High-Affinity Methane Oxidation by a Soil Enrichment Culture Containing a Type II Methanotroph

Abstract

ABSTRACT Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH 4 ). After 4 years of growth and periodic dilution (>10 20 times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [ K m(app) ] for CH 4 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, the K m(app) increased when the culture was transferred to higher mixing ratios of CH 4 (1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH 4 revealed a single gene product of pmoA , 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% CH 4 and phylogenetically positioned based on its 16S rRNA, pmoA , and mxaF gene sequences as a type II strain of the Methylocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH 4 , had a K m(app) (129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH 4 varies with growth conditions and that the oxidation of atmospheric CH 4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.