Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

Abstract

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the "forest sequence cluster" (USC ␣), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel "upland soil cluster ␥" (USC ␥) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with 13 CH 4 at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of 13 C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC ␥ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC ␣ sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.Methane (CH 4 ) is present in the atmosphere at a mixing ratio of about 1.7 ppm of volume (ppmv). An estimated 30 Tg of CH 4 from the atmosphere year Ϫ1 is oxidized by aerobic methanotrophic bacteria (MB) in upland soils, accounting for about 6% of the global atmospheric CH 4 sink (21, 31). Bender and Conrad (2) suggested that MB active in upland soils are specialized oligotrophs adapted to the trace level of atmospheric CH 4 and possess a methane monooxygenase (MMO) with a higher substrate affinity than that of cultivated MB. It was later demonstrated that the application of single-reactant Michaelis-Menten kinetics to MMO is not always appropriate and that the apparent affinity for CH 4 varies depending on the cultivation conditions (13). Nevertheless, recent studies indicate that MB in at least some soils that oxidize atmospheric CH 4 are indeed taxonomically distinct from known MB (28).The 13 recognized genera of MB are divided into two groups, type I (further divided into types I and X) and type II. These differ in phylogenetic affiliation (Gammaproteobacteria versus Alphaproteobacteria) and in diverse biochemical characteristics (21). Identification of MB in soils is often performed by the cultivation-independent detection of a fragment of pmoA, a gene encoding the active-site subunit of particulate MMO (22,26,30,35,38). This marker gene is ...

An active atmospheric methane sink in high Arctic mineral cryosols

“…The low percentage of pmo genes confirmed that MOB are a minority in the cryosol community (o1%) (Supplementary Table S3A). The phylogeny of our pan-pmoA, pmoB and pmoC genes (Supplementary Figures S1-S3) suggests that the prevalent pMMO-possessing MOB in our cryosol samples are closely related to USCα, which appears to be the dominant MOB in acidic soils where atm CH 4 uptake was reported (Knief et al, 2003;Ricke et al, 2005;Martineau et al, 2014), such as our samples (pH 5.5-6).…”

“…Soils consuming atmospheric CH 4 , for example, forest soils, show low Michaelis-Menten constants (K m ), ranging from 2 to 348 p.p.m.v. (Knief et al, 2003;Bender and Conrad, 1992). As genetic information on atmMOB has been limited to a collection of pmoA genes encoding for the α subunit of pMMO (Degelmann et al, 2010;Martineau et al, 2014;Bárcena et al, 2011) and a 42-kb-long sequence of the genotype USCα (Ricke et al, 2005), the search for pmo genes has been the only way to detect the yet-to-be cultured atmMOB whose phylogenetic identity of 16S rRNA gene has remained elusive.…”

Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2

“…In contrast to methanotrophs in the aerobic interfaces of methanogenic environments, the methanotrophs active in dry, well aerated upland soils, for example, forest soils, consume methane with high apparent affinity (K m(app) values of 0.01-0.28 M CH 4 ) (6). Molecular studies have indicated the presence of type II methanotrophs in most upland soils (10,11) and have provided increasing evidence that, in these soils, the most abundant methanotrophs belong to several uncultivated groups, including upland soil cluster ␣ (USC␣) and upland soil cluster ␥ (USC␥) (10,12,13). Members of USC␣ and USC␥ are presumed to be specialized oligotrophs adapted to living solely on atmospheric methane.…”

Edaphobacter modestus gen. nov., sp. nov., and Edaphobacter aggregans sp. nov., acidobacteria isolated from alpine and forest soils

“…7H 2 O, 0.04 g CaCl 2 , 0.001 g sequestrene Fe [ethylenediaminedi(o-hydroxyphenylacetic) acid (Fe EDDHA)], 0.1 ml trace elements (Whittenbury et al, 1970) and 10 ml sterile-filtered 100 mM NaH 2 PO 4 / Na 2 HPO 4 buffer (pH 7.0). Incubation at 20 u C in a gastight chamber containing 20 % (v/v) methane in air yielded large, slimy, semi-transparent masses composed primarily of the methane-oxidizing proteobacterium Methylocella silvestris (Dunfield et al, 2003) around the soil crumbs on the plates. After several transfers, colonies were streaked on R2A agar (Oxoid) for the isolation of accompanying bacteria.…”

“…The upper 8-13 cm of the A h horizon were sampled in July 2001 (Knief et al, 2003) and soil crumbs were placed on agar plates of dilute ammonium mineral salts medium containing (per litre) 0.1 g NH 4 Cl, 0.2 g MgSO 4 . 7H 2 O, 0.04 g CaCl 2 , 0.001 g sequestrene Fe [ethylenediaminedi(o-hydroxyphenylacetic) acid (Fe EDDHA)], 0.1 ml trace elements (Whittenbury et al, 1970) and 10 ml sterile-filtered 100 mM NaH 2 PO 4 / Na 2 HPO 4 buffer (pH 7.0).…”