Methanol biosynthesis by covalently immobilized cells of Methylosinus trichosporium: Batch and continuous studies

Abstract

The DEAE-cellulose linked cells of Methylosinus trichosporium displaying high specific methane mono-oxygenase activity (66 μmol methane oxidized/h mg cells) were used for methanol biosynthesis from biogas derived methane in a batch and a continuous cell reactor. The optimum cell-to-carrier ratio was determined to be 0.5 g cells/g dry weight cellulose. Batch experiments indicated that 100 mM phosphate ion concentration was necessary to inhibit further oxidation of methanol; excess oxygen supply favored methanol accumulation with an increase in methane conversion efficiency to 27%. A pulse of 40 mM sodium formate at the end of 6 h resulted in restoration of methanol accumulation by regenerating NADH₂ required for the sustained activity of methane mono-oxygenase. Maximum methanol level of 50 μmol/mg cells was obtained in the batch reactor. In a standard 50-mL ultrafiltration continuous reactor, the covalently linked cells produced methanol at a continuous rate of 100 μmol/h for the first 10 h, after which the methanol accumulation rate fell low due to the depletion of NADH₂. The methanol accumulation could be stimulated by supplying sodium formate (40 mM) in either 20 or 100 mM phosphate buffer. Maximum methanol accumulation rate of 267 μmol/h was obtained when 20 mM formate was supplied in the feed stream containing 100 mM phosphate ions, and this level of biosynthesis was maintained for over 72 h. The stoichiometric balance made at various points of formate addition indicated that the molar amount of methanol generated at steady state is dependent on the equimolar addition of sodium formate to the feed. The half-life t_1/2 and thermal denaturation rate constant K_d were computed to be 108 h and 6.42 × 10⁻³ h⁻¹, respectively.