The kinetics of inhibition of CH 4 oxidation by NH 4 ؉ , NO 2 ؊ , and NO 3 ؊ in a humisol was investigated. Soil slurries exhibited nearly standard Michaelis-Menten kinetics, with half-saturation constant [K m(app) ] values for CH 4 of 50 to 200 parts per million of volume (ppmv) and V max values of 1.1 to 2.5 nmol of CH 4 g of dry soil ؊1 h ؊1. With one soil sample, NH 4 ؉ acted as a simple competitive inhibitor, with an estimated K i of 8 M NH 4 ؉ (18 nM NH 3). With another soil sample, the response to NH 4 ؉ addition was more complex and the inhibitory effect of NH 4 ؉ was greater than predicted by a simple competitive model at low CH 4 concentrations (<50 ppmv). This was probably due to NO 2 ؊ produced through NH 4 ؉ oxidation. Added NO 2 ؊ was inherently more inhibitory of CH 4 oxidation at low CH 4 concentrations, and more NO 2 ؊ was produced as the CH 4-to-NH 4 ؉ ratio decreased and the competitive balance shifted. NaNO 3 was a noncompetitive inhibitor of CH 4 oxidation, but inhibition was evident only at >10 mM concentrations, which also altered soil pHs. Similar concentrations of NaCl were also inhibitory of CH 4 oxidation, so there may be no special inhibitory mechanism of nitrate per se.

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Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH4). After 4 years of growth and periodic dilution (>1020 times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [Km(app) ] for CH4 of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, theKm(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene product ofpmoA, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH4 and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of theMethylocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH4, had a Km(app) (129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH4 varies with growth conditions and that the oxidation of atmospheric CH4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.

A novel species is proposed for two strains of methanotrophic bacteria (H2 T and Sakb1) isolated from an acidic (pH 4.3) Sphagnum peat bog lake (Teufelssee, Germany) and an acidic (pH 4.2) tropical forest soil (Thailand), respectively. Cells of strains H2 T and Sakb1 were aerobic, Gramnegative, non-motile, straight or curved rods that were covered by large polysaccharide capsules and contained an intracytoplasmic membrane system typical of type II methanotrophs. They possessed both a particulate and a soluble methane monooxygenase and utilized the serine pathway for carbon assimilation. They were moderately acidophilic organisms capable of growth between pH 4.4 and 7.5 (optimum 5.8-6.2). The most unique characteristic of these strains was the phospholipid fatty acid profile. In addition to the signature fatty acid of type II methanotrophs (18 Aerobic methanotrophic bacteria are capable of utilizing methane as the sole source of carbon and energy. They have been divided into two groups, types I and II, belonging to the Gamma-and Alphaproteobacteria, respectively. These types differ in several phenotypic, chemotaxonomic and genotypic features, including the arrangement of intracytoplasmic membranes (ICMs), the predominant phospholipid fatty acids (PLFAs) and the pathway used for carbon assimilation. Methanotrophic bacteria inhabit a wide range of natural environments of diverse temperature, salinity andAbbreviations: DMDS, dimethyldisulfide; FAME, fatty acid methyl ester; ICM, intracytoplasmic membrane; MDH, methanol dehydrogenase; PLFA, phospholipid fatty acid; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and partial sequences of the mxaF, mmoX and pmoA genes of Methylocystis heyeri strain H2 T are AM283543-AM283546, respectively.A supplementary figure showing mass spectra of DMDS adducts of 16 : 1 PLFA of strain H2