Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (α subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the α and γ subclasses of Proteobacteria : the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10 ¹ and 10 ² target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus , Methylomicrobium album , and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus -specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) × 10 ⁶ pmoA molecules g ⁻¹ for all methanotrophs. The Methylosinus group was predominant (2.7 × 10 ⁶ ± 1.1 × 10 ⁶ target molecules g ⁻¹ ). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 × 10 ⁶ ± 0.9 × 10 ⁶ target molecules g of soil ⁻¹ ). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 × 10 ⁴ target molecules g of soil ⁻¹ . Our results showed that pmoA -targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.

Summary: The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 µg CH ₄ m ⁻² h ⁻¹ ) of atmospheric CH ₄ at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real‐time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia‐oxidizing bacteria. As yet uncultured methanotrophs of a group previously named ‘forest soil cluster’ or ‘USCα’ were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group ‘Cluster I’ were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10 ⁶ pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia‐oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH ₄ uptake rates due to their low cell‐specific CH ₄ oxidation activity. Based on CH ₄ flux and bacterial abundance data, estimated cell‐specific CH ₄ oxidation rates of the detected methanotrophic bacteria were 540–800 × 10 ⁻¹⁸ mol cell ⁻¹ h ⁻¹ , which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell‐specific CH ₄ oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH ₄ alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USCα methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA trancripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USCα and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell‐specific CH ₄ oxidation activities.

The objective of the study was to elucidate the depth distribution and community composition of Archaea in a temperate acidic forest soil. Numbers of