HPO4 ^.12H2O, 0.45 g/l MgSO4 ^.7H2O, 3.3 mg/l CaCl2 ~ 2H2O, 1.3 mg/l FeSO4 ^.7H2O, 130 µg/l MnSO4 ^.4H2O, 130 µg/l ZnSO4 ^.4H2O, 40 µg/l CuSO4 ^.5H2O, 40 µg/l Na2MoO4 ^.4H2O, 40 µg/l CoCl2 . 6H2O, and 30 µg/l H3BO.

The medium for measuring the specific growth rates of ammonia, phosphate, and minerals sources was a modified growth medium containing 7.56 mM ammonia solution, 15.6 mM phosphoric acid, 9.53 potassium chloride, 11.8 mM sodium chloride, 1.8 mM magnesium chloride, 4.7 µM ferric sulphate, and 22 µM calcium chloride. The methanol concentration in the modified growth medium was 0.3%. The nitrogen source in the modified growth medium was ammonia solution instead of ammonium sulphate to reduce the effect of other salts. For the same reason, the phosphate source was phosphoric acid instead of sodium and potassium phosphate.

Fed-batch culture used the growth medium containing 0.3% methanol. The methanol concentration was controlled at a constant level of 0.3% during the fed-batch culture. The supplement of phosphate and minerals was coupled with methanol feeding and the ratio of components to methanol was constant. The phosphate line contained KH2PO4 36.5 g and Na2HPO4^.12H2O 26.0 g, the mineral line contained MgSO4 ^.7H2O 33 g, CaCl2 ^.2H2O 110 mg, FeSO4 ^.7H2O 45 mg, MnSO4 ^.4H2O 13 mg, ZnSO4 ^.4H2O 13 mg, CuSO4

A single colony was inoculated into 250 ml Erlenmeyer flask containing 50 ml of growth medium, and cultivated for 24 h in a rotary shaker (Inova 4330, NBS, USA) at 30 °C and 200 rev/min. The cells were subcultured once in liquid medium for12 h, and then inoculated into the main culture medium.

Fed-batch culture of methanol was performed using the direct on-line monitoring system with porous teflon-tubing (Dairaku & Yamane 1979) linked with a home-made feedback control system of a computer-interfaced pump, as previously reported (Kim et al. 1996). Phosphate and otherminerals were fed fed coupled with methanol feeding via different lines to prevent precipitation. The pH of phosphate and mineral solution was adjusted at 7.0 and 2.0, respectively. Ammonia solution was supplied as a nitrogen source by pH-stat method in the growth stage, and it was replaced with a mixture of sodium hydroxide (2 M) and potassium hydroxide (2 M) to induce the nitrogen-limited condition forpoly- fl-hydroxybutyrate accumulation. Temperature and pH were maintained at 30 °C and 7.0, respectively. Aeration and agitation was controlled manually to prevent limitation by dissolved oxygen.
 

Determination of initial specific growth rate

The initial specific growth rate was determined from the slope of the logarithm of optical density (600 nm) versus time curve by using Bioscreen C (LabSystem, Sweden). Actively growing cells were inoculated into the modified growth medium containing various concentrations of each component to observe substrate inhibition. The initial pH of the medium was set to 7.0.
 

Biomass was determined by optical density (600 nm) and dry cell weight. The concentration of poly-#-hydroxybutyrate was estimated by gas chromatography (DS6200, Donam, Korea) according to the modified Braunegg method (Braunegg et al. 1978), and the concentration of ammonia was measured by the indophenol method (Bolleter et al. 1961) with ammonium sulphate as a standard. The concentrations of phosphate and minerals were determined by using inductively coupled plasma emission spectrophotometer (ICP/ES, ICPS-1000III, Shimadzu, Japan).

RESULTS AND DISCUSSION

Effect of the concentration of carbon source on cell growth

The specific growth rates were measured for various concentrations of each medium component in order to investigate the inhibitory effect of the concentrations of medium component on the growth of Methylobacterium sp. forpoly- fl-hydroxybutyrate production. To achieve high cell density from methanol as a carbon source, the effect of methanol concentration on the cell growth was first studied by measuring the initial specific growth rate of the cells at various concentrations of methanol.

Figure 1. Inhibitory effect of methanol as a carbon source on the growth of Methylobacterium sp.

Figure 2. Effect of phosphate concentration on initial specific growth rate.

Figure 3. Initial specific growth rate with variable concentration of cations.

Figure 4. Fed-batch culture by reducing substrate inhibition. Arrow mark indicates the time of exchange of ammonia with NaOH (2 M) and KOH (2 M) to induce nitrogen limitation for PHB accumulation.

When the concentration of methanol was increased, the specific growth rate decreased (Figure 1). As the concentration of methanol was increased to 3% (v/v), the specific growth rate decreased to half of the maximal specific growth rate and was strongly inhibited above a methanol concentration of 6%. The specific growth rate was found to have a maximal value of 80 at 0.5% methanol concentration. The methanol concentration should be maintained below 0.5% by fed-batch culture of methanol in order to obtain a high-growth rate.

Effect of the concentrations of nitrogen and phosphate sources on the cel growth

The specific growth rates were measured by varying the concentration of ammonia solution as a nitrogen source from 7.6 to 76 mM. The concentration of medium component was changed from 1- to 10-fold of component concentration of the modified growth medium to determine the range of concentration with a low inhibition of medium component in fed-batch culture for high-density cells. The specific growth rate did not decrease markedly on increasing the concentration of ammonia solution. These results suggest that the cell growth was not inhibited at concentrations of ammonia from 7.6 to 76 mM, and it was not necessary to adjust the concentration of ammonia solution within 10-fold in fed-batch culture.

The effect of the concentration of phosphate on the cell growth was investigated using phosphoric acid (Figure 2). The specific growth rate was maximal at a phosphoric acid concentration of 31.2 mM and above this concentration, it decreased markedly. Cell growth below the phosphate concentration of 47 mM (3-fold) was not inhibited. The concentration of phosphate should be carefully controlled below 47 mM.

High concentrations of phosphate and its related compounds inhibited the growth of Phytophthora (Griffith et al. 1993) and antibiotic production by Streptomyces (Lobbe et al. 1984). Adenine nucleotides (ADP and ATP) as well as phosphate in some methylotrophs showed an inhibitory effect on the growth of cells (Mehta et al. 1987, 1989). Excess phosphate chelated the divalent ion of methanol dehydrogenase, thereby inhibiting enzyme activity (White et al. 1993). Because methanol dehydrogenase is an enzyme mediating the first step of methanol metabolism, the inhibition of methanol dehydrogenase causes inhibition of cell growth. The concentration of phosphate must be controlled carefully below the inhibitory level to achieve high-density cells.

Effect of the concentration of minerals on cell growth

The inhibitory effect of cations such as potassium, sodium, magnesium, calcium, and iron ions at high concentrations was investigated similarly. The initial specific growth rate was measured in media containing different concentrations of each component. As Figure 3 designates, calcium and iron ions did not significantly inhibit the cell growth. Sodium, potassium, and magnesium ions had some inhibitory effects as the concentration increased. Above certain concentrations, however, precipitates were formed, resulting in the inhibitory effects disappearing, and the growth rate slightly recovered.

High concentrations of methanol and phosphate gave inhibitory effects on the growth of the methylotroph. To obtain a high cell density by reducing substrate inhibition, methanol and phosphate were maintained below 0.5% and 45 mM, respectively. Fed-batch culture was carried out with an automatic methanol feeding and pH-stat system. Methanol feeding was automated by using the porous teflon-tubing method with computer-interfaced pump. Ammonia solution was fed with pH-stat.

Figure 4 represents the time courses of cell mass, poly-b-hydroxybutyrate, phosphate, and minerals. During the culture, the methanol concentration was kept within 0.3%. Minerals were also maintained around optimal concentration values and the phosphate concentration was not allowed to exceed 30 mM. The control of their concentrations within the desired level could reduce the inhibition of medium components and the final dry cell weight reached to 172 g/l at 84 h.

Table 1 shows the summary of the process parameters determined from Figure 4 and comparison with other results in methylotrophs grown on methanol. Suzuki et al. (1986), working with Pseudomonas sp. K, have obtained cell densities up to 206 g/l after175 h h but, unlike our process, oxygen-enriched air was used during the fermentation. Our results obtained with Methylobacterium sp. indicated the much improved results in the process time and the volumetric productivity of cells were 84 h, 2.05 g/l-h, respectively.

The medium components such as methanol, ammonia, phosphorus, magnesium, potassium, sodium, etc. should be carefully designed and be fed with proper ratio so as not to be accumulated or be depleted during the fed-batch operation for high-density cell culture of methylotrophs.

REFERENCES

Bolleter, W.T., Bushman, C.J. & Tidwell, P.W. 1961 Spectrophotometric determination of ammonia as indophenol. Analytical Chemistry 33, 592–594.

Bourque, D., Pomerleau, Y. & Groleau, D. 1995 High-cell-density production of poly-b-hydroxybutric acid (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB. Applied Microbiology and Biotechnology 44, 367–376.

Braunegg, G., Sonnleitner, B. & Rafferty, R.M. 1978 A rapid gas chromatographic method for the determination of poly-b-hydroxybutyric acid in microbial biomass. European Journal of Applied Micobiology 6, 29–37.

Dairaku, K. & Yamane, T. 1979 Use of porous teflon tubing method to measure gaseous or volatile substances dissolved in fermentation liquid. Biotechnology and Bioengineering 21, 1671–1676.

Faust, U. & Prave, P. 1983 Biomass from methane and methanol. In Biotechnology, ed. Dellweg, H. vol. 3, pp. 83–103. New York: Verlag Chemie Gmbh. ISBN 3-527-25765-3.

Griffith, J.M., Coffey, M.D. & Grant, B.R. 1993 Phosphonate inhibition as a function of phosphate concentration in isolates of Phytophthora palmivora. Journal of General Microbiology 139, 2109–2116.

Hou, C.T. 1984 Microbiology and biochemistry of methylotrophic bacteria. In Methylotrophs: microbiology, biochemistry, and genetics, ed. Hou, C.T. pp. 1–53. Florida: CRC Press, Inc. ISBN 0-8493-5992-2.

Kim, P., Kim, S.W., Lee, G.M., Lee, H.S. & Kim, J.H. 1995 Isolation and characterization of a methylotroph producing 3-hydroxybutyrate-3-hydroxyvalerate copolymer. Journal of Microbiology and Biotechnology 5, 167–171.

Kim, S.W., Kim, P. & Kim, J.H. 1996 High production of poly-b-hydroxybutyrate (PHB) from Methylobacterium organophilum underpotassium limitation. Biotechnology Letters 18, 25–30.

Lobbe, C., Jensen, S.E. & Demain, A.L. 1984 Prevention of phosphate inhibition of cepharosphorin synthetases by ferrous ion. FEMS Microbiology Letters 25, 75–79.

Mehta, P.K., Mishra, S. & Ghose, T.K. 1987 Methanol accumulation by resting cells of Methylosinus trichosporium. Journal of General Applied Microbiology 33, 221–229.

Mehta, P.K., Mishra, S. & Ghose, T.K. 1989 Growth kinetics and methanol oxidation in Methylosinus trichosporium NCIB 11131. Biotechnology and Applied Biochemistry 11, 328–335.

Ogata, K., Izumi, Y., Kawamori, M., Asano, Y. & Tani, Y. 1977 Amino acid formation methanol-utilizing bacteria. Journal of Fermentation Technology 55, 444–451.

Oki, T., Kitai, A., Kouno, K. & Ozaki, A. 1973 Production of L-glutamic acid by methanol-utilizing bacteria. Journal of General Applied Microbiology 19, 79–83.

Powell, K.A. & Rogers, B.L.F. 1984 Single-cell protein. In Methylotrophs: microbiology, biochemistry, and genetics, ed. Hou, C.T. pp. 1–53. Florida: CRC Press, Inc. ISBN 0-8493-5992-2.

Suzuki, T., Yamane, T. & Shimizu, S. 1986 Mass production of polyb-hydroxybutric acid by fully automatic fed-batch culture of methylotroph. Applied Microbiology and Biotechnology 23, 322– 329.

White, S., Boyd, G., Mathew, F.S., Xia, Z., Dai, W., Zhang, Y. & Davison, V.L. 1993 The active site structure of the calcium-containing quinoprotein methanol dehydrogenase. Biochemistry 32, 12955–12958.

Yamane, T. & Shimidzu, S. 1986 Fed-batch techniques in microbial process. In Advances in Biochemical Engineering, ed. Fiechter, A. vol. 30, pp. 146–194. New York: Springer-Verlag. ISBN 3-540-13539-1.

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