Journal of Microbiology and Biotechnology

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

DOI QR Code

Optimization of Culture Medium for the Production of an Exopolysaccharide (p-CY02) with Cryoprotective Activity by Pseudoalteromonas sp. RosPo-2 from the Antarctic Sea

  • Pilsung Kang (Development of Biomaterials from Polar Region, Korea Polar Research Institute) ;
  • Sung Jin Kim (Development of Biomaterials from Polar Region, Korea Polar Research Institute) ;
  • Ha Ju Park (CRYOTECH Inc.) ;
  • Il Chan Kim (Development of Biomaterials from Polar Region, Korea Polar Research Institute) ;
  • Se Jong Han (Development of Biomaterials from Polar Region, Korea Polar Research Institute) ;
  • Joung Han Yim (Development of Biomaterials from Polar Region, Korea Polar Research Institute)
  • Received : 2024.02.22
  • Accepted : 2024.03.08
  • Published : 2024.05.28
Download PDF
⟨ Previous Next ⟩

Abstract

When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.

Keywords

Acknowledgement

This work was supported by a Korea Polar Research Institute (KOPRI) grant funded by the Ministry of Oceans and Fisheries (PE24160). Fig. 1A was reproduced from Reference [35] with permission from the Royal Society of Chemistry.

References

  1. Yong SH, Park KB, Seol YW, Kim DH, Jeong MJ, Choi MS. 2023. Optimization conditions for cryopreservation of Nelumbo nucifera Gaertn., aquatic plant. Plant Biotechnol. Rep. 17: 625-635.
  2. Jesus AR, Duarte ARC, Paiva A. 2022. Use of natural deep eutectic systems as new cryoprotectant agents in the vitrification of mammalian cells. Sci. Rep. 12: 8095.
  3. Awan M, Buriak I, Fleck R, Fuller B, Goltsev A, Kerby J, et al. 2020. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen. Med. 15: 1463-1491.
  4. Zhu M, Wang X, Zhou Y, Tan J, Zhou Y, Gao F. 2022. Small RNA sequencing revealed that miR4415, a legume-specific miRNA, was involved in the cold acclimation of Ammopiptanthus nanus by targeting an L-ascorbate oxidase gene and regulating the redox state of apoplast. Front. Genet. 13: 870446.
  5. Horner R, Ackley SF, Dieckmann GS, Gulliksen B, Hoshiai T, Legendre L, et al. 1992. Ecology of sea ice biota: 1. Habitat, terminology, and methodology. Polar Biol. 12: 417-427.
  6. Krembs C, Eicken H, Junge K, Deming J. 2002. High concentrations of exopolymeric substances in Arctic winter sea ice: implications for the polar ocean carbon cycle and cryoprotection of diatoms. Deep Sea Res. I: Oceanogr. Res. Pap. 49: 2163-2181.
  7. Aletsee L, Jahnke J. 1992. Growth and productivity of the psychrophilic marine diatoms Thalassiosira antarctica Comber and Nitzschia frigida Grunow in batch cultures at temperatures below the freezing point of sea water. Polar Biol. 11: 643-647.
  8. Nichols CM, Guezennec J, Bowman J. 2005. Bacterial exopolysaccharides from extreme marine environments with special consideration of the southern ocean, sea ice, and deep-sea hydrothermal vents: a review. Mar. Biotechnol. 7: 253-271.
  9. Palmisano AC, Sullivan CW. 2018. Growth, metabolism, and dark survival in sea ice microalgae, pp. 131-146. Sea ice biota, Ed. CRC Press.
  10. Cooksey K, Wigglesworth-Cooksey B. 1995. Adhesion of bacteria and diatoms to surfaces in the sea: a review. Aquatic Microb. Ecol. 9: 87-96.
  11. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. 1995. Microbial biofilms. Ann. Rev. Microbiol. 49: 711-745.
  12. Kim SJ, Yim JH. 2007. Cryoprotective properties of exopolysaccharide (P-21653) produced by the Antarctic bacterium, Pseudoalteromonas arctica KOPRI 21653. J. Microbiol. 45: 510-514.
  13. Stoderegger K, Herndl GJ. 1998. Production and release of bacterial capsular material and its subsequent utilization by marine bacterioplankton. Limnol. Oceanogr. 43: 877-884.
  14. Connor W, Ashwood-Smith M. 1973. Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms. Cryobiology 10: 488-496.
  15. Korber C, Scheiwe M, Boutron P, Rau G. 1982. The influence of hydroxyethyl starch on ice formation in aqueous solutions. Cryobiology 19: 478-492.
  16. Whittaker RJ, Araujo MB, Jepson P, Ladle RJ, Watson JE, Willis KJ. 2005. Conservation biogeography: assessment and prospect. Divers. Distrib. 11: 3-23.
  17. Liu SB, Chen XL, He HL, Zhang XY, Xie BB, Yu Y, et al. 2013. Structure and ecological roles of a novel exopolysaccharide from the Arctic sea ice bacterium Pseudoalteromonas sp. strain SM20310. Appl. Environ. Microbiol. 79: 224-230.
  18. Poli A, Anzelmo G, Nicolaus B. 2010. Bacterial exopolysaccharides from extreme marine habitats: production, characterization and biological activities. Mar. Drugs 8: 1779-1802.
  19. Iyer A, Mody K, Jha B. 2006. Emulsifying properties of a marine bacterial exopolysaccharide. Enzyme Microb. Technol. 38: 220-222.
  20. Raza ZA, Khan MS, Khalid ZM, Rehman A. 2006. Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant. Z. Naturforsch. C. J. Biosci. 61: 87-94.
  21. Song Y, Li S, Gong H, Yip RCS, Chen H. 2023. Biopharmaceutical applications of microbial polysaccharides as materials: a review. Int. J. Biol. Macromol. 239: 124259.
  22. Pornsunthorntawee O, Wongpanit P, Chavadej S, Abe M, Rujiravanit R. 2008. Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil. Bioresour. Technol. 99: 1589-1595.
  23. Murray KA, Gibson MI. 2022. Chemical approaches to cryopreservation. Nat. Rev. Chem. 6: 579-593.
  24. Amiri MJ, Bahrami M, Dehkhodaie F. 2019. Optimization of Hg (II) adsorption on bio-apatite based materials using CCD-RSM design: characterization and mechanism studies. J. Water Health 17: 556-567.
  25. Yilmaz S, Zengin A, Sahan T. 2021. Bentonite grafted with poly (N-acryloylglycineamide) brush: A novel clay-polymer brush hybrid material for the effective removal of Hg (II) and As (V) from aqueous environments. Colloids Surf. A: Physicochem. Eng. Asp. 612: 125979.
  26. Sheikhmohammadi A, Dahaghin Z, Mohseni SM, Sarkhosh M, Azarpira H, Atafar Z, et al. 2018. The synthesis and application of the SiO2@ Fe3O4@ MBT nanocomposite as a new magnetic sorbent for the adsorption of arsenate from aqueous solutions: modeling, optimization, and adsorption studies. J. Mol. Liq. 255: 313-323.
  27. Choudhury AR, Bhattacharyya M, Prasad G. 2012. Application of response surface methodology to understand the interaction of media components during pullulan production by Aureobasidium pullulans RBF-4A3. Biocatal. Agric. Biotechnol. 1: 232-237.
  28. Yu X, Wang Y, Wei G, Dong Y. 2012. Media optimization for elevated molecular weight and mass production of pigment-free pullulan. Carbohydr. Polym. 89: 928-934.
  29. Wu S, Chen J, Pan S. 2012. Optimization of fermentation conditions for the production of pullulan by a new strain of Aureobasidium pullulans isolated from sea mud and its characterization. Carbohydr. Polym. 87: 1696-1700.
  30. Ahmed Z, Mehmood T, Ferheen I, Noori AW, Almansouri M, Waseem M. 2023. Optimization of exopolysaccharide produced by L. Kefiranofaciens ZW3 using response surface methodology. Int. J. Food Prop. 26: 2285-2293.
  31. Li Y, Kan J, Liu F, Lian K, Liang Y, Shao H, et al. 2024. Depth shapes microbiome assembly and network stability in the Mariana Trench. Microbiol. Spectr. 12: e02110-02123.
  32. Yang SH, Park MJ, Oh HM, Park YJ, Kwon KK. 2024. Flavivirga spongiicola sp. nov. and Flavivirga abyssicola sp. nov., isolated from marine environments. J. Microbiol. 62: 11-19.
  33. Sekiguchi T, Kitani Y, Ogiso S. 2024. Observation of marine invertebrates in the Noto Peninsula, pp. 75-86. Field Work and Laboratory Experiments in Integrated Environmental Sciences, Ed. Springer.
  34. Costa N, Hannon J, Guinee T, Auty M, McSweeney P, Beresford T. 2010. Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese. J. Dairy Sci. 93: 3469-3486.
  35. Kim SJ, Youn UJ, Kang P, Kim TK, Kim IC, Han SJ, et al. 2023. A novel exopolysaccharide (p-CY01) from the Antarctic bacterium Pseudoalteromonas sp. strain CY01 cryopreserves human red blood cells. Biomater. Sci. 11: 7146-7157.
  36. Klbik I, Cechova K, Milovska S, Svajdlenkova H, Mat'ko I, Lakota J, et al. 2023. Polyethylene glycol 400 enables plunge-freezing cryopreservation of human keratinocytes. J. Mol. Liq. 379: 121711.
  37. Kehe K, Abend M, Kehe K, Ridi R, Peter RU, van Beuningen D. 1999. Tissue engineering with HaCaT cells and a fibroblast cell line. Arch. Dermatol. Res. 291: 600-605.
  38. Baek K, Lee YM, Hwang CY, Park H, Jung Y-J, Kim M-K, et al. 2015. Psychroserpens jangbogonensis sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment. Int. J. Syst. Evol. Microbiol. 65: 183-188.
  39. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, et al. 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int. J. Syst. Evol. Microbiol. 62: 716-721.
  40. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. 2014. Ribosomal database project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42: D633-D642.
  41. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
  42. Jukes TH, Cantor CR. 1969. Evolution of protein molecules. Mammalian Protein Metab. 3: 21-132.
  43. Fitch WM. 1971. Toward defining the course of evolution: minimum change for a specific tree topology. Syst. Biol. 20: 406-416.
  44. Felsenstein J. 1981. Evolutionary trees from gene frequencies and quantitative characters: finding maximum likelihood estimates. Evolution 35: 1229-1242.
  45. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.
  46. Plackett RL, Burman JP. 1946. The design of optimum multifactorial experiments. Biometrika 33: 305-325.
  47. Myers RH, Montgomery DC, Anderson-Cook CM. 2016. Response surface methodology: process and product optimization using designed experiments, Ed. John Wiley & Sons.
  48. London L, Price N, Ryan P, Wang L, Auty M, Fitzgerald G, et al. 2014. Characterization of a bovine isolate Lactobacillus mucosae DPC 6426 which produces an exopolysaccharide composed predominantly of mannose residues. J. Appl. Microbiol. 117: 509-517.
  49. Onogi S, Matsumoto T, Warashina Y. 1973. Rheological properties of dispersions of spherical particles in polymer solutions. Transact. Soc. Rheol. 17: 175-190.
  50. Liu JZ, Weng LP, Zhang QL, Xu H, Ji LN. 2003. Optimization of glucose oxidase production by Aspergillus niger in a benchtop bioreactor using response surface methodology. World J.Microbiol. Biotechnol. 19: 317-323.
  51. Caruso C, Rizzo C, Mangano S, Poli A, Di Donato P, Nicolaus B, et al. 2018. Extracellular polymeric substances with metal adsorption capacity produced by Pseudoalteromonas sp. MER144 from Antarctic seawater. Environ. Sci. Pollut. Res. 25: 4667-4677.
  52. Rizzo C, Perrin E, Poli A, Finore I, Fani R, Lo Giudice A. 2022. Characterization of the exopolymer-producing Pseudoalteromonas sp. S8-8 from Antarctic sediment. Appl. Microbiol. Biotechnol. 106: 7173-7185.
  53. Mancuso Nichols C, Garon S, Bowman J, Raguenes G, Guezennec J. 2004. Production of exopolysaccharides by Antarctic marine bacterial isolates. J. Appl. Microbiol. 96: 1057-1066.
  54. Nichols CM, Bowman JP, Guezennec J. 2005. Effects of incubation temperature on growth and production of exopolysaccharides by an Antarctic sea ice bacterium grown in batch culture. Appl. Environ. Microbiol. 71: 3519-3523.
  55. Donot F, Fontana A, Baccou J, Schorr-Galindo S. 2012. Microbial exopolysaccharides: main examples of synthesis, excretion, genetics and extraction. Carbohydr. Polym. 87: 951-962.
  56. Decho AW. 1990. Microbial exopolymer secretions in ocean environments: their role (s) in food webs and marine processes. Oceanogr. Mar. Biol. Annu. Rev. 28: 73-153.
  57. Anchordoguy TJ, Cecchini CA, Crowe JH, Crowe LM. 1991. Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers. Cryobiology 28: 467-473.
  58. Chen G, Yue A, Ruan Z, Yin Y, Wang R, Ren Y, et al. 2016. Comparison of the effects of different cryoprotectants on stem cells from umbilical cord blood. Stem Cells Int. 2016: 1396783.
  59. Jang TH, Park SC, Yang JH, Kim JY, Seok JH, Park US, et al. 2017. Cryopreservation and its clinical applications. Integr. Med. Res. 6: 12-18.
  60. Pegg D. 2002. Presented at the Seminars in reproductive medicine.
  61. Sui L, Nomura R, Dong Y, Yamaguchi F, Izumori K, Tokuda M. 2007. Cryoprotective effects of D-allose on mammalian cells. Cryobiology 55: 87-92. 
  62. Naaldijk Y, Johnson AA, Friedrich-Stockigt A, Stolzing A. 2016. Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants. BMC Biotechnol. 16: 85.
  63. Arcarons N, Vendrell-Flotats M, Yeste M, Mercade E, Lopez-Bejar M, Mogas T. 2019. Cryoprotectant role of exopolysaccharide of Pseudomonas sp. ID1 in the vitrification of IVM cow oocytes. Reprod. Fertil. Dev. 31: 1507-1519.
  64. Hughes ZE, Malajczuk CJ, Mancera RL. 2013. The effects of cryosolvents on DOPC- β-sitosterol bilayers determined from molecular dynamics simulations. J. Phys. Chem. B. 117: 3362-3375.
  65. Brinkmeyer R, Knittel K, Jurgens J, Weyland H, Amann R, Helmke E. 2003. Diversity and structure of bacterial communities in Arctic versus Antarctic pack ice. Appl. Environ. Microbiol. 69: 6610-6619.
  66. Delille D. 1992. Marine bacterioplankton at the Weddell Sea ice edge, distribution of psychrophilic and psychrotrophic populations. Polar Biol. 12: 205-210.
  67. Bowman JP, McCammon SA, Brown MV, Nichols DS, McMeekin TA. 1997. Diversity and association of psychrophilic bacteria in Antarctic sea ice. Appl. Environ. Microbiol. 63: 3068-3078.
  68. Bowman JP. 1998. Pseudoalteromonas prydzensis sp. nov., a psychrotrophic, halotolerant bacterium from Antarctic sea ice. Int. J. Syst. Evol. Microbiol. 48: 1037-1041.
  69. Mahapatra S, Banerjee D. 2013. Optimization of a bioactive exopolysaccharide production from endophytic Fusarium solani SD5. Carbohydr. Polym. 97: 627-634.
  70. Song YR, Jeong DY, Baik SH. 2013. Optimal production of exopolysaccharide by Bacillus licheniformis KS-17 isolated from kimchi. Food Sci. Biotechnol. 22: 417-423.
  71. Lu MK, Lee MH, Chao CH, Hsu YC. 2024. Sodium sulfate addition increases the bioresource of biologically active sulfated polysaccharides from Antrodia cinnamomea. Int. J. Biol. Macromol. 257: 128699.
  72. Epstein W. 2003. The roles and regulation of potassium in bacteria. Progress Nucleic Acid Res. Mol. Biol. 75: 293-320.
  73. Jiang Y, Zhang M, Zhang Y, Zulewska J, Yang Z. 2021. Calcium (Ca2+)-regulated exopolysaccharide biosynthesis in probiotic Lactobacillus plantarum K25 as analyzed by an omics approach. J. Dairy Sci. 104: 2693-2708.
  74. Cheng JJ, Chao CH, Chang PC, Lu MK. 2016. Studies on anti-inflammatory activity of sulfated polysaccharides from cultivated fungi Antrodia cinnamomea. Food Hydrocoll. 53: 37-45.
  75. Suresh Kumar A, Mody K, Jha B. 2007. Bacterial exopolysaccharides-a perception. J. Basic Microbiol. 47: 103-117.