• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.140-146
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• 1999

When methanol was the sole carbon source, Methylobacterium organophilum NCIB 11278, a facultative methylotroph, accumulated Poly-$\beta$-hydroxybutyrate (PHB) as 59% (w/w) of dry cell weight under potassium limitation, 37% under sulfate limitation, and 33% under nitrogen limitation. Based on a stoichiometric analysis of PHB synthesis from methanol, it was suspected that PHB synthesis is accompanied by the overproduction of energy, either 6-10 ATP and 1 $FADH_2$ or 6 ATP and 3 NADPH to balance the NADH requirement, per PHB monomer. This was confirmed by observation of increased intracellular ATP levels during PHB accumulation. The intracellular ATP with limited potassium, sulfate, and ammonium increased to 0.185, 0.452, and 0.390 $\mu$moles ATP/g Xr (residual cell mass) during PHB accumulation, respectively. The intracellular ATP level under potassium limitation was similar to that when there was no nutrient limitation and no PHB accumulation, 0.152- 0.186 $\mu$moles ATP/g Xr. We propose that the maximum PHB accumulation observed when potassium was limited is a result of the energy balance during PHB accumulation. Microorganisms have high energy requirements under potassium limitation. Enhanced PHB accumulation, in ammonium and sulfate limited conditions with the addition of 2,4-dinitrophenol, which dissipates surplus energy, proves this assumption. With the addition of 1 mM of 2,4-dinitrophenol, the PHB content increased from 32.4% to 58.5% of dry cell weight when nitrogen limited and from 15.1 % to 31.0% of dry cell weight when sulfate limited.

• 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.

• 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 $^1H-NMR$.

• 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.6 no.2
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• pp.120-127
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• 1996

The characteristics of cell growth and poly-$\beta$-hydroxybutyrate biosynthesis of Alcaligenes latus ATCC 29713 were investigated. The PHB accumulation pattern of A. latus followed a growth-associated type where the cell growth and PHB accumulation were carried out simultaneously. Various intermediate compounds such as metabolites involved in the TCA cycle, amino acids, and saturated and unsaturated fatty acids were added to examine their effect on cell growth and PHB accumulation. Citrate, tyrosine, and palmitic acid showed the most significant increase both on cell growth and PHB accumulation. Maximum PHB concentrations were noticeably increased about 1.4 to 1.6 times higher than that of control, corresponding to 5.54, 6.45, and 6.45 g/l for citrate, tyrosine, and palmitic acid, respectively. The stimulatory effects of the supplemented metabolites were analyzed in terms of the increment of enzyme activities related to sugar catabolism and PHB biosynthesis.

• 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.

• 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 Microbiology and Biotechnology
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• v.8 no.4
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• pp.301-304
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• 1998

Methylobacterium organophilum can use nitrogen in the form of ammonium ions ($($NH_4$)_2SO_4\;and\;NH_4Cl) and from nonammonium sources such as glycine, alanine, peptone, and yeast extract. When potassium was limited, significantly more PHB was produced when the ammonium ion was the nitrogen source rather than a nonammonium form. With ammonium, the amount of PHB produced was 0.50-0.53 g PHB/l or $52.0~53.2\%$ of the dry cell weight. If nitrogen was from a nonammonium source, the respective values were 0.04~0.06 g PHB/1 or $8.1~11.3\%$ of dry cell weight. When ammonium sulfate was the sole source of nitrogen under potassium-limited conditions, cell growth and PHB accumulation increased as the pH increased from 6.0 to 7.5. Cell growth and PHB amount at pH 7.5 were 2.50 g dry cell weight/1 and 1.40 g PHB/1, respectively.

• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.147-149
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• 1996

Poly(3-hydroxybutyrate) (PHB) was synthesized and accumulated intracellularly to a high concentration (7 g/l) by cultivating recombinant Escherichia coli XL1-Blue (pSYLl05) in a complex medium containing 20 g/l glucose. The morphology of PHB granules was examined by transmission electron microscopy. The PHB granules synthesized in recombinant E. coli were much larger than reported values for wild type microorganisms, and were often irregularly shaped. Some cells were apparently extruding PHB into the medium, which suggests that PHB granules maintain some fluidity and cells become fragile due to PHB accumulation.

• KSBB Journal
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• v.9 no.5
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• pp.475-482
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• 1994

To find out the optimum conditions for the cell growth and the synthesis of PHB in A. eutrophus, the effects of culture conditions and extracellular by-products were investigated. Glucose and$>(NH_4)_2HP0_4$were optimum carbon and nitrogen sources, respectively for cell growth of A. eutrophus. PHB accumulation was stimulated by deficiency of nutrients such as $>NH_4^{3-}, P0_4^{3-}, and Mg^{2+}$ in the medium. $>NH_4^+$, deficiency was the most suitable for PHB accumulation and PHB accumulation ratio was reached 42% of dry cell weight. The specific growth rate was increased 1.5 times by addition yeast extract in the medium, and proteins and vitamins are supposed a main factor of that effect. The extracellular products such as ethanol and butanediol were excreted under anaerobic conditions. And ethanol was found to decrease the specific growth rate.