• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.363-370
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• 1992

Microorganisms capable of accumulating poly-p-hydroxybutyric acid(PHB) were isolated from soil by enrichment culture technique. Among them, the strain designated as FL-027 had high PHB productivity and was identified as Alcaligenes. The optimal medium composition for cell growth was 8.0 $g/\ell$ of fructose and 3.0 $g/\ell$ of $(NH_4)_2S0_4$, equivalent to C/N ratio 5.04 at pH 7.0 and $30^{\circ}C$. To investigate the optimal conditions for the PHB accumulation, we divided the process into two stages; the first stage for the growth of the cell in nutrient-rich medium and the second stage for the PHB accumulation in nutrient-deficiency medium. The optimal conditions for PHB accumulation were 8.0 $g/\ell$ of fructose and 0.25 $g/\ell$ of $(NH_4)_2S0_4$, equivalent to C/N ratio 60 at pH 6.5 and $30^{\circ}C$. PHB accumulation was stimulated by deficiency of nutrients such as $NH_4^+$, $Ca^{2+}$, $SO_4^{2+}$ in medium. Among them. $NH_4^+$ deficiency was chosen because of its effectiveness. We found the inhibition of cell growth by fructose in batch culture. In order to keep the fructose concentration at an optimal level, intermittent feeding fed-batch culture was employed, and the cell concentration as high as 10.83 $g/\ell$ whose PHB content was responsible for 43% of the dry cell weight. The purified PHB was identified as homopolymer of 3-hydroxybutyric acid by using IR and $^1H-NMR$.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.273-279
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• 1990

For poly- $\beta$ -hydroxybutyrate (PHB) production, a pink-pigmented facultative methylotrophic bacterium (PPFM) P-10 was newly isolated from soils through methanol-enrichment culture. The optimal medium composition for cell growth was 1.0% (vlv) of methanol as carbon source and l.Og/l of ,TEX>$NH_4Cl$, equivalent to C/N ratio of 13.2 at pH 7.0 and $30^{\circ}C$. To investigate the optimal condition for YHB accumulation, two-stage culture technique was adopted; first stage for cell growth and second stage for accumulation of PHB providing unbalanced growth conditions. The optimal PHB accumulation was 1.0% (vIv) of methanol and 0.26gll of $NH_4Cl$, C/N of 50.8 at pH 6.0. To overcome methanol inhibition on cell growth, intermittent feeding fed-batch culture technique was employed, and the cell concentration as high as 14gll with 40% of PHB was achieved. The purified PHB was identified using IR and $1^H NMR$ as homopolymer of 8hydroxybutyric acid. The absorption spectrum of extracted pink colored microbial pigment was alsa investigated.

• Applied Biological Chemistry
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• v.34 no.3
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• pp.273-278
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• 1991

Methanol assimilating bacterium, Pseudomonas sp. ILS-003 was used to investigate the optimum conditions for the production of $poly-{\beta}-hydroxybutyric$ acid from methanol. For PHB production, the optimum initial pH was 6.4 and the optimum temperature was $30^{\circ}C$. Also the optimum methanol concentration was found to be 1.0%(v/v). In the PHB production, $(NH_4)_2SO_4$ was the most effective nitrogen source and the optimum concentration was 0.8 g/l, which was eqivalent to 17.4 in C/N ratio. Also, deficiency of the 2 valence metal ions in the medium had stimulating effect on PHB accumulation. Under the optimum substrate concentration, successive feeding of 0.25%(v/v) methanol was the most effective on PHB production. Under the optimum conditions, 1.94 g/l of PHB and 2.78 g/l of dry biomass were produced in 96 hours, and the yield was 69.8%(w/w).

• KSBB Journal
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• v.9 no.4
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• pp.365-371
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• 1994

The effects of ratios of initial concentration of carbon source to the initial concentration of nitrogen source in the fermentation media on both the yields of PHB fermentation variables and the accumulation of poly-${\beta}$-hydroxybutyric acid(PHB) were investigated. The fermentation media were composed of the combination of varing glucose concentrations, 10, 20, 25, 30, 40, $50g/\ell$ and the NH4Cl concentrations 0.33, 0.4, 0.5, 1.5, 3, $5g/\ell$. The yield of biomass on glucos, Yx/s, decreased very slowly according to the increase of the ratio of C to N. And the yield became constant at 0.35(g biomass/g glucose) with the ratio higher than 70. The yield of residual biomass, Yx/s, also decreased with the ratio of C to N and finally showed a constant value of 0.065(g residual biomass/g glucose) when the ratio was higher than 65. In accordance with the augmentation of the ratio, the yield of PHB, YPHB/S, however, increased and showed the maximum value of 0.35 (g PHB/g glucose) between 40 and 60 of the ratio. The maximum yield of PHB to the change of biomass, YPHB/S, was 0.87(g PHB/g biomass), and the yie1d YPHB/RX, was 4.2(g PHB/g residual biomass). The maximum accumulation percent of PHB to the final biomass was 81% when the ratio was higher than 67.

• KSBB Journal
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• v.6 no.1
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• pp.35-43
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• 1991

The production of poly-$\beta$-hydroxybutyrate(PHB) from methanol by batch and fed-batch cultivations of Methylobacterium sp. GL-10 was studied. PHB accumulation was stimulated by the nutrients deficiency including, NH4+, SO42-, and K+. The nitrogen deficiency was the most critical factor for PHB accumulation. In batch cultivation, the maximum cell concentration and PHB content were 1.86g/l and 0.62g/l, respectively, with 1.0%(v/v) of methanol and 0.5g/1 of ammonium sulfate. The mass doubling time of Methylobacterum sp. GL-10 was in the range of 4-5 hrs. The cell growth and PHB accumulation were severely inhibited at the methanol concentration over than 2% (v/v). To overcome methanol Inhibition, constant feeding and intermittent feedillg fed-batch cultivations were adopted, using C/N molar ratio as a control factor. In constant feeding fed-batch process, cell concentration was increased up to 2.67g/1, and PHB yield was enhanced from 0.33 of batch culture to 0.53. The relatively low cell concentration was caused by methanol accumulated in culture broth at late growth phase. To prevent methanol accumulation and to maximize PHB production, DO-state intermittent fed-batch cultivation was attempted. The cell and PHB concentration was reached up to 4.55g/1 and 1.80g/1, respectively. It was possible to maintain methanol concentration low and also to feed nutrient of desired C/N molar ratio.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.333-336
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• 2001

An isolated phbC gene from Alcaligenes latus was reintroduced into the parent A. latus through the transformation process, and the effect of the amplified phbC gene on the biosynthesis of P(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] and P(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] in the transformant A. latus was investigated. The biosynthesis rate and content of the above copolymers increased up to 1.3-fold after enforcing its own phbC gene, and the molar fractions of 3HV and 4HB in P(3HB-3HV) and P(3HB-4HB) also changed remarkably from 35.0 to 48.0% and from 34.0 to 56.0%, respectively, showing a critical role of PHB synthase which catalyzes the polymerizing reactions between eiher 3HV or 4HB from precursor compounds and 3HB.

• 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.

• 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.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.220-228
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• 1995

For polyhydroxyalkanoic acid (PHA) production, several microorganisms were isolated from sewage sludge. One of them, GB-77 strain, was chosen from its PHB/HV copolymer production on only fructose without cosubstrate. The isolated strain GB-77 was identified as the genus Alcaligenes. Optimal temperature and pH for cell growth were 36C and 6.8. Optimal medium composition was 10 g/l of fructose and 5 g/l of polypeptone, 1 $\times$ 10$^{-2}$M Na$^{2}$HP0$^{4}$, 1.3 $\times$ 10$^{-2}$M KH$^{2}$PO$^{4}$. To investigate the optimal condition for polyhydroxyalkanoic acid production two-stage culture technique was used; first stage for cell growth and second stage for PHA production on unbalanced growth conditions. Optimal conditions for high PHA production were C/N ratio 50, temperature 36$\circ$C and pH 6.8. To overcome fructose inhibition on cell growth, intermittent feeding fed-batch culture technique was used. Total cell concentration was 17.4 g/l with 9.1 g/l of PHA. The purified PHA was identified PHB/HV copolymer by NMR analysis.