Alpha- and Gammaproteobacterial Methanotrophs Codominate the Active Methane-Oxidizing Communities in an Acidic Boreal Peat Bog

Abstract

The objective of this study was to characterize metabolically active, aerobic methanotrophs in an ombrotrophic peatland in the Marcell Experimental Forest, in Minnesota. Methanotrophs were investigated in the field and in laboratory incubations using DNA-stable isotope probing (SIP), expression studies on particulate methane monooxygenase (pmoA) genes, and amplicon sequencing of 16S rRNA genes. Potential rates of oxidation ranged from 14 to 17 μmol of CH4g dry weight soil(-1)day(-1) Within DNA-SIP incubations, the relative abundance of methanotrophs increased from 4% in situ to 25 to 36% after 8 to 14 days. Phylogenetic analysis of the(13)C-enriched DNA fractions revealed that the active methanotrophs were dominated by the genera Methylocystis(type II;Alphaproteobacteria),Methylomonas, and Methylovulum(both, type I;Gammaproteobacteria). In field samples, a transcript-to-gene ratio of 1 to 2 was observed for pmoA in surface peat layers, which attenuated rapidly with depth, indicating that the highest methane consumption was associated with a depth of 0 to 10 cm. Metagenomes and sequencing of cDNA pmoA amplicons from field samples confirmed that the dominant active methanotrophs were Methylocystis and Methylomonas Although type II methanotrophs have long been shown to mediate methane consumption in peatlands, our results indicate that members of the genera Methylomonas and Methylovulum(type I) can significantly contribute to aerobic methane oxidation in these ecosystems.

FIG 1

pmoA gene and transcript abundance in copies per gram of dry weight at the indicated depths from the S1 peatland. Samples tested were from duplicate soil cores collected in July 2013. Error bars represent standard deviations.

FIG 2

Methanotroph community composition at the indicated depths in the SPRUCE S1 peat bog based on metagenomic and cDNA analysis of pmoA. Methanotrophs detected included Methylocystis, Methylomonas, and Methylosinus. Samples for metagenomic analysis were collected at SPRUCE in July 2012 (SP0712), and samples for cDNA analysis were collected at SPRUCE in July 2013 (SP0713). Values are percentages.

FIG 3

The consumption of methane with time in the stable isotope probing incubations. Circles represent ¹²CH₄ treatments whereas triangles represent ¹³CH₄-amended treatments. The observed methane consumption rates ranged between 13.85 and 17.26 μmol of CH₄ g dwt⁻¹ day⁻¹ (calculated based on a three-point linear region within each sample distribution). Peat utilized was from a depth of 0 to 10 cm in hollows from the S1 bog, collected in July 2012.

FIG 4

The relative abundance of alphaproteobacterial (type II) and gammaproteobacterial (type I) methanotrophs based on 16S rRNA genes in ¹³C-enriched fractions (H) and light fractions (L) after 8 days (T1) and 14 days (T2) of incubation. The difference between methanotrophic communities in heavy and light fractions was significant based on ANOVA (F = 7.144, df = 3, P = 0.0439).

FIG 5

Phylogeny of methanotrophs within SIP fractions from 8- and 14-day incubations (diamonds) showing organisms within the Alphaproteobacteria, Methylocystis sp., and the Gammaproteobacteria, Methylomonas and Methylovulum spp. based on 16S rRNA gene analysis. This phylogenetic tree was prepared with the maximum-likelihood method with bootstrap analysis of nucleic acid sequences (1,000 replications).