• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.714-718
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• 1998

Various mutants either lacking or having decreased levels of light-harvesting complexes and reaction center complex were obtained with a high frequency by an increased formation of poly-3-hydroxybutyrate (PHB) in Rhodobacter sphaeroides. One of the photosynthesis-defective mutants, PY-17, which was devoid of any of the light-harvesting complexes (B800-850, B875) as well as the reaction center complex, was analyzed further. The mutant showed substantial transcription of the puhA, pufKBALMX, and pucBAC operons coding for the structural proteins of the photosynthetic complexes although each of the activities was lower than that of the wild type. Translation of the pufKBALMX and pucBAC operons were also active in the mutant although with activities different from the corresponding one of the wild type. From these results the mutation appears to exert its effect at the post-translational level of the photosynthetic complex assembly. Complementation of the photosynthesis-defective phenotype of the mutant was achieved with an about 12-kb DNA region containing the puhA gene. The relationship between the formation of PHB and photosynthetic complexes is discussed.

• Macromolecular Research
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• 제10권3호
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• pp.150-157
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• 2002

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a microbial storage polymer with biodegradable properties. In order to improve the cell compatibility of PHBV surfaces, the physicochemical treatments have been demonstrated. In this study, physical method was corona discharge treatment and chemical method was chloric acid mixture solution treatment. The physicochemically treated PHBV film surfaces were characterized by the measurement of water contact angle, electron spectroscopy for chemical analysis, and scanning electron microscopy (SEM). The water contact angle of the physicochemically treated PHBV surfaces decreased from 75 to 30～40 degree, increased hydrophilicity. due to the introduction of oxygen-based functional group onto the PHBV backbone chain. The mouse NIH/3T3 fibroblasts cultured onto the physicochemically treated PHBV film surfaces with different wettability. The effect of the PHBV surface with different wettability was determined by SEM as counts of cell number and [$^3$H]thymidine incorporation as measures of cell proliferation. As the surface wettability increased, the number of the cell adhered and proliferated on the surface was increased. The result seems closely related with the serum protein adsorption on the physicochemically treated PHBV surface. In conclusion, this study demonstrated that the surface wettabilily of biodegradable polymer as the PHBV plays an important role for cell adhesion and proliferation behavior for biomedical application.

• KSBB Journal
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• 제13권2호
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• pp.187-194
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• 1998

For mass production of polyhydroxyalkanoates (PHA), high cell density cultures of Alcaligenes eutrophus by fed-batch culture under phosphate-limitation condition has been investigated. PHA accumulation by the regulation by the regulation of initial phosphate concentration could be automatically induced, and high density cell culture above 200 g/L also could be successfully produced. The production of Poly-$\beta$-hydroxybutyrate (PHB) and dry cell weight increased with increasing the initial phosphate concentration. When the initial concentrations of phosphate were in the ranges of 1.5~4.5 g-PO$_4$/L, PHB and dry cell weight obtained were 83~266 g/L and 61~216 g/L, respectively, and PHB productivity was in the ranges of 1.35~3.10 g/L.h. When a mixture of glucose and propionic acid is used as carbon sources, poly(3-hydroxybutyrate-co-poly-3-hydroxyvalerate), P(3HB-co-3HV), could be also successfully produced under phosphate limitation condition. When the mole ratio of propionic acid to glucose in the feeding solution is 0.22, a final dry cell weight of 150 g/L and a P(3-HB-co-3HV) of 90 g/L were produced. Morphological changes and size distribution of PHB granules synthesized in A. eutrophus under phosphate-limitation condition are determined by TEM during the course of fed-batch. Mean granule diameters of PHB produced are in the range of 0.36~0.39 $\mu$m, and mean cell size was elongated from 0.54~0.59 $\mu$m$\times$ 1.3~1.5 $\mu$m to 0.83~0.89 $\mu$m $\times$2.0~2.3 $\mu$m. Phosphate concentration in media did not affect size distribution of PHB granule and cell.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1330-1336
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• 2005

Polyhydroxyalkanoic acid (PHA) inclusion bodies were analyzed in situ by $^{13}C$-nuclear magnetic resonance ($^{13}C$-NMR) spectroscopy. The PHA inclusion bodies studied were composed of poly(3-hydroxybutyrate) or poly(3hydroxybutyrate-co-4-hydroxybutyrate), which was accumulated in Hydrogenophaga pseudoflava, and medium-chain-length PHA (MCL-PHA), which was accumulated in Pseudomonas fluorescens BM07 from octanoic acid or 11-phenoxyundecanoic acid (11-POU). The quantification of the $^{13}C$-NMR signals was conducted against a standard compound, sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The chemical shift values for the in vivo NMR spectral peaks agreed well with those for the corresponding purified PHA polymers. The intracellular degradation of the PHA inclusions by intracellular PHA depolymerase(s) was monitored by in vivo NMR spectroscopy and analyzed in terms of first-order reaction kinetics. The H. pseudoflava cells were washed for the degradation experiment, transferred to a degradation medium without a carbon source, but containing 1.0 g/l ammonium sulfate, and cultivated at $35^{\circ}C$ for 72 h. The in vivo NMR spectra were obtained at $70^{\circ}C$ for the short-chain-length PHA cells whereas the spectra for the aliphatic and aromatic MCL-PHA cells were obtained at $50^{\circ}C\;and\;80^{\circ}C$, respectively. For the H. pseudoflava cells, the in vivo NMR kinetics analysis of the PHA degradation resulted in a first-order degradation rate constant of 0.075/h ($r^{2}$=0.94) for the initial 24 h of degradation, which was close to the 0.050/h determined when using a gas chromatographic analysis of chloroform extracts of sulfuric acid/methanol reaction mixtures of dried whole cells. Accordingly, it is suggested that in vivo $^{13}C$-NMR spectroscopy is an important tool for studying intracellular PHA degradation in terms of kinetics.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.382-391
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• 2019

Many poultry eggs are discarded worldwide because of infection (i.e., avian flu) or presence of high levels of pesticides. The possibility of adopting egg yolk as a source material to produce polyhydroxyalkanoate (PHA) biopolymer was examined in this study. Cupriavidus necator Re2133/pCB81 was used for the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) or poly(3HHx), a polymer that would normally require long-chain fatty acids as carbon feedstocks for the incorporation of 3HHx monomers. The optimal medium contained 5% egg yolk oil and ammonium nitrate as a nitrogen source, with a carbon/nitrogen (C/N) ratio of 20. Time course monitoring using the optimized medium was conducted for 5 days. Biomass production was 13.1 g/l, with 43.7% co-polymer content. Comparison with other studies using plant oils and the current study using egg yolk oil revealed similar polymer yields. Thus, discarded egg yolks could be a potential source of PHA.

• 생명과학회지
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• 제29권1호
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• pp.76-83
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• 2019

본 연구에서는 생분해성 고분자인 poly(3-hydroxbutyrate) (PHB)의 생산 비용 절감을 위해, 탄소원으로 폐식용유(waste frying oil, WFO)을 사용하여 분리 균주 Pseudomonas sp. EML2의 최적 생장 및 PHB 생합성 조건을 확립하였다. WFO와 새 식용류(fresh frying oil, FFO)의 지방산을 분석한 결과 FFO의 지방산 함량은 불포화지방산 82.6%, 포화지방산 14.9%를 차지하는 것으로 나타났으나 WFO의 경우 불포화지방산 56.3%와 포화지방산 33.5%로 FFO와 비교할 때 지방산 조성의 변화를 확인할 수 있었으며, 이러한 불포화지방산의 조성 변화는 가열, 산화반응 및 가수분해에 의한 화학적, 물리적 특성의 변화 때문인 것으로 사료된다. 분리 균주 Pseudomonas sp. EML2의 최대 건조세포중량과 PHB 생합성량(g/l)을 확인하기 위해 플라스크를 이용하여 탄소원 농도, 질소원 종류 및 배양 pH와 온도 및 시간을 확립하였다. 그 결과 30 g/l의 WFO과 0.5 g/l의 $NH_4Cl$를 질소원으로 사용하여 pH 7 및 $20^{\circ}C$의 배양 조건에서 96시간 배양 시 최적의 건조세포중량과 PHB 생합성량을 확인하였다. 이 결과를 바탕으로 3 l jar fermenter를 이용하여 Pseudomonas sp. EML2의 생장 및 PHB 수율을 확인하였다. 그 결과 30 g/l의 WFO를 단일 탄소원으로 사용하여 96시간 배양 시 3.6 g/l의 건조세포중량을 얻었으며 73.0 wt%의 PHB 축적률을 확인하였다. 이 경우 PHB 생합성량 2.6 g/l로 나타났다. FFO를 대조군으로 사용하여 대량배양 한 결과 WFO를 사용한 경우와 비슷한 건조세포중량(3.4 g/l), PHB 축적률(70.0 wt%), 그리고 PHB 생합성량(2.4 g/l)을 확인하였다. 본 연구에서 분리한 Pseudomonas sp. EML2는 WFO를 효과적으로 이용하여 PHB를 생합성 하였으며 이 균주와 WFO는 PHB의 산업적 생산을 위한 새로운 생산 후보자 및 탄소원으로서 이용될 수 있음을 확인하였다.

• 생명과학회지
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• 제18권12호
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• pp.1625-1630
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• 2008

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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권6호
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• pp.371-374
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• 2002

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)［P(3HB/V)］, by fed-batch culture of recombinant Escherichia coli harboring a plasmid containing the Alcaligenes latus polyhy-droxyalkanoate (PHA) biosynthesis genes, was examined in two pilot-scale fermentors with air supply only, In a 30 L fermentor having a XLa value of 0.11 S­$^1$, the final P(3HB/V) concentration and the P(3HB/V) content obtained were 29.6 g/L and 70.1 wt%, respectively giving a productivity of 1.37 g P(3HB/V)/L-h. In a 300 L fermentor having a XLa of 0.03 S­$^1$, the P(3HB/V) concentration and the P(3HB/V) content were 20.4 g/L and 69 wt%, respectively giving a productivity of 1.06g P(3HB/V)/L-h. These results suggest that economical production of P(3HB/V) is possible by fed-batch culture of recombinant E. coli in a large-scale fermentor having low KLa value.

• Journal of Microbiology
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• 제39권1호
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• pp.79-82
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• 2001

The influence of levulinic acid (LA) on the production of copolyester consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) by Ralstonia eutropha was investigated. Addition of LA into the culture medium greatly increased the molar fraction of 3HV in the copolyester, indicating that LA can be utilized as a precursor of 3HV. In shake flask culture, the 3HV content in the copolyester increased from 7 to 75 mol% by adding 0.5 to 4.0 g/L LA to the medium containing fructose syrup as a main carbon source. A maximal copolyester concentration of 3.6 g/L (69% of dry cell weight) was achieved with a 3HV content of 40 mo1% in a jar fermentor culture containing 4.0 g/L of LA. When LA (total concentration, 4 g/L) was added repeatedly into a fermentor culture to maintain its concentration at a low level, the copolyester content and the 3HV yield from LA reached up to 85% of dry cell weight and 5.0 g/g, respectively, which were significantly higher than those when the same concentration of the LA was supplied al1 at once. The present results indicated that LA is more effective than propionate or valerate as a cosubstrate fur the production of copolyesters with varying molar fractions of 3HV by R. eutropha.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.735-738
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• 2001

Metabolically engineered Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) [P(3HB-co-3HHx)] from dodecanoic acid. By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt%, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 g/L/h. The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol%. The 3HHx fraction in the copolymer could be increased by three fold when the Aeromonas hydrophila orfl gene was co-expressed with the PHA biosynthesis genes.