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

Abstract

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 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, the Km(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 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% 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 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.

FIG. 1

Oxidation of CH₄ at mixing ratios of <200 ppmv (○) in closed vials (125 ml) containing 50 ml of NMS inoculated with the enrichment culture at day 0. Methane was added to the vials at 34 and 46 days. The data are the means of results with six vials ± 1 standard error of the mean. □, mean of results with three uninoculated vials.

FIG. 2

Kinetic curve of CH₄ oxidation in a soil enrichment culture which was grown continuously on CH₄ at a gaseous mixing ratio of <275 ppmv. Each symbol represents the mean of results with two samples.

FIG. 3

Kinetic curve of CH₄ oxidation in a soil enrichment culture initially grown on CH₄ at a gaseous mixing ratio of <275 ppmv and later on 1% CH₄. Each symbol represents the mean of results with two samples. ■, samples diluted 1:1 with water.

FIG. 4

Phylogenetic tree, based on 16S rRNA gene sequences, showing the relationship of isolate LR1 to other type II methanotrophs of the Methylocystis/Methylosinus group and to representative members of the alpha subclass of the class Proteobacteria. The 16S rRNA gene sequence from Methylococcus capsulatus was used as an outgroup reference. The numbers indicate the bootstrap values (percentage of outcome) for the respective interior branch points from a neighbor-joining test. Only values above a threshold of 50% are shown. The scale bar represents the estimated number of base changes per nucleotide sequence position.

FIG. 5

Unrooted phylogenetic tree, based on derived amino acid sequences of pmoA gene fragments, showing the identity of the pmoA product from isolate LR1 and that from the high-affinity enrichment culture (grown on <275 ppmv of CH₄) and their relationship to other methanotrophic species. The distance bar represents percent dissimilarity.

FIG. 6

Unrooted phylogenetic tree, based on derived amino acid sequences of mxaF gene fragments, showing the identity of the mxaF product from isolate LR1 and that from the high-affinity enrichment culture (grown on <275 ppmv of CH₄) and their relationship to other methylotrophic species. The distance bar represents percent dissimilarity.

FIG. 7

DGGE patterns of DNA, amplified with primers for mxaF and pmoA, from selected methanotrophic strains (first four lanes), the high-affinity enrichment culture (grown on <275 ppmv of CH₄), the methanotrophic isolate LR1, a coculture of LR1 with a Variovorax sp., and a Hyphomicrobium sp. isolated from the enrichment culture.