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      Production of Poly-3-hydroxybutyrate from Xylose by Bacillus megaterium J-65

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      https://www.riss.kr/link?id=A76338127

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      국문 초록 (Abstract)

      본 연구는 생분해성 플라스틱인 poly-β-hydroxybutyrate (PHB)의 생산단가를 낮추기 위한 노력으로 저가의 기질로부터 PHB 대량생산을 위한 기초자료를 얻는데 그 목적을 두었다. Hemicellulose hydrolysate...

      본 연구는 생분해성 플라스틱인 poly-β-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℃, 초발 pH 8.0이었으며 2% xylose, 0.25% (NH₄)₂SO₄, 0.3% Na₂HPO₄ㆍ12H₂O, 0.1% KH₂PO₄였다. PHB 축적에 영향을 미치는 요인을 검토하기 위해 균체생육 최적배지에서 37℃, 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 공중합체를 생산하기 위하여 보조기질로 propionic acid를 첨가하였을 때, 0.1% propionic acid 농도에서 HV ㏖%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 26.52 g/l, PHB 9.28 g/l를 얻을 수 있었다. 생산된 PHB를 alkaline solution 처리와 chloroform을 이용한 유기용매 추출법을 이용하여 추출ㆍ정제한 후 Gas Chromatography로 정제를 확인하고 300㎒ 1H-NMR을 실시한 결과 3-hydroxybutyrate의 homopolymer임을 확인하였다.

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      다국어 초록 (Multilingual 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 characte...

      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℃ and 8.0, respectively. The optimum medium composition for the cell growth was 2% xylose, 0.25% (NH₄)₂SO₄, 0.3% Na₂HPO₄ㆍ12H₂O, and 0.1% KH₂PO₄. The optimum condition for PHB accumulation was same to the optimum condition for cell growth. Copolymer of β-hydroxybutyric and β-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.

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      목차 (Table of Contents)

      • Introduction
      • Materials and Methods
      • Results and Discussion
      • Acknowledgments
      • References
      • Introduction
      • Materials and Methods
      • Results and Discussion
      • Acknowledgments
      • References
      • 초록
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      참고문헌 (Reference)

      1 Yamane,T, "Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study" 41 : 165-170, 1993

      2 Liu, F., "Production of poly-β-hydroxybutyrates on molasses by recombinant Escherichia coli" 20 : 345-348, 1998

      3 Kim, B. S, "Production of poly-(β- hydroxybutyrates) from starch by Azotobacter chroococcum" 20 : 109-112, 1998

      4 Parshad, J., "Poly-3-hydroxybutyrate production by Azotobacter chroococcum" 46 : 315-320, 2001

      5 Lee,S.Y, "Poly-(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli" 18 : 397-399, 1998

      6 Lee,S.Y, "Plastic bacteria? progress and prospects for polyhydroxyalkanoates production in bacteria" 14 : 431-435, 1996

      7 Lee, I. Y., "Optimization of fed-batch fermentation for production of poly-β-hydroxybutyrate in Alcaligenes eutrophus" 4 : 146-150, 1994

      8 Tsuge, T., "Optimization of L-lactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture" 88 : 404-409, 1999

      9 Anderson, A. J, "Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates" 54 : 450-472, 1990

      10 Somogyi,M, "Notes on sugar determination" 195 : 19-23, 1952

      1 Yamane,T, "Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study" 41 : 165-170, 1993

      2 Liu, F., "Production of poly-β-hydroxybutyrates on molasses by recombinant Escherichia coli" 20 : 345-348, 1998

      3 Kim, B. S, "Production of poly-(β- hydroxybutyrates) from starch by Azotobacter chroococcum" 20 : 109-112, 1998

      4 Parshad, J., "Poly-3-hydroxybutyrate production by Azotobacter chroococcum" 46 : 315-320, 2001

      5 Lee,S.Y, "Poly-(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli" 18 : 397-399, 1998

      6 Lee,S.Y, "Plastic bacteria? progress and prospects for polyhydroxyalkanoates production in bacteria" 14 : 431-435, 1996

      7 Lee, I. Y., "Optimization of fed-batch fermentation for production of poly-β-hydroxybutyrate in Alcaligenes eutrophus" 4 : 146-150, 1994

      8 Tsuge, T., "Optimization of L-lactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture" 88 : 404-409, 1999

      9 Anderson, A. J, "Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates" 54 : 450-472, 1990

      10 Somogyi,M, "Notes on sugar determination" 195 : 19-23, 1952

      11 Anthony, G. O, "Nile blue A as a fluorescent stain for Poly-β-hydroxybutyrate" 44 : 238-241, 1982

      12 Young, F. K., "Microbial production of poly-β-hydroxybutyric acid from D-xylose and lactose by Psudomonas cepacia" 60 : 4195-4198, 1994

      13 Suzuki, T., "Mass production of poly-β-hydroxybutyric acid by fed-batch culture with controlled C/N feeding" 24 : 370-374, 1986

      14 Ryu, H. W., "Mass production of poly(3-hydroxybutyrate) by fed-batch cultures of Ralstonia eutropha with nitrogen and phosphate limitation" 9 : 751-756, 1999

      15 Yamane, T., "Increased PHB productivity by high-cell density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer" 50 : 197-202, 1995

      16 Page, W. J, "Hyperproduction of Poly-beta-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD" 55 : 1334-1339, 1989

      17 Preusting, H., "High cell density cultivation of Pseudomonas olevorans: Growthand production of poly(3-hydroxybutyrate) in two-liquid phase batch and fed-batch systems" 41 : 550-556, 1993

      18 Ramsay, J. A., "Hemicellulose as a potential substrate for production of poly-(β-hydroxybutyrates)" 41 : 262-266, 1995

      19 Thakur, P. S., "Growth-associated production of poly-3-hydroxybutyrate by Bacillus mycoides" 46 : 488-494, 2001

      20 Claus, D, "Genus Bacillus Cohn ; Bergey's manual of systematic bacteriology, Vol. 2" The Williams & Wilkins Co. 1105-1139, 1986

      21 Choi, J, "Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation" 51 : 13-21, 1999

      22 Kim, J. H, "Cloning, nucleotide sequence and expression of gene coding for poly-β-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1" 7 : 229-236, 1997

      23 Srienc, F., "Characterization of intracellular accumulation of poly-β-hydroxybutyrate in individual cells of A. eutrophus H16 by flow cytometry" 26 : 982-987, 1984

      24 Bertrand, J. L., "Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava" 56 : 3133-3138, 1990

      25 Lee,S.Y, "Bacterial Polyhydroxyalkanoates" 49 : 1-14, 1996

      26 Lee, I. Y., "A simple method for recovery of microbial poly-β-hydroxybutyrate by alkaline solution treatment" 5 : 238-240, 1995

      27 Braunegg, G., "A rapid gas chromatography method for the determination of polyhydroxybutric acid in microbial biomass" 6 : 29-37, 1978

      28 Nelson,N.J, "A photometric adaptation of the Somogyi method for the determination of glucose" 153 : 375-380, 1944

      29 Kim, J. M., "1992. Effect of propionic acid on poly(β-hydroxybutyric-co-β-hydroxyvaleric)acid production by Alcaligenes eutrophus" 14 : 903-906, 1992

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