• Applied Biological Chemistry
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• v.41 no.4
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• pp.209-212
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• 1998

Methylan, a polysaccharide produced from Methylobacterium organophilum, was chemically modified by adding diethylaminoethyl (DEAE) group to the backbone of methylan. The structure of DEAE-methylan was determined by measuring its nitrogen content obtained from an elemental analysis. From the analysis of mass spectrum, the DEAE group in DEAE-methylan was also confirmed by determining diethylaminoethene as a separate form of DEAE. Mercury adsorption of DEAE-methylan was higher than that of native methylan. This fact was valid for a variety of pH, reaction times, metal concentrations, and polysaccharide concentrations. In particular, native methylan and DEAE-methylan adsorbed 16% (w/w) and 18% (w/w) for mercury after 30 min at pH 7, respectively. The increase in mercury adsorption of DEAE-methylan may be resulted from mercury adsorption by the lone pair electron of nitrogen atom in DEAE group.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.397-402
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• 1989

A new extracellular biopolymer was produced by Methylobacterium organophilum from methanol as a sole carbon and energy source. The purified biopolymer was found to have a high molecular weight of about 4-5$\times$10$^6$ dalton and contained 66% (w/w) of carbohydrate but no polyhydro xybutyrate. Other organic constituents were consisted of protein, pyruvic acid, uronic acid, and acetic acid, whereas content of inorganic ash was 22%. Based on the chemical analysis of the biopolymer by TLC method, the polymer was consisted of glucose, galactose, and mannose with an approximate molar ratio of 2:3:2. The biopolymer solution showed a characteristics of pseudoplastic non-Newtonian fluid. The viscosity of the 1%-biopolymer solution was found to be 18,000 cp at a shear rate, 1 sec$^{-1}$, which was almost 10 times higher than that of a commercial xanthan gum.

• 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.

• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.244-249
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• 1998

An extracellular polysaccharide, methylan, was produced under the specific conditions by Methylobacterium organophilum from methanol. The specific growth rate of cells was approximately constant regardless of C/N ratio and the specific product yield was maximum at a C/N ratio of 30. Methylan production was suppressed by the deficiency of mineral ions such as Mn$^{++}$ or Fe$^{++}$ ion. The optimal pH for cell growth and methylan production was 7. Whereas the optimal temperature for cell growth was found to be 37$^{\circ}C$, that for methylan production was 3$0^{\circ}C$. The methanol concentration above 4% completely inhibited the cell growth. The initial methanol concentration for the maximal production of methylan was 0.5% (v/v) and above this concentration, methylan production was markedly inhibited. To overcome the substrate toxicity and inhibition for both cell growth and methylan production, a fed-bach culture of intermittent feeding within 5 g/l methanol was conducted under the optimal culture condition. Methylan production of was stimulated by nitrogen limitation and methylan was accumulated up to 8.7 g/1 and cell mass also increased up to 12.4 g/l.

• KSBB Journal
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• v.10 no.2
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• pp.176-182
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• 1995

The environmental and physiological factors affecting the production of exopolysaccharide (Methylan) and Poly-${\beta}$-hydroxybutyrate(PHB) by Methylobacterium organophilum were investigated. The maximum PHB content was obtained at $38^{\circ}C$ whereas maximum polysaccharide concentration was $3.54g/\ell$ at $30^{\circ}C$. Optimum pH was pH 7-8 for PHB production and pH 6-7 for polysaccharide production, respectively. Under the condition of $Mo^{2+}, Mg^{2+} or Mn^{2+}$ limitation with nitrogenlimitation, the PHB accumulation was increased, whereas the polysaccharide production was decreased as compared with that of solenitrogenlimitation. Under the condition of sole K+ limitation, cell growth was significantly inhibited and no polysaccharide was produced. However, the PHB content was as high as 60% of dry cell weight. Effect of C/N ratios (methanol/ammonium) in the feeding solution was examined for the simultaneous production of polysaccharide and PHB. The higher ratio of C/N showed the lower cell growth, higher content of PHB in cells, and higher yield of polysaccharide.

• 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.10 no.2
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• pp.170-175
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• 1995

The effect of nitrogen source on production of a high viscosity exopolysaccharide, methylan, from methanol by Mentylobacterium organophilum was investigated in fed-batch culture. During the fermentation, cells continued to grow even after the nitrogen source added to the medium was depleted and methylan production was stimulated under the condition which ammonium ion was depleted. Cell growth increased proportionally to the initial concentration of ammonium ion in the medium, but methylan production was significantly inhibited at the high concentration of ammonium ion. As the initial concentration of ammonium ion increased, the specific growth rate, the specific product formation rate and the specific substrate consumption rate decreased due to the inhibitory effect of excess ammonium ions. In order to reduce the inhibitory effect by high concentration of ammonium ion. The control of ammonium ion concentration within the desired level(usually $0.45g/\ell$) was necessary. When ammonium ion concentration was maintained below $0.15g/\ell$ by exponential feeding, methylan production could be increased up to $12.5g/\ell$.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.392-396
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• 1989

Methylobacterium organophilum, a facultative methylotroph was cultivated on a methanol as a sole carbon and energy source. The cell growth was affected by the various components of minimal synthetic medium and the medium composition was optimized with 0.5% (v/v) methanol at pH 6.8 and at 3$0^{\circ}C$. The maximum specific growth rate of M. organophilum was achieved to 0.26 hr$^{-1}$ in the optimized medium which has following composition: Methanol, 0.5% (v/v):(NH$_4$)$_2$SO$_4$, 1.0g/l:KH$_2$PO$_4$, 2.13g/l:KH$_2$PO$_4$, 1.305g/ι:MgSO$_4$.7$H_2O$. 45g/l and trace elements (CaCl$_2$.2$H_2O$, 3.3mg:FeSO$_4$.7$H_2O$, 1.3mg:MnSO$_4$.4$H_2O$, 130$\mu\textrm{g}$:ZnSO$_4$.5$H_2O$, 40$\mu\textrm{g}$:Na$_2$MoO$_4$.2$H_2O$, 40$\mu\textrm{g}$:CoCl$_2$.6$H_2O$, 40$\mu\textrm{g}$:H$_3$BO$_3$, 30$\mu\textrm{g}$ per liter). By the limitation of nitrogen and deficiency of Mn$^{+2}$ or Fe$^{+2}$, the cell growth was significantly repressed. Methanol greatly repressed the cell growth and the complete inhibition was observed at concentration above 4% (v/v). In order to overcome the methanol inhibition and to prevent the methanol limitation, intermittent feeding of methanol was conducted by a D.O.-stat technique. PHB production by M. organophilum was stimulated by deficiency of nutrients such as NH$_{4}^{+}$, SO$_{4}^{-2}$, $Mg^{+2}$, $K^{+}$, or PO$_{4}^{-3}$ in the medium. The maximum PHB content was obtained as 58% of dry cell weight under deficiency of potassium ion in the optimized synthetic medium.

• The Microorganisms and Industry
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• v.18 no.1
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• pp.59-63
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• 1992

값이 저렴한 메탈올로부터 새로운 고점도성 생물고분자인 다당류의 생산과 활용은 산업적으로도 매우 흥미있는 분야이다. 본고에서는 최근 본 연구실에서 개발한 메탄올 자화세균(facultative methylotroph)인 Methylobacterium organophilum이 생산하는 신규의 고점성 다당류인 메틸란(methylan)의 생산 및 그 특성과 응용에 관하여 고찰하고자 한다.

• Applied Biological Chemistry
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• v.38 no.3
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• pp.191-195
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• 1995

Production of a high viscosity exoploysaccharide, methylan, by Methylobacterium organophilum from methanol was carried out in fed-batch cultures and the rheological properties of methylan fermentation broth were studied. Bacterial biomass showed little influence on viscosity, but the accumulation of methylan caused the increase of viscosity. With proceeding fermention, the viscosity at the same concentration of methylan was significantly increased and methylan solution showed slightly higher pseudoplasticity. The composition changes of methylan were investigated at various fermentation times. Contents of total sugar, reducing sugar and methylan were decreased but contents of acids(pyruvic acid, uronic acid and acetic acid) were increased with the culture time. It was considered that the increased content of acids resulted in the increase of the hyrodynamic domain in the solution due to charge repulsion. Consequently, the solution viscosity increased in propotion to the acids contents of methylan. Cell growth and methylan production were severely decreased by the limitation of dissolved oxygen. However, the cellular activity for methylan production was almost constant regardless of the level of dissolved oxygen. As a result, the high speed of agitation increased the methylan production, the specific production rate of methylan, and the methylan yield of the cell.