Two bacterial strains that grow anaerobically on betaine were isolated from enrichment cultures and identified as strains of Eubacterium limosum. In a mineral medium supplemented with yeast extract and Casitone, the doubling time of E. limosum strain 11A on betaine was 6 h at 37°C. The molar growth yield amounted to 9 g of dry cell mass per mol. Betaine was fermented in accordance with the following equation: 7 betaine + 2 CO 2 → 7 N,N -dimethylglycine + 1.5 acetate + 1.5 butyrate. E. limosum also grew on methanol and choline. The former was converted to acetate and butyrate, and the latter was converted to N,N -dimethylethanolamine, acetate, and butyrate. The conditions for the quantitative determination of N,N -dimethylglycine by capillary tube isotachophoresis have been determined.

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Methanosarcina barkeri was grown on trimethylamine, methylamine, or methanol containing completely deuterated methyl groups. Methane was collected and analyzed in a mass spectrometer. It contained 79 to 83% CD3H and 14 to 18% CD2H2. This demonstrated that the methyl groups of the above compounds served primarily as direct precursors of methane.It was demonstrated many years ago that growth of Methanosarcina barkeri on deuterated acetate results in the formation of trideuteromethane (4) and that growth on methanol in D20 gives monodeuteromethane (5). Thus, Cl compounds more oxidized than methanol apparently do not occur as intermediates in methane fonnation. In addition, the intermediates which do occur are not subject to exchange reactions with the protons (or deuterons) of water. Since M. barkeri has been shown to grow on mono-, di-, and trimethylamines (2, 7), it was of interest to determine whether the methyl groups of these compounds were also direct precursors of methane.M. barkeri Fusaro (DSM804) was cultivated as described by Hippe et al. (2). Methanol, methylamine, and trimethylamine in final concentrations of 100 mM were used as growth substrates. The bacteria were cultivated in 5 ml of medium, using anaerobic culture tubes and a 5% (vol/vol) inoculum.To obtain trimethylamine containing completely deuterated methyl groups, we used the following procedure (3). A 5-ml amount of CD3I and 6 ml of 25% NH4OH were combined in a 100-ml glass bottle, the bottle was stoppered, and the mixture was incubated at 600C overnight. The precipitated (CD3)4N+Iwas separated from the residual liquid by filtration on filter paper. The crystals were washed with 10 ml of ice-cold water, and the remaining material was recrystallized in about 10 ml of water. The purified tetramethylammonium iodide was dissolved in 20 ml of water and heated to 600C.About 1 g of wet Ag20 was then added with stirring, and the precipitating AgI was quickly removed, by filtration. The filtrate was then dried under an infrared lamp, carefully avoiding high temperatures. The resulting very hygroscopic tetramethylammonium hydroxide was 37:transferred into a round-bottom flask and slowly heated with stirring for 3 h under a continuous stream of gaseous hydrogen. The trimethylamine formed was trapped in 50 ml of 0.1 M hydrochloric acid. For the removal of traces of methanol, the liquid was concentrated to dryness under an infrared lamp, dissolved in 1 ml of water, and stored at -20°C. The chemical purity of the trimethylamine synthesized was confirmed by gas chromatography according to Hippe et al. (2). The isotopic purity was checked by mass spectrometry. The spectrum showed a prominent peak for the completely deuterated compound and peaks for the fragments derived therefrom. A small peak was present at an m/e value of the parent compound minus 1. It corresponded to an H content of the deuterotrimethylamine of 4.2%.Isotopic analysis was done with a Varian MAT model CH5 high-resolution mass spectrometer.The cathode emission was set at 300 ,AA, and the ioni...

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