Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

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

DOI QR Code

Isolation, Identification and Mutant Development of Butanol Tolerance Bacterium

부탄올 내성 미생물의 분리, 동정 및 변이주의 개발

  • Jung, Hyesook (Department of Food Science & Biotechnology, Kyungsung University) ;
  • Lee, Jinho (Department of Food Science & Biotechnology, Kyungsung University)
  • 정혜숙 (경성대학교 식품생명공학과) ;
  • 이진호 (경성대학교 식품생명공학과)
  • Received : 2012.10.23
  • Accepted : 2012.12.05
  • Published : 2013.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

Butanol-resistant bacteria were isolated from butanol solvent. The cell growth of isolated strains declined with increasing concentrations of butanol, and isolated strain BRS02 displayed more resistance to 12.5 g/L of butanol than other isolated strains. In addition, strain BRS251, which was resistant to even higher concentrations of butanol, was developed by the mutation of BRS02 using UV. BRS251 could grow in LB medium containing up to 17.5 g/L of butanol, 32.5 g/L of propanol, or 6 g/L of pentanol, whereas the control strain Escherichia coli was found to be tolerant to 7.5 g/L of butanol, 20 g/L of propanol, or 2 g/L of pentanol. The isolated BRS02, a Gram(+) bacterium seen to have a cocci form under the microscope, grew in 6.5% NaCl. According to biochemical tests, BRS02 can metabolize and produce acid with D-galactose, D-maltose, D-mannitol, D-mannose, methyl-${\beta}$-Dglucopyranoside, D-ribose, sucrose, or D-trehalose, as carbon sources. Also, this strain showed resistance to bacitracin, vibriostatic agent O/129, and optochin, alongside positive activities for arginine dihydrolase, ${\alpha}$-glucosidase, and urease. The BRS02 strain was identified as Staphylococcus sp. by analyses of the 16S rRNA gene, phylogenetic tree, and biochemical tests.

부탄올 용매에서 생존하는 부탄올 내성 미생물을 분리하였다. 분리된 미생물들의 세포성장은 부탄올 농도가 증가함에 따라 감소하였으며, 그 중에서 BRS02가 12.5 g/L에서 가장 높은 내성도를 나타내었다. 또한, UV를 이용하여 BRS02균의 변이를 유도하여 고농도 부탄올 내성균 BRS251을 개발하였다. 부탄올 생산 모델균주로 대장균과 함께 부탄올, 프로판올 및 펜탄올에 대한 내성도를 비교한 결과, 대장균은 7.5 g/L 부탄올과 20 g/L 프로판올, 2 g/L 펜탄올 농도까지 생육이 가능한 반편, BRS251은 더 고농도인 17.5 g/L 부탄올과 32.5 g/L 프로판올, 6 g/L 펜탄올 농도까지 생육이 가능하였다. 분리된 세균을 동정하기 위해서 그람염색 후 광학현미경으로 관찰한 결과 그람양성의 구균으로 확인이 되었으며, 6.5% NaCl에서 생육이 가능하였다. 생화학적 특성을 분석한 결과, arginine dihydrolase, ${\alpha}$-glucosidase, urease 효소활성을 가지고 있었으며, 호기적인 조건에서 D-galactose, Dmaltose, D-mannitol, D-mannose, methyl-${\beta}$-D-glucopyranoside, D-ribose, sucrose, D-trehalose를 탄소원으로 자화하여 산을 생성할 수 있었으며, bacitracin, vibriostatic agent O/129 및 optochin에 대한 항생제 내성을 나타내었다. 16S rRNA 유전자 서열을 결정하고 계통발생도 분석을 통해 BRS02는 최종적으로 Staphylococcus sp.임을 동정하였다.

Keywords

References

  1. Alsaker, K. V., C. Paredes, and E. T. Papoutsakis. 2010. Metabolite stress and tolerance in the production of biofuels and chemicals: Gene-expression-based systems analysis of butanol,butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotechnol. Bioeng. 105: 1131-1147.
  2. Atsumi, S., A. F. Cann, M. R. Connor, C. R. Shen, K. M. Smith, M. P. Brynildsen, K. J. Y. Chou, T. Hanai, and J. C. Liao. 2007. Metabolic engineering of Escherichia coli for 1-butanol production. Metab. Eng. 10: 305-311.
  3. Atsumi, S., T. Hanai, and J. C. Liao. 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451: 86-90. https://doi.org/10.1038/nature06450
  4. Blombach, B., T. Riester, S. Wieschalka, C. Ziert, J. W. Youn, V. F. Wendisch, and B. J. Eikmanns. 2011. Corynebacterium glutamicum tailored for efficient isobutanol production. Appl. Environ. Microbiol. 77: 3300-3310. https://doi.org/10.1128/AEM.02972-10
  5. Cann, A. F. and J. C. Liao. 2008. Production of 2-methyl-1- butanol in engineered Escherichia coli. Appl. Microbiol. Biotechnol. 81: 89-98. https://doi.org/10.1007/s00253-008-1631-y
  6. Ezeji, T., C. Milne, N. D. Price, and H. P. Blaschek. 2010. Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms. Appl. Microbiol. Biotechnol. 85: 1697-1712. https://doi.org/10.1007/s00253-009-2390-0
  7. Fischer, C. R., D. K. Marcuschamer, and G. Stephanopoulos. 2008. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 10: 295-304. https://doi.org/10.1016/j.ymben.2008.06.009
  8. Grkovic, S., M. H. Brown, K. M. Hardie, N. Firth, and R. A. Skurray. 2003. Stable low-copy-number Staphylococcus aureus shuttle vectors. Microbiology 149: 785-794. https://doi.org/10.1099/mic.0.25951-0
  9. Knoshaug, E. P. and M. Zhang. 2008. Butanol tolerance in a selection of microorganisms. Appl. Biochem. Biotechnol. 153: 13-20.
  10. Kumar, S., R. Jansen, F. Sasse, and G. Hofle. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5: 150-163. https://doi.org/10.1093/bib/5.2.150
  11. Lee, J. H. 2012. Isolation and genetic characterization of protease- producing halophilic bacteria from fermenting anchovy. J. Life Science 22: 167-176. https://doi.org/10.5352/JLS.2012.22.2.167
  12. Lee, S. Y., J. H. Park, S. H. Jang, L. K. Nielsen, J. Kim, and K. S. Jung. 2008. Fermentative butanol production by Clostridia. Biotechnol. Bioeng. 101: 209-228. https://doi.org/10.1002/bit.22003
  13. Li, J., J. B. Zhao, M. Zhao, Y. L. Yang, W. H. Jiang, and S. Yang. 2010. Screening and characterization of butanol-tolerant micro-organisms. Lett. Appl. Microbiol. 50: 373-379. https://doi.org/10.1111/j.1472-765X.2010.02808.x
  14. Lin, Y. and S. Tanaka. 2006. Ethanol fermentation from biomass resources: current state and prospects. Appl. Microbiol. Biotechnol. 69: 627-642. https://doi.org/10.1007/s00253-005-0229-x
  15. Liu, S. and N. Qureshi. 2009. How microbes tolerate ethanol and butanol. New Biotech. 26: 117-121. https://doi.org/10.1016/j.nbt.2009.06.984
  16. Park, H. J. and J. H. Lee. 2012. Transcriptional analysis responding to propanol stress in Escherichia coli. J. Life Science 22: 417-427. https://doi.org/10.5352/JLS.2012.22.3.417
  17. Qureshi, N. and H. P. Blaschek. 2001. Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101. J. Ind. Microbiol. Biotechnol. 27: 287-291. https://doi.org/10.1038/sj.jim.7000114
  18. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for constructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
  19. Sambrook, J. and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  20. Shen, C. R. and J. C. Liao. 2008. Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways. Metab. Eng. 10: 312-320. https://doi.org/10.1016/j.ymben.2008.08.001
  21. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  22. Xu, M., P. Wang, F. Wang, and X. Xiao. 2005. Microbial diversity at a deep-sea station of the Pacific nodule province. Biodivers. Conserv. 14: 3363-3380. https://doi.org/10.1007/s10531-004-0544-z
  23. Yan, Y. and J. C. Liao. 2009. Engineering metabolic systems for production of advanced fuels. J. Ind. Microbiol. Biotechnol. 36: 471-479. https://doi.org/10.1007/s10295-009-0532-0