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Dissimilation of [¹³C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 °C studied by ¹³C NMR and isotope-ratio mass spectrometry

BioTechnology Institute and Department of Chemical Engineering and Materials Science¹, and BioTechnology Institute and Department of Biochemistry, Molecular Biology and Biophysics², University of Minnesota, Saint Paul, MN 55108, USA

Author for correspondence: Michael C. Flickinger. Tel: +1 612 624 9259. Fax: +1 612 625 1700. e-mail: mflick{at}biosci.umn.edu

Using a continuous culture of Bacillus methanolicus MGA3 limited by 100 mM methanol in the feed and growing at a dilution rate D=0·25 h^-1, transients in dissolved methanol were studied to determine the effects of methanol toxicity and the pathway of methanol dissimilation to CO₂. Steady-state cultures were disturbed by pulses of methanol resulting in a rapid change in concentration of 6·4–12·8 mM. B. methanolicus MGA3 responded to a sudden increase in available methanol by a transient decline in the biomass concentration in the reactor. In most cases the culture returned to steady state between 4 and 12 h after pulse addition. However, at a methanol pulse of 12·8 mM, complete biomass washout occurred and the culture did not return to steady state. Integrating the response curves of the dry biomass concentration over a 12 h time period showed that a methanol pulse can cause an average transient decline in the biomass yield of up to 22%. ¹³C NMR experiments using labelled methanol indicated that the transient partial or complete biomass washout was probably caused by toxic accumulation of formaldehyde in the culture. These experiments also showed accumulation of formate, indicating that B. methanolicus possesses formaldehyde dehydrogenase and formate dehydrogenase activity resulting in a methanol dissimilation pathway via formate to CO₂. Studies using isotope-ratio mass spectrometry provided further evidence of a methanol dissimilation pathway via formate. B. methanolicus MGA3, growing continuously under methanol limitation, consumed added formate at a rate of approximately 0·85 mmol l^-1 h^-1. Furthermore, significant accumulation of ¹³CO₂ in the reactor exhaust gas was measured in response to a pulse addition of [¹³C]formic acid to the bioreactor. This indicates that B. methanolicus dissimilates methanol carbon to CO₂ in order to detoxify formaldehyde by both a linear pathway to formate and a cyclic mechanism as part of the RuMP pathway.

Keywords: Gram-positive methylotroph, ribulose monophosphate cycle, formaldehyde oxidation, methanol pulse, formaldehyde toxicity

Abbreviations: RuMP, ribulose monophosphate

^a Present address: Pfizer Inc., Groton, CT, 06340, USA.

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