• KSBB Journal
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• v.25 no.3
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• pp.239-243
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• 2010

Six strains of Ralstonia eutropha were grown to investigate characteristics of polyhydroxyalkanoates (PHA) synthesis from vegetable oils or glycerol. Poly(3-hydroxybutyrate) homopolymer was formed using soybean oil, olive oil, or glycerol as carbon source, while poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or poly(3-hydroxybutyrateco-3-hydroxyvalerate) copolymers were synthesized by co-feeding $\gamma$-butyrolactone or pentanoic acid, respectively. Optimum strain was determined as R. eutropha KCTC 2662 in terms of final cell concentration and PHA content. From 20 g/L of soybean oil (optimum substrate), cell concentration and PHA content at 72 h ranged 1.7~9.2 g/L and 70~92 wt%, respectively.

• Journal of Korean Society of Environmental Engineers
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• v.28 no.11
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• pp.1222-1227
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• 2006

In this study, nine strains were isolated from heavy metal-contaminated soil in a mine. The high efficiency bacteria, JH1, to be able removal cadmium and copper, was selected by the screen test. JH1 was identified as Ralstonia eutropha by 16S rDNA analysis, fatty acid analysis, and its morphological and biochemical characteristics. After the cadmium-contaminated soil was washed with citric acid solution(pH 6, 10 mM), Ralstonia eutropha JH1 was inoculated in the soil washing water. In order to determine the optimal cell concentration for inoculation, cell concentrations were considered in 0.5, 1.0, 2.0, 4.0 g/L, respectively. The removal efficiencies for cadmium in each cell concentration of Ralstonia eutropha JH1 were 49.9, 84.4, 89.7% and 89.9% of 110 mg/L(Cd), after 5 days culture in soil washing water. When Ralstonia eutropha JH1 was inoculated in soil washing water containing each cadmium(110 mg/L) and copper(100 mg/L), each of them was removed completely during 6 days culture. The completely removing time for cadmium and copper in each low concentration, 10, 30 and 60 mg/L were 12, 18 and 48 hrs, respectively.

• Journal of Microbiology
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• v.40 no.4
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• pp.340-343
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• 2002

A PCB degrader, Ralstonia eutropha H850 was shown to induce bphC gene encoding 2,3-dihydroxy-biphenyl-1,2-dioxygenase in a carvone-amended pure culture in our previous study (Park et al.,1999). The present study was carried out to examine how plant terpenes, as natural substrates, would cause an expression of a PCB degradative gene in soil that was amended with terpenes. The population of Ralstonia eutropha H850 was maintained at least around 10$\^$8/ (CFU/g fresh soil) in the soil amended with carvone or limonene in the presence of succinate as a growth substrate at 50 th day. The gene expression was monitored by RT-PCR using total RNA directly extracted from each soil and bphC gene primers. The bphC gene expression of the seeded strain H850 was observed in the soil amended with biphenyl (4 days) but not with succinate, carvone and limonene. These results indicate that terpenes widely distributed in nature could be a potential inducing substrate for effective PCB biodegration in the soil but their bioavailability and specific induction behavior should be taken into account before PCB bioremediation implementation.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.260-266
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• 2002

In this study, chromosomal DNA fragment related to the regulation of phenol metabolism in Ralstonia eutropha JMP 134 was cloned and sequenced. The result has shown that two open reading frames (ORF1 and ORF2) exist on this regulatory region. ORF1, which initiates from 454 bp downstream of the stop codon of the phenol hydroxylase genes, was found to be composed of 501 amino acids. ORF2, whose start codon is overlapped with the stop codon of ORFl, was found to contain 232 amino acids. The comparison of amino acid sequences with other proteins has revealed that ORF1 belongs to the family of NtrC transcriptional activator, whereas ORF2 shares high homology with the family of GntR protein, which is known to be a negative regulator. ORF1 and ORF2 were designated as a putative positive regulator, phlR2 and a negative regulator phlA, respectively. Possible regulatory mechanisms of phenol metabolism in this strain was discussed.

• Microbiology and Biotechnology Letters
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• v.28 no.2
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• pp.71-79
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• 2000

Two typess of copolyesters, poly(3-hydroxybutyric acid-co-4-hydroxy-butyric acid)[P(3HB-co-4HB] and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)[P(3HB-co-3HV)], with various monomer ratios and different degree of microstructural heterogeneity were synthesized from Ralstonia eutropha H16 and Hydrogenophaga pseudoflava by using ${\gamma}$-butyrolactone and ${\gamma}$-valerolactone, respectively. The two bacteria showed a large difference in the utilization of ${\gamma}$-butyrolactone for cell growth and PHA synthesis. H. pseudoflava synthesized P(3HB-co-4HB) copolyesters with a wide range of 4HB content from 13 to 96 mol% depending on culture conditions, whiel R. eutropha H16 was able to synthesize the copolyesters containing less than 20 mol% of 4HB. An increase in the 4HB content in the P(3HB-co-4HB) copolyesters synthesized by H. pseud-oflava induced an lowering of their melting temperatures as well as their enthalpies of fusion. The increase in the 4HB content, however, increased the rate of degradation by an extracellular P(3HB) depolymerase. NMR spectros-copy and differential scanning calorimetry showed that the P(3HB-co-4HB) copolyesters from H. pseudoflava were generally microstructurally heterogeneous. The P(3HB-co-4HB) copolyesters) synthesized by R. eutropha H16 were rather random copolymers showing less microstructural heterogeneity than those synthesized by H. pseudoflava. The NMR D value analysis suggested that the monomer distribution of the P(3HB-co-3HV) copolymers from the two bacteria were relatively random.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.603-606
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• 2005

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures of R. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the ${\gamma}-butyrolactone$ concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures of R. eutropha NCIMB 11599, glucose and ${\gamma}-butyrolactone$ were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104g/L, with glucose fed in the first step and constant feeding of ${\gamma}-butyrolactone$, at 6g/h, in the second, final cell concentration at 67h was 106g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7mol%. When the same feeding strategy was applied to the fedbatch culture of R. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and ${\gamma}-butyrolactone$ (1.5g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74h were 51g/L, 35% and 32 mol%, respectively. In summary, R. eutropha ATCC 17699 was better than R. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.

• 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.

• Journal of Microbiology and Biotechnology
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• v.29 no.10
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• pp.1656-1664
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• 2019

Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutropha H16 and examined the strains for isoprene production. We optimized codons of all the genes involved in isoprene synthesis by the mevalonate pathway and manipulated the promoter regions using pLac and pJ5 elements. Our results showed that isoprene productivity was higher using the J5 promoter ($1.9{\pm}0.24{\mu}g/l$) than when using the lac promoter ($1.5{\pm}0.2{\mu}g/l$). Additionally, the use of three J5 promoters was more efficient ($3.8{\pm}0.18{\mu}g/l$) for isoprene production than a one-promoter system, and could be scaled up to a 5-L batch-cultivation from a T-flask culture. Although the isoprene yield obtained in our study was insufficient to meet industrial demands, our study, for the first time, shows that R. eutropha can be modified for efficient isoprene production and lays the foundation for further optimization of the fermentation process.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.771-774
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• 2001

NADPH has been known as a regulating factor the biosynthesis of polyhydroxyalkanote(PHA), and the flux of NADPH for PHA biosynthesis could be enforced by the amplification of zwf gene encoding glucose 6-phosphate dehydrogenase. The recombinant plasmid pCZWF harboring PHA synthase, phbC from R. eutropha and zwf from E. coli were constructed, and were transformed to R. eutropha by electroporation. The biosynthesis of P(3HB-3HV) copolymer were carried out in transformant R. eutropha through the two-stage cultivation method using valerate as a precursor. The biosynthesis rate and PHA content of transformant R. eutropha harboring pCZWF were increased compared with transformant R. eutropha harboring only phbC. Especially, the molar fraction of 3HV was increased from 68% to 74% due to amplification of zwf gene. And the biosynthesis P(3HB-3HV) and P(3HB-4HB) carried out using propionate and ${\gamma}-butyrolactone$ as a precursor, respectively. But the rate, content, and molar fraction of biosynthesis copolymers were not influenced appreciably. This may be due to the reduced availability of NADPH.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.751-756
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• 1999

For mass production of poly(3-hydroxybutyrate) (PHB), high cell density cultures of Ralstonia eutropha were carried out in 2.5-1 and 60-1 fermentors by two fed-batch culture techniques of nitrogen and phosphate limitation. When the nitrogen limitation technique was employed using both an on-line glucose monitoring and control system, a high concentration level of PHB (121g/l) was obtained in the small-scale fermentor of 2.5 1. However, the PHB concentration obtained in a large-scale fermentor of 60 1 only turned out to be 60g/l. In contrast, when another fed-batch culture technique of the phosphate-limitation employing dissolved oxygen (DO) stat glucose feeding was used, a large amount of PHB was successfully produced in both 60-1 and 2.5-1 fermentors. In a 2.5-1 fermentor, concentrations of PHB and cells obtained in 58 h were 175 and 210 g/l, respectively, which corresponded to the PHB productivity level of 3.02 g/l/h. In a 60-1 fermentor, a final cell concentration of 221 g/l and a PHB concentration of 180 g/l with PHB productivity level of 3.75 g/l/h were obtained in 48h. PHB content and yield from glucose were 81% and 0.38g PHB/g glucose, respectively. These data suggest that the phosphate limitation technique is more effective compared to nitrogen limitation in the mass production of PHB by R. eutropha of a large scale.