An obligate methanotrophic bacterium, strain MTS, was isolated from a methane-fed microaerobic denitrifying bioreactor. 16S rRNA and DNA-DNA hybridization analysis revealed that this organism was most closely related to Methylocystis parvus, a Type II methanotroph, belonging to the α-subclass of the Proteobacteria. The metabolism of the bacterium under microaerobic and anaerobic conditions was studied by (13) C-NMR. (13) C-labelled poly-β-hydroxybutyrate (PHB) formation occurred in cell suspensions incubated with (13) C-labelled methane at low (5-10%) oxygen concentration. Under these conditions low levels of succinate, acetate and 2,3-butanediol were formed and excreted into the culture medium. Intracellular PHB degradation was observed in intact cells under anaerobic conditions in the absence of an exogenous carbon source during a long-term incubation of 90 days. Multiple (13) C-labelled β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were identified as products in in vivo(13) C-NMR spectra and in the spectra of culture medium during the dynamic PHB degradation. The isolated obligate methanotroph clearly shows a fermentative metabolism of PHB under anaerobic conditions. The excreted products may serve as substrates for denitrifying bacteria.
Poly-beta-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [(13)C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by (13)C NMR. During PHB synthesis in the presence of labelled acetate, low levels of beta-hydroxybutyrate, butyrate, acetone, isopropanol, 2,3-butanediol and succinate were observed. Subsequent anaerobic PHB breakdown showed enhanced levels of these products at the expense of PHB. Fermentative metabolism occurring during anaerobic PHB degradation was confirmed in experiments with fully (13)C-enriched cells, which were grown on (13)C-labelled methane. beta-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were detected as multiple (13)C-labelled compounds in the culture medium. Our results suggest that intracellular PHB degradation can be used as a reserve energy source by methanotrophs under anoxic conditions.
13C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35-25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of (13)C-labelled methanol to carbon dioxide was observed at low (1-5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically.
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