Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Production of Poly-3-hydroxybutyrate from Xylose by Bacillus megaterium J-65

Bacillus megaterium J-65에 의한 xylose로부터 poly-3-hydroxybutyrate 생산

  • Jun, Hong-Ki (Department of Microbiology, College of Natural Science, Pusan National University) ;
  • Jin, Young-Hi (Department of Microbiology, College of Natural Science, Pusan National University) ;
  • Kim, Hae-Nam (Department of Beauty Care, Masan College) ;
  • Kim, Yun-Tae (Seoul Medical Science Institute and Seoul Clinical Laboratories) ;
  • Kim, Sam-Woong (Department of Microbiology, College of Natural Science, Pusan National University) ;
  • Baik, Hyung-Suk (Department of Microbiology, College of Natural Science, Pusan National University)
  • 전홍기 (부산대학교 자연과학대학 미생물학과) ;
  • 진영희 (부산대학교 자연과학대학 미생물학과) ;
  • 김해남 (마산대학 뷰티케어과) ;
  • 김윤태 (서울의과학연구소) ;
  • 김삼웅 (부산대학교 자연과학대학 미생물학과) ;
  • 백형석 (부산대학교 자연과학대학 미생물학과)
  • Published : 2008.12.30
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Abstract

A microorganism capable of producing high level of poly-3-hydoxybutyrate (PHB) from xylose was isolated from soil. The isolated strain J-65 was identified as Bacillus megaterium based on the morphological, biochemical and molecular biological characteristics. The optimum temperature and pH for the growth of B. megaterium J-65 were $37^{\circ}C$ and 8.0, respectively. The optimum medium composition for the cell growth was 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, and 0.1% $KH_2PO_4$. The optimum condition for PHB accumulation was same to the optimum condition for cell growth. Copolymer of ${\beta}$-hydroxybutyric and ${\beta}$-hydroxyvaleric acid was produced when propionic acid was added to shake flasks containing 20 g/l of xylose. Fermenter culture was carried out to produce the high concentration of PHB. In batch culture, cell mass was 9.82 g/l and PHB content was 35% of dry cell weight. PHB produced by B. megaterium J-65 was identified as homopolymer of 3-hydoxybutyric acid by GC and NMR.

본 연구는 생분해성 플라스틱인 poly-${\beta}$-hydroxybutyrate (PHB)의 생산단가를 낮추기 위한 노력으로 저가의 기질로부터 PHB 대량생산을 위한 기초자료를 얻는데 그 목적을 두었다. Hemicellulose hydrolysate는 지구상에 풍부하게 존재하는 저가의 waste by-product로서 xylose가 많이 포함되어 있다. 본 연구에서는 xylose로부터 PHB를 생산할 수 있는 균주를 토양에서 분리하여, 분류학적 위치를 밝히고, 균체 생육 최적 조건, PHB 생산을 위한 최적 발효 배양 조건, PHB의 구조 확인 등을 검토 하였으며, 그 결과는 다음과 같다. 토양으로부터 분리한 균주 J-65는 형태학적, 배양적, 생화학적 및 partial 16S rRNA sequence에 근거하여 Bacillus megaterium로 동정하였다. B. megaterium J-65의 균체 생육 최적 조건은 온도 $37^{\circ}C$, 초발 pH 8.0이었으며 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, 0.1% $KH_2PO_4$였다. PHB 축적에 영향을 미치는 요인을 검토하기 위해 균체생육 최적배지에서 $37^{\circ}C$, 24시간 1차 배양한 후, 균체를 회수하여 각종 영양분이 결핍된 배지에 2차 배양을 실시한 결과 B. megaterium J-65는 균형생육조건(balanced-growth condition)에서 PHB를 합성하는 균주로 나타났다. PHB보다 물성이 향상된 PHB/HV 공중 합체를 생산하기 위하여 보조기질로 propionic acid를 첨가하였을 때, 0.1% propionic acid 농도에서 HV mol%가 14%인 PHB/HV 공중합체가 합성되었다. 5 l 용량의 발효조에 B. megaterium J-65를 회분배양하였을 때 배양 21시간에 건조균체량 10 g/l, PHB 3.5 g/l를 얻을 수 있었고, 유가배양을 실시한 결과 배양 48시간에 건조균체량 26.52g/l, PHB 9.28 g/l를 얻을 수 있었다. 생산된 PHB를 alkaline solution 처리와 chloroform을 이용한 유기용매 추출법을 이용하여 추출.정제한 후 Gas Chromatography로 정제를 확인하고 300MHz 1H-NMR을 실시한 결과 3-hydroxybutyrate의 homopolymer임을 확인하였다.

Keywords

References

  1. Anderson, A J. and E. A Dawes. 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450-472
  2. Anthony, G O. and J. G Holt. 1982. Nile blue A as a fluorescent stain for Poly-$\beta$-hydroxybutyrate. Appl. Environ. Microbiol. 44, 238-241
  3. Bertrand, J. L., B. A Ramsay, J. A Ramsay and C. Chavarie. 1990. Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava. Appl. Environ. Microbiol. 56, 3133-3138
  4. Braunegg, G, B. Sonneleitner and R. M. Lafferty. 1978. A rapid gas chromatography method for the determination of polyhydroxybutric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol. 6, 29-37 https://doi.org/10.1007/BF00500854
  5. Choi, J. and S. Y. Lee. 1999. Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl. Microbiol. Biotechnol. 51, 13-21 https://doi.org/10.1007/s002530051357
  6. Claus, D. and R. C. W. Berkeley. 1986. Genus Bacillus Cohn, pp. 1105-1139, In Sneath, P. H. A., N. S. Mair, M. E. Sharpe, and J. G. Holt (eds.), Bergey's manual of systematic bacteriology, Vol. 2, The Williams & Wilkins Co., Baltimore, Maryland.
  7. Kim, B. S. and H. N. Chang. 1998. Production of poly-($\beta$-hydroxybutyrates) from starch by Azotobacter chroococcum. Biotechnol. Lett. 20, 109-112. https://doi.org/10.1023/A:1005307903684
  8. Kim, J. H. and J. K. Lee. 1997. Cloning, nucleotide sequence and expression of gene coding for poly-$\beta$-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1. J. Microbial. Biotechnol. 7, 229-236
  9. Kim, J. M., B. G Kim and C. Y. Choi. 1992. Effect of propionic acid on poly($\beta$-hydroxybutyric-co-$\beta$-hydroxyvaleric) acid production by Alcaligenes eutrophus. Biotechnol. Lett. 14, 903-906 https://doi.org/10.1007/BF01020626
  10. Lee, I. Y., E. S. Choi, G J. Kim, S. W. Nam, Y. C. Shin, H. N. Chang and Y. H. Park. 1994. Optimization of fed-batch fermentation for production of poly-$\beta$-hydroxybutyrate in Alcaligenes eutrophus. J. Microbial. Biotechnol. 4, 146-150
  11. Lee, I. Y., H. N. Chang and Y. H. Park. 1995. A simple method for recovery of microbial poly-$\beta$-hydroxybutyrate by alkaline solution treatment. J. Microbial. Biotechnol. 5, 238-240
  12. Lee, S. Y. 1996. Bacterial Polyhydroxyalkanoates. Biotechnol. Bioeng. 49, 1-14 https://doi.org/10.1002/(SICI)1097-0290(19960105)49:1<1::AID-BIT1>3.3.CO;2-1
  13. Lee, S. Y. 1996. Plastic bacteria? progress and prospects for polyhydroxyalkanoates production in bacteria. Trends Biotechnol. 14, 431-435 https://doi.org/10.1016/0167-7799(96)10061-5
  14. Lee, S. Y. 1998. Poly-(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli. Bioprocess Eng. 18, 397-399 https://doi.org/10.1007/s004490050462
  15. Liu, F., W. Li, D. Ridgway and T. Gu. 1998. Production of poly-$\beta$-hydroxybutyrates on molasses by recombinant Escherichia coli. Biotechnol. Lett. 20, 345-348 https://doi.org/10.1023/A:1005367011378
  16. Nelson, N. J. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. BioI. Chem. 153, 375-380
  17. Page, W. J. and O. Knosp. 1989. Hyperproduction of Poly-beta-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD. Appl. Environ. Microbiol. 55, 1334-1339
  18. Parshad, J., S. Suneja, K. Kukreja and K. Lakshminarayana. 2001. Poly-3-hydroxybutyrate production by Azotobacter chroococcum. Folia Microbiol. 46, 315-320 https://doi.org/10.1007/BF02815620
  19. Preusting, H., R. van Houten, A Hoefs, E. K. van Langenberghe, O. Favre-Bulle and B. Witholt. 1993. High cell density cultivation of Pseudomonas olevorans: Growthand production of poly(3-hydroxybutyrate) in two-liquid phase batch and fed-batch systems. Biotechnol. Bioeng. 41, 550-556 https://doi.org/10.1002/bit.260410507
  20. Ramsay, J. A, M. C. Aly Hansan, and B. A Ramsay. 1995. Hemicellulose as a potential substrate for production of poly-($\beta$-hydroxybutyrates). Can. J. Microbiol. 41, 262-266 https://doi.org/10.1139/m95-195
  21. Ryu, H. W., K. S. Cho, B. S. Kim, Y. K. Chang, H. N. Chang, and H. J. Shim. 1999. Mass production of poly(3-hydroxybutyrate) by fed-batch cultures of Ralstonia eutropha with nitrogen and phosphate limitation. J. Microbial. Biotechnol. 9, 751-756
  22. Somogyi, M. 1952. Notes on sugar determination. J. BioI. Chem. 195, 19-23
  23. Srienc, F., B. Arnold and J. E. Bailey. 1984. Characterization of intracellular accumulation of poly-$\beta$-hydroxybutyrate in individual cells of A. eutrophus H16 by flow cytometry. Biotechnol. Bioeng. 26, 982-987 https://doi.org/10.1002/bit.260260824
  24. Suzuki, T., T. Yamane and S. Shimizu. 1986. Mass production of poly-$\beta$-hydroxybutyric acid by fed-batch culture with controlled C/N feeding. Appl. Microbiol. Biotechnol. 24, 370-374 https://doi.org/10.1007/BF00294592
  25. Thakur, P. S., B. Borah, S. D. Baruah and J. N. Nigam. 2001. Growth-associated production of poly-3-hydroxybutyrate by Bacillus mycoides. Folia Microbiol. 46, 488-494 https://doi.org/10.1007/BF02817991
  26. Tsuge, T., K. Tanaka, M. Shimoda and A Ishizaki. 1999. Optimization of L-Iactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture. J. Biosci. Bioeng. 88, 404-409 https://doi.org/10.1016/S1389-1723(99)80217-5
  27. Yamane, T. 1993. Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study. Biotechnol. Bioeng. 41, 165-170 https://doi.org/10.1002/bit.260410122
  28. Yamane, T., M. Fukunaga and Y. W. Lee. 1995. Increased PHB productivity by high-cell density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer. Biotechnol. Bioeng. 50, 197-202
  29. Young, F. K., J. R. Kastner and S. W. May. 1994. Microbial production of poly-$\beta$-hydroxybutyric acid from D-xylose and lactose by Psudomonas cepacia. Appl. Environ. Microbiol. 60, 4195-4198