Optimization of Culture Conditions for 1,3-propanediol Production from Glycerol Using Klebsiella pneumoniae

글리세롤로부터 1,3-propanediol 생산을 위한 Klebsiella pneumoniae 배양 조건 최적화

  • Jun, Sun-Ae (Center for Environmental Technology research, Korea Institute of Science and Technology) ;
  • Kong, Sean W (INWOO Corporation) ;
  • Sang, Byoung-In (Center for Environmental Technology research, Korea Institute of Science and Technology) ;
  • Um, Youngsoon (Center for Environmental Technology research, Korea Institute of Science and Technology)
  • 전선애 (한국과학기술연구원 환경기술연구단) ;
  • 공성욱 ((주)인우코퍼레이션) ;
  • 상병인 (한국과학기술연구원 환경기술연구단) ;
  • 엄영순 (한국과학기술연구원 환경기술연구단)
  • Received : 2009.08.03
  • Accepted : 2009.09.22
  • Published : 2009.12.31

Abstract

To improve the productivity of 1,3-propanediol(1,3-PD) with K. pneumoniae DSM4799 using pure glycerol and crude glycerol derived from the biodiesel process, optimizing fermentation conditions was performed by changing environmental factors such as anaerobic/aerobic condition, temperature, glycerol concentration, and pH. When anaerobic conditions were maintained, there was an improved 1,3-PD production compared with that from aerobic/anaerobic 2-stage fermentation. From the results with temperature $26{\sim}37^{\circ}C$, the higher 1,3-PD production yield was observed at $30{\sim}33^{\circ}C$. For an initial glycerol concentration higher than 60 g/L, cell growth and 1,3-PD production were inhibited. When crude glycerol was used, the initial 1,3-PD production appeared to be inhibited. After 48 hr of incubation, however, 1,3-PD production with crude glycerol was even higher than that with pure glycerol, demonstrating the feasibility of 1,3-PD production using crude glycerol as a substrate. Fed-batch fermentation was applied for the high concentration of 1,3-PD without substrate inhibition. By regulating pH at 7 during the fed-batch with glycerol lower than 40 g/L, the yield of 1,3-PD was 25% higher than that without pH regulation(0.56 g/g vs. 0.45 g/g). In conclusion, based on our results, anaerobic conditions, temperature at $30^{\circ}C$, pure or crude glycerol lower than 40 g/L, and pH regulation at 7 were the optimized conditions for 1,3-PD production using K. pneumoniae DSM4799, making it more feasible to produce 1,3-PD at higher concentration and a lower price.

본 연구는 통성 혐기성 미생물인 K. pneumoniae DSM4799을 이용하여 순수 글리세롤과 국내 바이오디젤 생산공정에서 발생된 폐글리세롤로부터 1,3-PD을 생산하기 위한 연구로서, 혐기 및 호기 조건, 배양온도, 글리세롤 농도, pH에 따른 1,3-PD 생산성 비교를 통해 최적 배양조건을 찾고자 하였다. K. pneumoniae DSM4799를 혐기조건과 호기/혐기 2단계 배양을 한 결과, 혐기조건에서 더 효율적인 1,3-PD 생산이 이루어졌다. 배양 온도를 $26{\sim}37^{\circ}C$로 변화시키면서 배양한 결과, $30{\sim}33^{\circ}C$에서 높은 1,3-PD 생산성을 나타내었고, 글리세롤 농도는 글리세롤의 종류에 상관없이 60 g/L 이상에서 균주의 성장 및 1,3-PD 생산이 저해되는 현상을 관찰할 수 있었다. 폐글리세롤 사용시 순수 글리세롤에 비해 초기 1,3-PD 생산은 감소하였으나, 48시간 후에는 오히려 더 높은 농도의 1,3-PD를 생산하였다. 유가식 배양으로 글리세롤 농도를 40 g/L 이하로 조절하면서 pH 조절유무에 따른 1,3-PD 및 부산물의 변화를 살펴본 결과, pH를 7.0으로 유지시켰을 때 pH 조절을 하지 않은 경우보다 25% 향상된 1,3-PD 수율을 나타내었다(0.56 g/g vs. 0.45 g/g). 본 연구를 통해 K. pneumoniae DSM4799를 이용하여 1,3-PD 생산시 혐기조건, 온도 $30^{\circ}C$, 순수 또는 폐글리세롤 40 g/L 이하, pH 조절 등의 배양조건이 적합함을 알 수 있었으며, 최적화된 배양조건을 통해 보다 가격경쟁력이 있는 생물학적 1,3-PD 생산이 가능할 것으로 기대된다.

Keywords

Acknowledgement

Supported by : 에너지관리공단, 교육과학기술부

References

  1. Biebl, H., Menzel, K., Zeng, A. P. and Deckwer, W. D., "Microbial Production of 1,3-propanediol," Appl. Microbiol. Biotechnol., 52, 289-297(1999) https://doi.org/10.1007/s002530051523
  2. Zeng, A.-P. and Biebl, H., "Bulk Chemicals from Biotechnology: The Case of 1,3-propanediol Production and the New Trends," Adv. Biochem. Eng./Biotechnol., 74, 239-259(2002) https://doi.org/10.1007/3-540-45736-4_11
  3. Knifton, J. F., James, T. G., Slaugh, L. H., Allen, K. D., Weider, P. R. and Powell, J. B., "One-step Production of 1,3-propanediol from Ethylene Oxide and Syngas with a Cobalt-iron Catalyst," US Patent No. 6,750,373(2004)
  4. Besson, M., Gallezot, P., Pigamo, A. and Reifsnyder, S., "Development of An Improved Continuous Hydrogenation Process for the Production of 1,3-propanediol Using Titania Supported Ruthenium Catalysts," Appl. Catal. A-Gen., 250, 117-124(2003) https://doi.org/10.1016/S0926-860X(03)00233-3
  5. Chaminand, J., Djakovitch, L. A., Gallezot, P., Marion, P., Pinel, C. and Rosier, C., "Glycerol Hydrogenolysis on Heterogeneous Catalysts," Green Chem., 6, 359-361(2004) https://doi.org/10.1039/b407378a
  6. Wang, K., Hawley, M. C. and DeAthos, S. J., "Conversion of Glycerol to 1,3-propanediol Via Selective Dehydroxylation, " Ind. Eng. Chem. Res., 42, 2913-2923(2003) https://doi.org/10.1021/ie020754h
  7. Mu, Y., Xiu, Z.-L. and Zhang, D.-J., "A Combined Bioprocess of Biodiesel Production by Lipase with Microbial Production of 1,3- propanediol by Klebsiella pneumoniae, " Biochem. Eng. J., 40, 537-541(2008) https://doi.org/10.1016/j.bej.2008.02.011
  8. Willke, T. and Vorlop, K., "Biotransformation of Glycerol Into 1,3-propanediol," Eur. J. Lipid Sci. Technol., 110, 831-840(2008) https://doi.org/10.1002/ejlt.200800057
  9. Chen, X., Zhang, D. J., Qi, W. T., Gao, S. J., Xiu, Z. L. and Xu, P., "Microbial Fed-batch Production of 1,3-propanediol by Klebsiella pneumoniae Under Micro-aerobic Conditions," Appl. Microbiol. Biotechnol., 63, 143-146(2003) https://doi.org/10.1007/s00253-003-1369-5
  10. Laffend, L. A., Nagarajan, V. and Nakamura, C. E., "Bioconversion of a Fermentable Carbon Source to 1,3-propanediol by Single Microorganism," WO Patent No. 9,635,796(1996)
  11. Hirschmann, S., Baganz, K., Koschik, I. and Vorlop, K. D. 'Development of an Integrated Bioconversion Process for the Production of 1,3-propanediol from Raw Glycerol Waters,' Landbauforsch. Volk., 55, 261-267(2005)
  12. Yazdani, S. S. and Gonzalez, R., 'Anaerobic Fermentation of Glycerol: a Path to Economic Viability for the Biofuels Industry,' Curr. Opin. Microbiol., 18, 213-219(2007)
  13. Jun, S.-A., Kang, C.-H., Kong, S. W., Sang, B.-I. and Um, Y., 'Biological Production of 1,3-propanediol Using Crude Glycerol Derived from Biodiesel Process,' Korean J. Biotechnol. Bioeng., 23, 413-418(2008)
  14. Nemeth, A., Kupcsulik, B. and Sevella, B., "1,3-propanediol Oxidoreductase Production with Klebsiella pneumoniae DSM2026," World J. Microbiol. Biotechnol., 19, 659-663(2003) https://doi.org/10.1023/A:1025116308484
  15. Cheng, K.-K., Liu, H.-J. and Liu, D.-H., "Multiple Growth Inhibition of Klebsiella pneumoniae in 1,3-propanediol Fermentation," Biotechnol. Lett., 27, 19-22(2005) https://doi.org/10.1007/s10529-004-6308-8
  16. Biebl, H., Zeng, A. P., Menzel, K. and Deckwer, W. D., "Fermentation of Glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae," Appl. Microbiol. Biotechnol., 50, 24-29(1998) https://doi.org/10.1007/s002530051251