Brand J (2005) Cryopreservation methods for maintaining microalgal cultures. Algal culturing techniques: 165-187
  12. Day JG, Fenwick C (1993) Cryopreservation of members of the genus Tetraselmis used in aquaculture. Aquaculture 118:151-160 https://doi.org/10.1016/0044-8486(93)90288-A
  13. Day JG, Fleck RA, Benson EE (2000) Cryopreservation-recalcitrance in microalgae: novel approaches to identify and avoid cryoinjury. Journal of Applied Phycology 12:369-377 https://doi.org/10.1023/A:1008107229005
  14. Durand PM, Rashidi A, Michod RE (2011) How an organism dies affects the fitness of its neighbors. The American Naturalist 177:224-232 https://doi.org/10.1086/657686
  15. Fahy GM, MacFarlane D, Angell C, Meryman H (1984) Vitrification as an approach to cryopreservation. Cryobiology 21:407-426 https://doi.org/10.1016/0011-2240(84)90079-8
  16. Fleck R (1998) The assessment of cell damage and recovery in cryopreserved freshwater protists. Ph.D. thesis. University of Abertay, Dundee. Scotland, U.K.
  17. Fukuda S-y, Iwamoto K, Atsumi M, Yokoyama A, Nakayama T, Ishida K-i, Inouye I, Shiraiwa Y (2014) Global searches for microalgae and aquatic plants that can eliminate radioactive cesium, iodine and strontium from the radio-polluted aquatic environment: a bioremediation strategy. Journal of plant research 127:79-89 https://doi.org/10.1007/s10265-013-0596-9
  18. Hirano T, Godo T, Mii M, Ishikawa K (2005) Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Reports 23:534-539 https://doi.org/10.1007/s00299-004-0893-9
  19. Kaczmarczyk A, Turner SR, Bunn E, Mancera RL, Dixon KW (2011) Cryopreservation of threatened native Australian species-what have we learned and where to from here? In Vitro Cellular & Developmental Biology-Plant 47:17-25
  20. Katkov II, Isachenko V, Isachenko E, Kim MS, Lulat AGI, Mackay AM, Levine F (2006) Low-and high-temperature vitrification as a new approach to biostabilization of reproductive and progenitor cells. International Journal of Refrigeration 29:346-357 https://doi.org/10.1016/j.ijrefrig.2005.11.004
  21. Kirsop BE, Doyle AC (1991) Maintenance of microorganisms and cultured cells. Academic Press Ltd.,London p 308
  22. Koh HY, Lee JH, Han SJ, Park H, Lee SG (2015) Effect of the antifreeze protein from the arctic yeast Leucosporidium sp. AY30 on cryopreservation of the marine diatom Phaeodactylum tricornutum. Applied biochemistry and biotechnology 175:677-686 https://doi.org/10.1007/s12010-014-1337-9
  23. Li Y, Wang F, Wang H (2010) Cell death along single microfluidic channel after freeze-thaw treatments. Biomicrofluidics 4:014111 https://doi.org/10.1063/1.3324869
  24. Meikle MN, Schlapp G, Menchaca A, Crispo M (2018) Minimum volume spatula MVD vitrification method improves embryo survival compared to traditional slow freezing, both for in vivo and in vitro produced mice embryos. Cryobiology
  25. Namrata K, Mukesh KG, Raghubansh KS (2016) Open encapsulationvitrification for cryopreservation of algae. Cryobiology 73(2):232-239 https://doi.org/10.1016/j.cryobiol.2016.07.005
  26. Nowshari M, Brem G (2001) Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos. Human Reproduction 16:2368-2373 https://doi.org/10.1093/humrep/16.11.2368
  27. Polge C, Smith AU, Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666 https://doi.org/10.1038/164666a0
  28. Priyadarshani I, Rath B (2012) Commercial and industrial applications of micro algae-A review. J algal biomass utln 3:89-100
  29. Rall WF, Fahy GM (1985) Ice-free cryopreservation of mouse embryos at- 196 C by vitrification. Nature 313:573 https://doi.org/10.1038/313573a0
  30. Redekar P, Wagh A (2000) Cryopreservation studies on the marine diatom Navicula subinflata Grun
  31. Rhodes L, Smith J, Tervit R, Roberts R, Adamson J, Adams S, Decker M (2006) Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology 52:152-156 https://doi.org/10.1016/j.cryobiol.2005.10.003
  32. Rhodes L, Smith J, Tervit R, Roberts R, Adamson J, Adams S, Decker M (2006) Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology 52:152-156 https://doi.org/10.1016/j.cryobiol.2005.10.003
  33. Shanab S, Essa A, Shalaby E (2012) Bioremoval capacity of three heavy metals by some microalgae species (Egyptian Isolates). Plant signaling & behavior 7:392-399 https://doi.org/10.4161/psb.19173
  34. Smith D, Onions AH (1994) The preservation and maintenance of living fungi. CAB international
  35. Tanniou A, Turpin V, Lebeau T (2012) Comparison of cryopreservation methods for the long term storage of the marine diatom Haslea ostrearia (simonsen). Cryobiology 65:45-50 https://doi.org/10.1016/j.cryobiol.2012.03.011
  36. Taylor R, Fletcher RL (1998) Cryopreservation of eukaryotic algae -a review of methodologies. Journal of Applied Phycology 10:481-501 https://doi.org/10.1023/A:1008094622412
  37. Tzovenis I, Triantaphyllidis G, Naihong X, Chatzinikolaou E, Papadopoulou K, Xouri G, Tafas T (2004) Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain. Aquaculture 230:457-473 https://doi.org/10.1016/S0044-8486(03)00444-7
  38. Wusteman MC, Simmonds J, Vaughan D, Pegg DE (2008) Vitrification of rabbit tissues with propylene glycol and trehalose. Cryobiology 56:62-71 https://doi.org/10.1016/j.cryobiol.2007.10.177
  39. Yang D, Li W (2016) Methanol-promoted lipid remodelling during cooling sustains cryopreservation survival of Chlamydomonas reinhardtii. PloS one 11:e0146255 https://doi.org/10.1371/journal.pone.0146255
  40. Zhang E, Zhang L, Wang B, Yan B, Wang Q (2009) Cryopreservation of marine diatom algae by encapsulation-vitrification. Cryoletters 30:224-231