KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Scaling Up Study of Exopolysaccharide Production through Mycelial Submerged Cultivation of Ganoderma lucidum

영지의 액체배양에 의한 세포외 다당 생산의 Scale Up 연구

  • Lee, Hak-Su (Technique Research Institute, Dongbang Future Chemical Co., Ltd.) ;
  • Lee, Shin-Young (Department of Bioengineering and Technology, Kangwon National University)
  • 이학수 (동방미래화학(주) 기술연구소) ;
  • 이신영 (강원대학교 생물공학과)
  • Published : 2009.06.29
Download PDF
⟨ Previous Next ⟩

Abstract

A scaling up study for the exopolysaccharide (EPS) production by submerged culture of Ganoderma lucidum was carried out in jar fermenter systems (2.6, 20 and 75 L) under bi-staged pH process. Profiles of dissolved oxygen (DO) and volumetric coefficient of oxygen transfer ($k_La$) as a function of operating variables (agitation speed and aeration rate) was investigated, and a correlation between $k_La$ and operating variables was analysed statistically. Under bi-staged pH process, no limitation of DO was observed at agitation speeds tested in the range of 200 and 600 rpm, and the highest EPS production was obtained at the level of DO of $40{\sim}80%$. From the regression analysis, the relation between $k_La$, gas velocity (Vs), stirrer speed (N) and impeller diameter (Di) could be expressed as : $$k_La=0.555{\times}Vs^{0.42}{\times}(N^3{\times}Di^2)^{0.33}\;(R^2=0.925,\;p<0.05)$$ It was found that under 2.6 L jar fermenter, the optimum agitation speed and aeration rate was 400 rpm and 1 vvm, respectively, obtaining the EPS production of 15.43 g/L. Under the submerged cultivation of G. lucidum in jar fermenters of $2.6{\sim}75\;L$, the similar EPS yields at each fermenter were achieved during scaling up based on $k_La$, and $k_La$ value for maximum EPS production was $85.4{\pm}26.70\;h^{-1}$.

Bi-staged pH process 하의 jar fermenter system에서 영지의 액체배양 시 효율적인 균사체 및 세포의 다당 생산을 위한 용존산소의 영향을 조사하고, 산소이동용량계수와 발효조 조작변수와의 상관관계를 해석하여 scale up의 조건을 검토, 확립하였다. 교반속도에 따른 용존산소의 농도변화를 살펴본 결과, hi-staged pH process에서 배양 $2{\sim}4$일에 용존 산소의 급격한 감소를 보였으나 용존 산소의 제한은 일어나지 않고, 다시 증가하는 경향을 보였으며, 교반속도가 증가할수록 감소 폭과 깊이는 증가하는 경향을 보였다. 다당 생성은 대수 증식기 이후의 용존 산소농도가 $40{\sim}80%$ 범위일 때 가장 높았으며, 이 보다 낮거나 높은 경우 다당의 생성은 저해되었다. 교반 및 통기에 따른 산소이동용량계수 ($k_La$)와 통기속도 (Vs), 교반속도 (N) 및 impeller 지름 (Di)의 자료로부터 중회귀분석한 결과, 이들 사이의 관계는 다음 식으로 표현할 수 있었다. $$k_La=0.555{\times}Vs^{0.42}{\times}(N^3{\times}Di^2)^{0.33}\;(R^2=0.925,\;p<0.05)$$ 2.6 L 발효조의 경우, 15.43 g/L의 최대 다당 생산량은 400 rpm 및 1 vvm에서 얻어졌으며, 2.6 L 발효조의 최적 산소이동용량계수 값을 적용한 20 및 75 L 발효조에서의 scale up 시험 결과는 서로 잘 일치하였다. 최대의 다당생성을 위한 산소전달계수의 감은 $85.4{\pm}26.70\;h^{-1}$이었다.

Keywords

References

  1. Jong, S. C. and J. M. Birmingham, and S. H. Pai (1991), Immunomodulatory substances of fungal origin. J. lmmunol. lmmunopharmacol. 11, 115-122
  2. Jong, S. C. and J. M. Birmingham (1992), Medicinal benefits of the mushroom Ganoderma. Adv. Appl. Microbiol. 37, 101-134 https://doi.org/10.1016/S0065-2164(08)70253-3
  3. Yang, H. L., T. X. Wu, and K. C. Zhang (2004), Enhancement of mycelial growth and polysaccharide production in Ganoderma lucidum by the addition of ethanol. Biotechnol. Lett. 26, 841-844 https://doi.org/10.1023/B:BILE.0000025888.93564.a0
  4. Hsieh, C., M H. Tseng, and C. J. Liu (2006), Production of polysaccharides from Ganoderma lucidum (CCRC 36041) under limitations of nutrients. Enzyme Microb. Technol. 38, 109-117 https://doi.org/10.1016/j.enzmictec.2005.05.004
  5. Yang, F. C. and C. B. Liau (1998), The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures. Process Biochem. 33, 547-553 https://doi.org/10.1016/S0032-9592(98)00023-5
  6. Tang, Y. J. and J. J. Zhong (2002), Exopolysaccharide biosynthesis and related enzyme activities of the medicinal fungus, Gan'Oderma lucidum, grown on lactose in a reactor. Biotechnol. Lett. 24, 1023-1026 https://doi.org/10.1023/A:1015677313598
  7. Fang, Q. H. and J. J. Zhong (2002), Effect of initial pH on production of ganoderic acid and polysaccharide by submerged fermentation of Gan'Oderma lucidum. Process Biochem. 37, 769-774 https://doi.org/10.1016/S0032-9592(01)00278-3
  8. Fang, Q. H., and J. J. Zhong (2002), Submerged fermentation of higher fungus Gan'Oderma lucidum for production of valuable bioactive metabolites-ganoderic acid and polysaccharide. Biochem. Eng. J. 10, 61-65 https://doi.org/10.1016/S1369-703X(01)00158-9
  9. Yang, B. K., S. C. Jeong, and C. H. Son (2002), Hypolipidemic effect of exo-and endo-biopolymers produced from submerged mycelial culture of Ganodermalucidum in rats. J. Microbiol. Biotechnol. 12, 872-877
  10. Wagner, R., D. A. Mitchell, G. L. Sassaki, M. A. L. de A. Amazonas, and M. Berovic (2003), Current techniques for the cultivation of G. lucidum for the production of biomass, ganoderic acid and polysaccharides. Food Technol. Biotechnol. 41, 371-382
  11. Tang, Y.J., L. W. Zhu, H. M. Li, and D. S. Li (2007), Submerged culture of mushrooms in bioreactor-Challenges, Current state-of-the-art, and Future prospects. Food Technol. Biotechnol. 45, 221-229
  12. Lee, S. Y., and T. S. Kang (1996), Production condition and characterization of exo-polysaccharide production produced by submerged cultivation of Ganoderma lucidum mycelium. Korean J. Appl. Microbiol. Biotechnol. 24, 111-118
  13. Lee, S. Y., T. S. Kang, S. O. Moon, I. D. Lew, and M. Y. Lee (1996), Fractionation and antitumor activity of the water-soluble exo-polysaccharide by submerged cultivation of Ganoderma lucidum mycelium. Korean cultivation of Ganoderma lucidum mycelium. Korean J. Appl. Microbiol. Biotechnol 24, 459-464
  14. Lee, S. Y., and T. S. Kang (1997). Optimization of antitumor active exo-polysaccharide production through the submerged cultivation of Ganoderma lucidum mycelium. Kor. J. Biotech. Bioeng. 12, 1391-135
  15. Lee, S. Y., T. S. Kang, and M. C. Lee (1998), Condition of exo-polysaccharide production from submerged mycelial culture of Ganoderma lucidum by using air-lift fermenter system. Korean J. Biotechnol. Bioeng. 13, 547-553
  16. Lee, K. M., S. Y. Lee, and H. Y. Lee (1999), Bistage Control of pH for improving exo-polysaccharide production from mycelia of Gan'Oderma lucidum in an air-lift fermentor. J. Biosci. Bioeng. 88, 646-650 https://doi.org/10.1016/S1389-1723(00)87094-2
  17. Lee, S. Y. and T. S. Kang (1999), Structural analysis of the antitumor active exo- polysaccharide produced by submerged cultivation of Ganoderma lucidum mycelium. Korean J. Mycol. 27, 76-81
  18. Lee, S. Y., and K. M. Lee (2000), Effect of different pH processes on branched $\beta$-1, 3-glucan production from submergεd mycelial culture of Ganoderma lucidum. Journal of lndustrial Technolgy (Kangwon Natl. Univ.). 20A, 45-50
  19. Lee, K. M. and S. Y. Lee (2001), Influence of ammonium phosphate on mycelial morphology during submerged cultivation of Ganoderma lucidum. The Korean Journal of Mycology 29, 91-98
  20. Lee, H. S., J. H. Jung, and S. Y. Lee (2001), Effects and batch kinetics of agitation and aeration on submerged cultivation of Ganoderma lucidum. Korean J. Biotechnol. Bioeng. 16, 307-313
  21. Lee, S. Y. and H. S. Lee (2001), Effect of polyacrylic acid addition on wall growth in submerged cultivation of Ganoderma lucidum. Journal of lndustrial Technolgy (Kangwon Natl. Univ.). 21A, 341-345
  22. Chung, W. T., S. H. Lee, J. D. Kim, Y. S. Park, B. Hwang, S. Y. Lee, and H. Y. Lee (2001), Effect of mycelial culture broth of Ganoderma lucidum on the growth characteristics of human cell lines. J. Biosci. Bioeng. 92, 550-555 https://doi.org/10.1263/jbb.92.550
  23. Lee, S. Y. and K. M. Lee (Jul. 9, 2002), Exopolysaccharide production from submerged mycelial culture of mushroom. US 6,416,978 B1
  24. Lee, S. Y., T. S. Kang, and S. O. Moon (2005), Antitumor activity and effect on cell proliferation and differenciation of exopolysaccharide produced by submerged cultivation of Ganoderma lucidum. Journal of lndustrial Technolgy (Kangwon Natl. Univ.). 25B, 241-251
  25. Rane, K. D. and K. A. Sims (1994), Oxygen uptake and citric acid production by Candida lipolytica Y1095, Biotechnol. Bioeng. 43, 131-137 https://doi.org/10.1002/bit.260430205
  26. Dronawat, S. N., C. K. Svihla, and T. Hanley (1995), The Effects of agitation and aeration on the production of gluconic Acid by Aspergillus niger, Appl. Biochem. Biotechnol. 51, 347-354 https://doi.org/10.1007/BF02933438
  27. Sone, Y., R. Okuda, A. Wada, A. Kishida, and A. Misaki (1985), Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderam lucidum. Agric. Biol. Chem. 49, 2642-2650
  28. Miller, G. L. (1959), Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chem. 31, 426-428 https://doi.org/10.1021/ac60147a030
  29. Bandyopadhysay, B. and A. E. Humphrey (1967), Dynamic of the volumetric oxygen transfer coefficient in fermentation systems. Biotechnol. Bioeng. 9, 533-544 https://doi.org/10.1002/bit.260090408
  30. Nho, H. J. (1998), Investigation Method and Statistical Analysis by Excell (in Korean), Pumunsa, Seoul, Korea, pp320-333
  31. Gibbs, P. A and R. J. Seviour (1998), The Production of exo-polysaccharide by Aureobasidium pullulans in fermenters with low-shear configurations. Appl. Microbiol. Biotechnol. 49, 168-174 https://doi.org/10.1007/s002530051154
  32. Gibbs, P. A. and R. J. Seviour (1996), Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture? Appl. Microbiol. Biotechnol. 46, 503-510 https://doi.org/10.1007/s002530050851
  33. Peter, H. U., H. Herbst, P. G. M. Hesselink, H. Lunsdorf, A. Schumpe, and W. D. Deckwer (1989), The influence of agitation rate on xanthan production by Xanthomonas campestris. Biotechnol, Bioeng. 34, 1393-1397 https://doi.org/10.1002/bit.260341108
  34. Rau, U., E. Gura, E. Olszewski, and F. Wagner (1992), Enhanced glucan formation of filamentous fungi by effective mixing, oxygen limitation and fed- batch processing. J. lndustrial Microbiol. 9, 19-26 https://doi.org/10.1007/BF01576364
  35. Lawford, H. and J. Rouseau (1989), Effect of oxygen on the rate of $\beta$-1, 3-Glucan microbial exopolysaccharide production. Biotechnol. Lett. 11, 125-130 https://doi.org/10.1007/BF01192188
  36. Rho, D., A. Mulchandini, J. H. T. Luong, and A. LeDuy (1988), Oxygen requirement in pullman fermentation. Appl. Microbiol. Biotechnol. 28, 361-366 https://doi.org/10.1007/BF00268196
  37. Brown, D. E. and M. A. Zainudeen (1978), Effect of inoculum size on the aeration pattem of batch cultures of a fungal microorganism. Biotechnol. Bioeng. 20, 1045-1061 https://doi.org/10.1002/bit.260200707
  38. Pessoa. P., M. Vitolo, and H. Hustedt (1996), Use of Kla as a criterion for scaling up the inulinase fermentation process. Appl. Biochem. Biotechnol. 57, 699-709 https://doi.org/10.1007/BF02941752
  39. Gaspar, A., L. Strodiot, and P. Tonart (1998), Improvement of oxygen transfer coefficient during Penicillium cancescens culture. Appl. Biochem Biotechnol. 70, 535-545 https://doi.org/10.1007/BF02920166