A gel‐stabilized system with counter gradients of CH4 and O2 was used to grow methanotrophs from wetland, agricultural and forest soils and lake sediment. Columns of semi‐solid nitrate‐ or ammonium‐minerai salts medium were continuously flushed at opposite ends with CH4 and O2 to create opposing concentration gradients of the two gases. Methanotrophs grew from all samples except forest soil, and were visible as thin bands after 5 to 15 days of incubation. The position of growth was CH4 and O2 concentration‐dependent and occurred at the point of maximum possible CH4 oxidation, where both substrates were completely consumed. Evidence was obtained for denitrification and nitrification activities concomitant with CH4 oxidation. This approach may be useful to isolate methanotrophs with different CH4 and O2 requirements and to study their interactions with other groups of bacteria in nature.

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The effect of pH on atmospheric methane (CH4) consumption was studied with slurries of forest soils and with bacteria extracted from the same soils. Soil samples were collected from a mixed hardwood stand in New Hampshire, from jackpine and aspen stands at the BOREAS (Boreal Ecosystem Atmosphere Study) site near Thompson, northern Manitoba, from sites in southern Québec, including a beech stand and a meadow, and from a site in Ontario (cultivated humisol). Consumption of atmospheric CH4 (concentration, approximately 1.8 ppm) occurred at depths of >5 cm in both acidic (pH 4.5 to 5.2) and alkaline (pH 7.2 to 7.8) soils. In slurries of acidic soils, maximum activity occurred at different pH values (pH 4.0 to 6.5). Bacteria extracted from these soils by high-speed blending and density gradient centrifugation showed pH responses different from the pH responses of the slurries. In all cases, these bacteria had a methanotrophy pH optimum of 5.8 and exhibited no activity at pH 6.8 to 7.0, the pH optimum range for known methanotrophs. This difference in pH responses could be useful in modifying media currently used for isolation of these organisms. Methanotrophic activity was induced in previously non-CH4-consuming soils by preincubation with 5% (vol/vol) CH4 (50,000 μl of CH4 per liter) or by liquid enrichment with 20% CH4. The bacteria showed pH responses typical of known methanotrophs and not typical of preexisting consumers of ambient CH4. Furthermore, methanotrophs induced by high CH4 levels were more readily extracted from soil than preexisting ambient CH4 consumers were. In the alkaline soils, preexisting activity either was destroyed or resisted extraction by the procedure used. The results support the hypothesis that consumers of ambient CH4 in soils are physiologically distinct from the known methanotrophs.

Comparisons between in situ CH4 concentration and potential factors controlling its net production were made in a temperate swamp. Seasonal measurements of water table level and depth profiles of pH, dissolved CH4, C02, 029 S042-, N03-, formate, acetate, propionate, and butyrate were made at two adjacent sites 1.5 to 2 m apart. Dissolved CH4 was inversely correlated to 02 and, in general, to N03-and so42-, potential inhibitors of methanogenesis. At low water table levels (August 1992), maximal CH4 (2 to 4 ,uM) occurred below 30 cm, whereas at high water table levels (October 1992) or under flooded conditions (May 1993), CH4 maxima (4 to 55 ,uM) occurred in the top 10 to 20 cm. Higher CH4 concentrations were likely supported by inputs of fresh organic matter from decaying leaf litter, as suggested by high acetate and propionate concentrations (25 to 100 ,uM) in one of the sites in fall and spring. Measurements of potential CH4 production (and consumption) showed that the highest rates generally occurred in the top 10 cm of soil. Soil slurry incubations confirmed the importance of organic matter to CH4 production but also showed that competition for substrates by nonmethanogenic microorganisms could greatly attenuate its effect.

Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (−)-α-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was;Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (−)-α-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (−)-α-pinene, γ-terpinene, or β-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (−)-α-pinene, (S)-(−)-limonene, (R)-(+)-limonene, and γ-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, α-pinene oxide, linalool, α-terpineol] and a noncyclic hydrocarbon (β-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature.