• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.100-104
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• 1995

Production of $poly-{\beta}-hydroxybutyrate$ (PHB) in a strain of Azotobacter chroococcum, a nitrogen-fixing bacteria, was investigated at various levels of nitrogen and oxygen. Feeding nitrogen source increased both cell growth and PHB accumulation. Oxygen supply appeared to be one of the most important operating parameters for PHB production. Both cell growth and PHB accumulation increased with the sufficient supply of air in the fed-batch fermentation of the strain. However, it was also noted that keeping the oxygen level under limited condition was critical to achieve high PHB productivity. A high titer of PHB (52 g/l) with a high cellular content (60%) was obtained after 48 hr of fed-batch operation by controlling the oxygen supply. Dual limitation of nitrogen and oxygen did not further increase the PHB accumulation probably due to the greater demand for reducing power and ATP for nitrogen fixation.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1031-1037
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• 2001

By a sequential action of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, two molecules of acetyl-CoA re converted into D-3-hydroxybutyryl-CoA, a substrate for PHB synthase to form poly-3-hydroxybutyryl-CoA, a substrate for PHB synthase to form poly-3-hydroxybutyrate (PHB) of rhodobacter sphaeroides. The ${\beta}$-ketothiolase gene, phbA, and acetoacetyl-CoA reductase gene, phbB, were cloned and analyzed for their expression. Enzyme activities of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase showed constitutive levels during aerobic and photoheterotrophic growth of R. sphaeroides. In addition, no difference of each enzyme activity was observed between cells grown aerobically and photoheterotrophically. The constitutive level of the enzyme activities are regulated according to the growth phases along with growth conditions. Thus, phbAB expression is not determinative in regulating the PB content. On the other hand, phbA-deleted cell AZI accumulated only $10\%$ PHB of the wild-type, and an elevated dosage of phbAB in trans in R. sphaeroides resulted in a higher content of PHB, indicating that phbAB codes for the enzymes responsible for providing the main supply of subsyrate for PHB synthase. PHB formation by an alternative pathway that does not does not depend on the phbA-and phbB-coding enzymes is also proposed.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.146-150
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• 1994

Production of poly-$\beta$-hydroxybutyrate (PHB) in fed-batch fermentation was studied. Utilization of carbon for PHB biosynthesis was investigated by using feeding solutions with different ratios of carbon to nitrogen (C/N). It was observed that at a high C/N ratio carbon source was more preferably utilized for PHB accumulation while its consumption for cellular metabolism appeared to be more favored at a low C/N value. A high cell concentration (184 g/l) was achieved when ammonium hydroxide solution was fed to control the pH, which was also utilized as the sole nitrogen source. For the mass production of PHB, two-stage fed-batch operations were carried out where PHB accumulation was observed to be stimulated by switching the ammonium feeding mode to the nitrogen limiting condition. A large amount of PHB (108 g/l) was obtained with cellular content of 80% within 50 hrs of operation.

• 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.5
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• pp.281-288
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• 2002

Recombinant Escherichia coli strain harboring the ${\lambda}$pR-pL promotor and heterologus poly-${\beta}$-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression of phb genes was induced by a temperature upshift from $33^{\circ}C\;to\;38^{\circ}C$. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lac-tate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.607-612
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• 1990

The biodegradation of Aroclor 1242 was investigated by the mixed cultivation of the natural bacterial isolates and a genetically engineered microorganism (GEM). The natural strain of MS-1003 degraded the Aroclor 1242 through the ortho-cleavage pathway, while the other strains through the meta-cleavage pathway. When the MS-1003 strain was additionally inoculated into the 1 day culture of the DJ-26 strain and then cultivated for 2 days, the Aroclor was degraded up to 86% and resulted in increase of the meta-cleavage product. But in the MS-1003 culture inoculated with the DJ-26, degradation of the Aroclor was limited to the level of each pure culture. By the mixed cultivation of the DJ-26 strain together with the DJ-12 or its GEM strain of DF-10, which degrades the Aroclor through the meta-cleavage pathway, degradation of the Aroclor as well as production of the meta-cleavage compound were lower than those of each pure culture. The degradation of Aroclor 1242 by the GEM strain was not improved over the parental strain. Therefore, a form of cometaboiism of Aroclor 1242 was found in the mixed culture of the DJ-26 and MS-1003 strains which degrade the Aroclor through the different metabolic pathway, but in the mixed culture of the DJ-26 and DJ-12 strains degrading Aroclor 1242 through the same pathway, a kind of competetion for the substrate was observed.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.5
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• pp.295-302
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• 2002

To understand the utilization property of light energy, Synechococcus sp. MA19, a poly-${\beta}$-hydroxybutyrate (PHB) producer, was cultivated at the different incident light intensities of 15.3, 50.0 and 78.2 W/$m^2$ using media with and without phosphate. From the results of metabolic flux analysis, it was found that the cell yield based on ATP synthesis was estimated as $3.5{\times}10^{-3}$ kg-biomass/mol-ATP in these cultures. Under the examined conditions, there were no significant differences in the efficiency of light energy conversion to chemical energies estimated as ATP synthesis and reducing potential (NADH + NADPH) formation whether the PHB synthesis took place or not. The energy converted from light to ATP was kept relatively high around the energy absorbed by the cells of $2.5-3.0{\times}10^{6} J\;h^{-1}\;kg^{-1}$, whereas the energy of reducing potential was hardly changed in the examined range of the energy absorbed by the cells.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.668-676
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• 1992

For the purpose of optimizing poly-l3-hydroxybutyrate (PHB) production from Alcaligenes eutrophus, two-stage fed-batch culture was adopted. In this system, specifk growth rate was maximized during the first stage whereas specific production rate was maximized during the second stage. The optimal concentrations of glucose and ammonium chloride were 16.6 and 0.54 g/I in the growth stage and 20.0 and 0.07 g/l in the production stage, respectively. Proportional feedback control considering time lag was suggested for PHB production process and a simulator was developed for real-time control purpose.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.71-77
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• 2010

The time, yield, and related genes expression of PHB accumulation of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios, 1.2, 2.4, 7.5, and 24. The results of time and yield of poly-$\beta$-hydroxybutyrate (PHB) accumulation show that the initial C/N ratio of 2.4 was optimum for strain DX1-1 to accumulate PHB, but both higher and lower initial C/N ratios did not favor that process. Based on the genome of Acidiphilium cryptum JF-5, 13 PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expressions of the 13 functional genes, in different C/N ratios as cited above, were then studied by real-time PCR. The results show that all the 13 genes were most upregulated when the initial C/N ratio was 2.4, and among which the gene Acry_3030 encoding poly-$\beta$-hydroxybutyrate polymerase and Aery_0626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which proved that they play the most important role for PHB accumulation, and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis showed that the genes related to PHB accumulation are regulated by different promoters and that the motif had weak similarity to the model promoters, suggesting that PHB metabolism in Acidiphilium cryptum may be mediated by a different mechanism.