Intermediate cellular regeneration counteracts the stop in methanol production from methane by Methylosinus trichosporium OB3b

• Methane hydroxylation by methanotrophs is attractive but yet limited. • Intermediate cell regeneration allowed to resume MeOH production. • This approach is a faster way than re-cultivating the methanotrophic bacteria. • Running two bioreactors (reaction and regeneration) is a technical solution. Methylosinus trichosporium OB3b stands out as a methanotrophic model strain, widely reported for its high productivity of exogenous methanol from methane. However, the development of this attractive bioprocess is hindered by a limited production time rounding 20h. The causes of MeOH production stop are not fully understood yet. This work investigated potential cytotoxic impacts of EDTA and NaCl, enzymatic inhibitors together added in the reaction medium to avoid the spontaneous methanol overoxidation. The tested NaCl concentrations did not refrain bacterial cells from proliferating normally, discarding osmotic stress phenomena. But EDTA addition appeared mandatory to yield an efficient MeOH production. Up to date, EDTA precise mode of action is not fully understood and several hypotheses have been reported. In this study, prior EDTA saturation with Cu 2+ ions had no effect on MeOH production, suggesting EDTA did not chelate MMO metallic cofactor. EDTA action was consequently assumed to target cell membrane compounds and affect cell integrity. Regenerating cells in their native culture conditions ( i.e. without inhibitors) appeared allowing them to recover their hydroxylating activity and produce MeOH with a productivity similar to the initial one, in a faster way than re-cultivation of the bacteria.