Abstract
Methylosinus trichosporium OB3b is a methanotrophic bacterium containing particulate methane monooxygenase (MMO), which catalyzes the hydroxylation of methane to methanol. The methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). We developed a novel compulsory circulation diffusion system for cell cultivation. A methane/air mixture (1:1, v/v) was prepared in a tightly sealed gas reservoir and pumped into a nitrate mineral salt culture medium under optimal conditions (5 μM CuSO4, pH 7.0, 30°C). Cells were harvested, washed, and resuspended (0.6 mg dry cells/mL) in a 500 mL flask in 100 mL of 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA as MDH inhibitors, and 20 mM sodium formate. A single 12 h batch reaction at 25°C yielded a final concentration of 13.2 mM methanol. The use of a repeated batch mode, in which the accumulated methanol was removed after each of three 8 h cycles over a 24 h period, showed a productivity of 2.17 μmol methanol/h/mg dry cell wt. Finally, a lab-scale reaction performed using a 3 L cylindrical reactor with a working volume of 1 L produced 13.7 mM methanol after 16 h. Our results identify a simple process for improving the productivity of biologically derived methanol and, therefore the utility of methane as an energy source.
Similar content being viewed by others
References
Anthony, C. (1986) Bacterial oxidation of methane and methanol. Adv. Microb. Physiol. 27: 113–210.
Takeguchi, M., T. Furuto, D. Sugimori, and I. Okura (1997) Optimization of methanol biosynthesis by Methylosinus trichosporium OB3b: An approach to improve methanol accumulation. Appl. Biochem. Biotechnol. 68: 143–152.
Furuto, T., M. Takeguchi, and I. Okura (1999) Semicontinuous methanol biosynthesis by Methylosinus trichosporium OB3b. J. Mol. Catal. A: Chem. 144: 257–261.
Lee, S. G., J. H. Goo, H. G. Kim, J. -I. Oh, Y. M. Kim, and S. W. Kim (2004) Optimization of methanol biosynthesis from methane using Methylosinus trichosporium OB3b. Biotechnol. Lett. 26: 947–950.
Mark, A. C., M. H. Kevin, A. P. Roy, and W. J. Colin (1984) The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus. Eur. J. Biochem. 138: 611–615.
Johannes, F. J., H. K. Simon, P. E. J. Verwiel, A. J. Jacob, C. M. Antonia, and A. D. Johannis (1989) On the mechanism of inhibition of methanol dehydrogenase by cyclopropane-derived inhibitors. Eur. J. Biochem. 184: 187–195.
Shimoda, M. and I. Okura (1991) Selective inhibition of methanol dehydrogenase from Methylosinus trichosporium (OB3b) by cyclopropanol. J. Mol. Catal. 64: L23–L25.
Kim, H. G. and S. W. Kim (2006) Purification and characterization of a methanol dehydrogenase derived from Methylomicrobium sp. HG-1 cultivated using a compulsory circulation diffusion system. Biotechnol. Bioproc. Eng. 11: 134–139.
Burrows, K. J., A. Cornish, D. Scott, and I. J. Higgins (1984) Substrate specificities of the soluble and particulate methane mono-oxygenases of Methylosinus trichosporiumm OB3b. J. Gen. Microbiol. 130: 3327–3333.
Cox, J. M., D. J. Day, and C. Anthony (1992) The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria. Biochim. Biophys. Acta 1119: 97–106.
Kim, H. G., G. H. Han, C. Y. Eom, and S. W. Kim (2008) Isolation and taxonomic characterization of a novel type I methanotrophic bacterium. J. Microbiol. 46: 45–50.
Dales, S. L. and C. Anthony (1995) The interaction of methanol dehydrogenase and its cytochrome electron acceptor. Biochem. J. 312: 261–265.
Jensen, S., A. Prieme, and L. Bakken (1998) Methanol improves methane uptake in starved methanotrophic microorganisms. Appl. Environ. Microbiol. 64: 1143–1146.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kim, H.G., Han, G.H. & Kim, S.W. Optimization of lab scale methanol production by Methylosinus trichosporium OB3b. Biotechnol Bioproc E 15, 476–480 (2010). https://doi.org/10.1007/s12257-010-0039-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-010-0039-6