Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Characterization of Filamentous Cyanobacteria Encapsulated in Alginate Microcapsules

알긴산염 마이크로캡슐 내부에 동결보존된 사상체 남세균의 특성 연구

  • Park, Mirye (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources) ;
  • Kim, Z-Hun (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources) ;
  • Nam, Seung Won (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources) ;
  • Lee, Sang Deuk (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources) ;
  • Yun, Suk Min (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources) ;
  • Kwon, Dae Ryul (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources) ;
  • Lee, Chang Soo (Algae Research Team, Microbial Research Department, Nakdonggang National Institute of Biological Resources)
  • 박미례 (국립낙동강생물자원관 미생물연구실 조류연구팀) ;
  • 김지훈 (국립낙동강생물자원관 미생물연구실 조류연구팀) ;
  • 남승원 (국립낙동강생물자원관 미생물연구실 조류연구팀) ;
  • 이상득 (국립낙동강생물자원관 미생물연구실 조류연구팀) ;
  • 윤석민 (국립낙동강생물자원관 미생물연구실 조류연구팀) ;
  • 권대률 (국립낙동강생물자원관 미생물연구실 조류연구팀) ;
  • 이창수 (국립낙동강생물자원관 미생물연구실 조류연구팀)
  • Received : 2019.12.17
  • Accepted : 2020.01.23
  • Published : 2020.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

Cyanobacteria are microorganisms which have important roles in the nitrogen cycle due to their ability to fix nitrogen in water and soil ecosystems. They also produce valuable materials that may be used in various industries. However, some species of cyanobacteria may limit the use of water resources by causing harmful algal blooms in water ecosystems. Many culture collection depositories provide cyanobacterial strains for research, but their systematic preservation is not well-developed in Korea. In this study, we developed a method for the cryopreservation of the cyanobacteria Trichormus variabilis (syn. Anabaena variabilis), using alginate microcapsules. Two approaches were used for the experiments and their outputs were compared. One of the methods involved the cryopreservation of cells using only a cryoprotectant and the other used the cryoprotectant within microcapsules. After cryopreservation for 35 days, cells preserved with both methods were successfully regenerated from the initial 1.0 × 105 cells/ml to a final concentration of 6.7 × 106 cells/ml and 1.1 × 107 cells/ml. Irregular T. variabilis shapes were found after 14 days of regeneration. T. variabilis internal structures were observed by transmission electron microscopy (TEM), revealing that lipid droplets were reduced after cryopreservation. The expression of the mreB gene, known to be related to cell morphology, was downregulated (54.7%) after cryopreservation. Cryopreservation using cryoprotectant alone or with microcapsules is expected to be applicable to other filamentous cyanobacteria in the future.

남세균은 수계나 토양 생태계에서 질소 고정 능력으로 인해 질소순환 과정에 중요한 역할을 하는 미생물로 다양한 산업분야에서 활용될 수 있는 유용 물질을 생산할 수 있다. 또한 일부 종은 수생태계에서 유해조류 번성을 일으켜 수자원의 이용을 제한한다. 일반적으로 생물소재은행은 관련 연구를 위해 남세균 배양주를 제공하는 역할을 한다. 하지만 국내 생물소재은행에서는 남세균의 체계적인 보존이 미비한 실정이다. 본 연구에서는 국내에서 분리한 Trichormus variabilis (syn. Anabaena variabilis)를 대상으로 알긴산 마이크로캡슐을 이용한 동결보존기술을 개발하였다. 동결보존제만 사용하는 일반 동결보존법과 세포를 마이크로캡슐에 포집한 후 동결보존제를 혼합하는 두 가지 방법으로 실험을 수행하였고 각각의 효율을 비교하였다. T. variabilis 동결보존 후 35일간 재배양하여 세포수를 비교한 결과 두 가지 동결보존법 모두 재생에 성공하였으며, 초기 농도 1.0 × 105 cells에서 마이크로캡슐 방법은 6.7 × 106 cells로, 일반 동결 보존법은 1.1 × 107 cells로 증가되었다. 또한 배양 14일 후 T. variabilis 세포의 부정형 형태를 발견하였다. 세포 소기관을 관찰하기 위해 투과전자현미경(TEM) 분석을 하였고 lacunae와 lipid body의 크기가 줄어든 것을 확인하였다. 세포 형태와 관련된 mreB 유전자가 동결보존 전에 비해 동결 보존 후 발현량이 54.7%로 감소된 것을 확인하였다. 본 연구결과를 통해 향후 사상성 남조류에 대하여 보존제와 마이크로캡슐을 이용한 동결보존이 가능할 것으로 기대된다.

Keywords

References

  1. Brock TD. 1973. Evolutionary and ecological aspects of the cyanophytes. Botanical Monographs. 9: 487-500.
  2. Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wunschiers R, Lindblad P. 2002. Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol. Mol. Biol. Rev. 66: 1-20. https://doi.org/10.1128/MMBR.66.1.1-20.2002
  3. Fay P. 1992. Oxygen relations of nitrogen fixation in cyanobacteria. Microbiol. Mol. Biol. Rev. 56: 340-373.
  4. Milledge JJ. 2010. Commercial application of microalgae other than as biofuels: a brief review. Rev. Environ. Sci. Biotechnol. 10: 31-41.
  5. Mourelle M, Gomez C, Legido J. 2017. The potential use of marine microalgae and cyanobacteria in cosmetics and thalassotherapy. Cosmetics 4: 46. https://doi.org/10.3390/cosmetics4040046
  6. Vijayakumar S, Menakha M. 2015. Pharmaceutical applications of cyanobacteria-A review. J. Acute Med. 5: 15-23. https://doi.org/10.1016/j.jacme.2015.02.004
  7. Wang B, Zhang E, Gu Y, Ning S, Wang Q, Zhou J. 2011. Cryopreservation of brown algae gametophytes of undaria pinnatifida by encapsulation-vitrification. Aquaculture 317: 89-93. https://doi.org/10.1016/j.aquaculture.2011.04.014
  8. Hallegraeff GM. 1993. A review of harmful algal blooms and their apparent global increase. Phycologia 32: 79-99. https://doi.org/10.2216/i0031-8884-32-2-79.1
  9. Wood SA, Rhodes LL, Adams SL, Adamson JE, Smith KF, Smith JF, et al. 2008. Maintenance of cyanotoxin production by cryopreserved cyanobacteria in the New Zealand culture collection. New Zeal J. Mar. Fresh 42: 277-283. https://doi.org/10.1080/00288330809509955
  10. Bartram J, Chorus I. 1999. Toxic cyanobacteria in water - A guide to their public health consequences, monitoring and management. In: Redman M, Crompton TR, Welch EB, Bau J, Henriques JD, Oliviera Raposo de J, Lobo Ferreira JP, Best G, Niemirycz E, Bogacka T, Lester JN, Birkett JW, Gleeson C, Gray N, Pereira LS, Gowing J, Bardolet R, Kay B, Smith LED, Franks T, Howsam P, Carter RC, Helmer R, Hespanhol I, Kimstach V, Meybeck M, Baroudy E, Chapman D, Bartram J, Balance R, Kay B, Howsan PE & FN Spon, pp. 416. London.
  11. Rastoll MJ, Ouahid Y, Martín-Gordillo F, Ramos V, Vasconcelos V, Campo FFd. 2013. The development of a cryopreservation method suitable for a large cyanobacteria collection. J. Appl. Phycol. 25: 1483-1493. https://doi.org/10.1007/s10811-013-0001-z
  12. Day JG, Benson EE, Harding K, Knowles B, Idowu M, Bremner D, et al. 2005. Cryopreservation and conservation of microalgae: the development of a Pan-European scientific and biotechnological resource (the COBRA project). Cryo Letters 26: 231-238.
  13. Day JG. 2007. Cryopreservation of microalgae and cyanobacteria. pp. 141-151. Cryopreservation and Freeze-Drying Protocols. Humana Press, Totowa, NJ.
  14. Cavalcante SC, Freitas RS, Vidal MSM, Dantas KC, Levi JE, Martins JEC. 2007. Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of cryptococcus neoformans strains. Mem. Inst. Oswaldo Cruz 102: 41-47. https://doi.org/10.1590/S0074-02762007000100006
  15. Romo S, Becares E. 1992. Preservation of filamentous cyanobacteria cultures under low temperatures. J. Microbiol. Methods. 16: 85-89. https://doi.org/10.1016/0167-7012(92)90028-3
  16. Benson EE. 2008. Cryopreservation of phytodiversity: a critical appraisal of theory & practice. CRC Crit. Rev. Plant Sci. 27: 141-219. https://doi.org/10.1080/07352680802202034
  17. Harding K, Day JG, Lorenz M, Timmermann H, Friedl T, Bremner DH, et al. 2004. Introducing the concept and application of vitrification for the cryo-conservation of algae-a mini-review. Nova Hedwigia. 79: 207-226. https://doi.org/10.1127/0029-5035/2004/0079-0207
  18. Day JG. 2004. Cryopreservation: fundamentals, mechanisms of damage on freezing/thawing and application in culture collections. Nova Hedwigia. 79: 191-205. https://doi.org/10.1127/0029-5035/2004/0079-0191
  19. 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
  20. Day JG, Fleck RA. 2015. Cryo-injury in algae and the implications this has to the conservation of micro-algae. Microalgae Biotechnol. 1: 1-11.
  21. Gwo JC, Chiu JY, Chou CC, Cheng HY. 2005. Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae). Cryobiology 50: 338-343. https://doi.org/10.1016/j.cryobiol.2005.02.001
  22. Mori F, Erata M, Watanabe MM. 2002. Cryopreservation of cyanobacteria and green algae [Chlorophyceae] in the NIES-collection [Japan]. Microbiology and Culture Collections (Japan). 18: 45-55.
  23. Holm-Hansen O. 1963. Viability of blue-green and green algae after freezing. Physio plant. 16: 530-540. https://doi.org/10.1111/j.1399-3054.1963.tb08330.x
  24. Morris GJ. 1976. The cryopreservation of Chlorella 1. interactions of rate of cooling, protective additive and warming rate. Arch Microbiol. 107: 57-62. https://doi.org/10.1007/BF00427867
  25. Paulet F, Engelmann F, Glaszmann J-C. 1993. Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration. Plant Cell Rep. 12: 525-529. https://doi.org/10.1007/BF00236101
  26. Sakai A, Engelmann F. 2007. Vitrification, encapsulation-vitrification and droplet-vitrification: a review. Cryoletters 28: 151-172.
  27. Kumari N, Gupta MK, Singh RK. 2016. Open encapsulation-vitrification for cryopreservation of algae. Cryobiology 73: 232-239. https://doi.org/10.1016/j.cryobiol.2016.07.005
  28. Park H-K. 2006. Long-term Preservation of bloom-forming cyanobacteria by cryopreservation. Algae 21: 125-131. https://doi.org/10.4490/ALGAE.2006.21.1.125
  29. Day JG, DeVille MM. 1995. Cryopreservation of algae. in: Day J.G., Pennington M.W. (eds) Cryopreservation and Freeze-Drying Protocols., pp. 141-151, Methods in Molecular $Biology^{TM}$, Ed. Humana Press, Totowa, NJ.
  30. Nam SW, Shin W. 2016. Ultrastructure of the flagellar apparatus in cryptomorphic Cryptomonas curvata (Cryptophyceae) with an emphasis on taxonomic and phylogenetic implications. Algae 31: 117-128. https://doi.org/10.4490/algae.2016.31.6.13
  31. Popova A, Kemp R. 2007. Effects of surfactants on the ultrastructural organization of the phytoplankton, Chlamydomonas reinhardtii and Anabaena cylindrica. Fund Appl. Limnol. 169: 131-136. https://doi.org/10.1127/1863-9135/2007/0169-0131
  32. Al-Tebrineh J, Mihali TK, Pomati F, Neilan BA. 2010. Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR. Appl. Environ. Microbiol. 76: 7836-7842. https://doi.org/10.1128/AEM.00174-10
  33. Hu B, Yang G, Zhao W, Zhang Y, Zhao J. 2007. MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120. Mol. Microbiol. 63: 1640-1652. https://doi.org/10.1111/j.1365-2958.2007.05618.x
  34. Purohit GK, Mahanty A, Mohanty BP, Mohanty S. 2016. Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel channa striatus under high-temperature stress. Fish Physiol. Biochem. 42: 125-135. https://doi.org/10.1007/s10695-015-0123-0
  35. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $22^{-{\Delta}{\Delta}CT}$ Method. Methods. 25: 402-408. https://doi.org/10.1006/meth.2001.1262
  36. Garrido-Cardenas JA, Han X, Alonso DL, Garcia-Maroto F. 2019. Evaluation and optimization of a methodology for the long-term cryogenic storage of Tetradesmus obliquus at $-80^{\circ}C$. Appl. Microbiol. Biotechnol. 103: 2381-2390. https://doi.org/10.1007/s00253-019-09650-0
  37. Lukesova A, Hrouzek P, Harding K, Benson EE, Day JG. 2008. Deployment of the encapsulation/dehydration protocol to cryopreserve diverse microalgae held at the Institute of Soil Biology, Academy of Sciences of the Czech Republic. Cryoletters 29: 21-26.
  38. Sakr S, Jeanjean R, Zhang CC, Arcondeguy T. 2006. Inhibition of cell division suppresses heterocyst development in Anabaena sp. strain PCC 7120. J. Bacteriol. 188: 1396-1404. https://doi.org/10.1128/JB.188.4.1396-1404.2006
  39. Braune W. 1980. Structural aspects of aknete germination in the cyanobacterium Anabaena variabilis. Arch Microbiol. 126: 257-261. https://doi.org/10.1007/BF00409929
  40. Black K, Buikema WJ, Haselkorn R. 1995. The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 177: 6440-6448. https://doi.org/10.1128/JB.177.22.6440-6448.1995
  41. Hagen C, Siegmund S, Braune W. 2002. Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation. Eur. J. Phycol. 37: 217-226. https://doi.org/10.1017/S0967026202003669
  42. Chekanov K, Vasilieva S, Solovchenko A, Lobakova E. 2018. Reduction of photosynthetic apparatus plays a key role in survival of the microalga Haematococcus pluvialis (Chlorophyceae) at freezing temperatures. Photosynthetica 56: 1268-1277. https://doi.org/10.1007/s11099-018-0841-5
  43. Singh SM, Elster J. 2007. Cyanobacteria in antarctic lake environments. Algae and cyanobacteria in extreme environments. pp. 303-320. Ed. Springer, Dordrecht.
  44. Potts M. 1994. Desccation tolerance of prokaryotes. Microbiol Mol. Biol. Rev. 58: 755-805.
  45. Bewley JD. 1979. Physiological aspects of desiccation tolerance. Annu. Rev. Plant Physiol. 30: 195-238. https://doi.org/10.1146/annurev.pp.30.060179.001211
  46. Singh SP, Montgomery BL. 2011. Determining cell shape: adaptive regulation of cyanobacterial cellular differentiation and morphology. Trends Microbiol. 19: 278-285. https://doi.org/10.1016/j.tim.2011.03.001
  47. Gaballah A, Kloeckner A, Otten C, Sahl HG, Henrichfreise B. 2011. Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae. PLoS One 6: e25129. https://doi.org/10.1371/journal.pone.0025129