Oxidation von atmosphärischem Methan in Böden: Ein neuer quantitativer Ansatz zur Erfassung der Struktur und Aktivität methanotropher Gilden

Methane (CH4) is the most important greenhouse gas after CO2. Its atmospheric mixing ratio is increasing by 1 % y-1 due to anthropogenic impact. The main sources of CH4 are wetlands (e.g. peat bogs or rice paddies) where methanogenesis takes place. Stratospheric destruction of CH4 by hydroxyl radicals, stratospheric loss into space, and CH4 uptake by upland soils are main sinks for atmospheric methane. In upland soils methane-oxidizing bacteria (MOB) are responsible for this acitivity. The aim of this work was to assess quantitatively the composition of this functional group of bacteria, and the acitivity of predominant taxa. Due to the lack of an applicable culture-independent method for quantitation of methanotrophic bacteria in soils, a novel molecular method was developed. Functional marker genes (pmoA and mmoX) that reflect the phenotype and phylogeny of methanotrophs were chosen as targets for quantitation by real-time PCR. These genes encode subunits of methane monooyxgenases. A comparison of 16S rRNA-based phylogenies to phylogenies based on these functional marker genes revealed distinct groups of methanotrophic genera (main groups). Real-time PCR assays based on pmoA and mmoX detection were developed to target these main groups. Amplicon detection was achieved by using SybrGreen. The introduction of an additional temperature step after elongation (4-step protocol) used for data acquisition led to detection limits comparable to probe-based assays (e.g. TaqMan or hybridization probes). Thus, a quantitative detection of methanotrophs by targeting the genes encoding key enzymes was possible. The cell number estimates obtained by application of this technique to soil were reproducable and comparable to estimates obtained by cultivation-dependent methods in other studies. Two upland forest soils in Germany that oxidized atmospheric methane were investigated. MF was an acidic (pH 4.3) sandy soil developed from sandstone, and GF was a neutral (pH 7.7) clay-textured soil developed from limestone. Both soils displayed high rates of atmospheric methane uptake (MF: 95[±32] mg CH4 m-2 d-1; GF: 50[±7] mg CH4 m-2 d-1). The dominant methanotrophic guilds were represented by two pmoA sequence groups from as-yet uncultivated methanotrophs. USC a (6,4[±0,2]x106 target molecules g-1 soil [dw], soil MF) and USC g (11,6[±3,3]x106 target molecules g-1 soil [dw], soil GF) contributed at least 85 % to the total detectable abundance of methanotrophic bacteria. Cell-specific activity of the taxon USC a in MF (17,1x10-5 fmol cell-1 h-1) was higher than the cell-specific activity of USC g in GF (2,77x10-5 fmol cell-1 h-1) despite the fact that the abundances of USC g were higher. Supporting this difference in metabolic activity, only mRNA of USC a (pmoA-Genes) was detectable above detection limit in soil MF, while no mRNA was detectable in soil GF. In total, these findings led to the hypothesis that these two main pmoA groups represent ?high affinity methanotrophs? adapted to atmospheric methane concentration.