• Journal of Microbiology and Biotechnology
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• v.8 no.6
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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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• v.10 no.3
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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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• v.13 no.2
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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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• v.15 no.6
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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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• v.29 no.3
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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.

• Journal of Life Science
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• v.29 no.1
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• pp.76-83
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• 2019

In this study, the optimal growth and poly(3-hydroxybutyrate) (PHB) biosynthesis of Pseudomonas sp. EML2 were established using waste frying oil (WFO) as a cheap carbon source. The fatty acid composition of WFO and fresh frying oil (FFO) were analyzed by gas chromatography. The unsaturated and saturated fatty acid contents of the FFO were 82.6% and 14.9%, respectively. These contents changed in the WFO. The compositional change in the unsaturated fatty acid content in the WFO was due to a change in its chemical and physical properties resulting from heating, an oxidation reaction, and hydrolysis. The maximum dry cell weight (DCW) and PHB yield (g/l) of the isolated strain Pseudomonas sp. EML2 were confirmed under the following culture conditions: 30 g/l of WFO, 0.5 gl of $NH_4Cl$, pH 7, and $20^{\circ}C$. Based on this, the growth and PHB yield of Pseudomonas sp. EML2 were confirmed by 3 l jar fermentation. After the cells were cultured in 30 g/l of WFO for 96 h, the DCW, PHB content, and PHB yield of Pseudomonas sp. EML2 were 3.6 g/l, 73 wt%, and 2.6 g/l, respectively. Similar results were obtained using 30 g/l of FFO as a carbon source control. Using the FFO, the DCW, PHB content, and PHB yield were 3.4 g/l, 70 wt%, and 2.4 g/l, respectively. Pseudomonas sp. EML2 and WFO may be a new candidate and substrate, respectively, for industrial production of PHB.

• Journal of Life Science
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• v.18 no.12
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• pp.1625-1630
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• 2008

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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.6
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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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• v.39 no.1
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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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• 2001.11a
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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.