High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph

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 (>10(20) times the initial soil inoc ulum), a mixed culture was obtained which displayed an apparent half-satura tion constant [K-m(app)] for CH4 of 56 to 186 nM (40 to 132 ppmv), This val ue was the same as that measured in the soil itself and about 1 order of ma gnitude lower than reported values for pure cultures of methane oxidizers. However, the K-m(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%), Denaturing gradient gel electroph oresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene p roduct of pmoA, which codes for a subunit of particulate methane monooxygen ase. This suggested that only one methanotroph species was present. This or ganism 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 the Methylocystis/Methylosinus group. A c oculture of this strain with a Variovorax sp., when grown on <275 ppmv of C H4, had a K-m(app) (129 to 188 nM) similar to that of the initial enrichmen t culture. The data suggest that the affinity of methanotrophic bacteria fo r CH4 varies with growth conditions and that the oxidation of atmospheric C H4 observed in this soil is carried out Pv type II methanotrophic bacteria which are similar to characterized species.