Methanotrophy across a natural permafrost thaw environment

Caitlin M. Singleton, Carmody K. McCalley, Ben J. Woodcroft, Joel A. Boyd, Paul N. Evans, Suzanne B. Hodgkins, Jeffrey P. Chanton, Steve Frolking, Patrick M. Crill, Scott R. Saleska, Virginia I. Rich & Gene W. Tyson

Abstract
The fate of carbon sequestered in permafrost is a key concern for future global warming as this large carbon stock is rapidly becoming a net methane source due to widespread thaw. Methane release from permafrost is moderated by methanotrophs, which oxidise 20–60% of this methane before emission to the atmosphere. Despite the importance of methanotrophs to carbon cycling, these microorganisms are under-characterised and have not been studied across a natural permafrost thaw gradient. Here, we examine methanotroph communities from the active layer of a permafrost thaw gradient in Stordalen Mire (Abisko, Sweden) spanning three years, analysing 188 metagenomes and 24 metatranscriptomes paired with in situ biogeochemical data. Methanotroph community composition and activity varied significantly as thaw progressed from intact permafrost palsa, to partially thawed bog and fully thawed fen. Thirteen methanotroph population genomes were recovered, including two novel genomes belonging to the uncultivated upland soil cluster alpha (USCα) group and a novel potentially methanotrophic Hyphomicrobiaceae. Combined analysis of porewater δ13C-CH4 isotopes and methanotroph abundances showed methane oxidation was greatest below the oxic–anoxic interface in the bog. These results detail the direct effect of thaw on autochthonous methanotroph communities, and their consequent changes in population structure, activity and methane moderation potential.

Permafrost, perennially frozen ground ranging in age from two to over 700,000 years old [1], is a significant carbon reservoir, storing 1330–1580 Pg or nearly half the world’s soil carbon [2, 3]. However, rising global temperature is leading to rapid permafrost thaw and release of greenhouse gases (GHGs), resulting in a positive feedback to warming [2]. Current thaw projections under the IPCC’s (Intergovernmental Panel on Climate Change) representative concentration pathway 8.5 predict that between 30 and 99% of near surface (<3.5 m) permafrost will disappear by 2100 [4]. Within this century, 37–174 Pg of carbon is projected to be released to the atmosphere as carbon dioxide (CO2) and the more potent greenhouse gas methane (CH4), due to the microbial degradation of newly thawed soil [2].

Methane flux in thawing permafrost is controlled by microbial methane producers (methanogens) and consumers (methanotrophs), however the environmental factors controlling the distribution of these microorganisms are poorly understood. The next key step towards improving methane emissions models is the incorporation of microbial community data [5,6,7], which is currently limited by knowledge of the population structure and activity of these communities in the changing natural environment. Aerobic and anaerobic methanotrophs can prevent a large proportion of methane from reaching the atmosphere, with oxidation estimates of 20–60% recorded in permafrost-associated environments [8, 9]. In these systems, aerobic methanotrophs are typically responsible for ameliorating methane release [10], as anaerobic methanotroph abundances are generally below detection levels with very low rates of methane oxidation [11].

Aerobic methanotrophs oxidise methane to methanol using the particulate (pMMO) and/or soluble (sMMO) methane monooxygenase enzymes [12], and are currently restricted to four major lineages, including well-studied members of the Gamma- and Alphaproteobacteria, the intra-aerobic Candidatus Methanomirabilis oxyfera within the NC10 phylum [13], and the verrucomicrobial genera Methylacidiphilum [14, 15] and Methylacidimicrobium [16]. Gammaproteobacterial methanotrophs are organised into 18 characterised genera within the families Methylococcaceae and Methylothermaceae [17,18,19], whereas alphaproteobacterial methanotrophs are less diverse comprising five genera within the Methylocystaceae and Beijerinckiaceae [18, 20]. The environmental distribution of aerobic methanotrophs is typically examined using the pmoA gene, which encodes subunit A of pMMO. However, Methyloferula and Methylocella within the Beijerinckiaceae lack pMMO, so mmoX, encoding subunit A of the sMMO, must be used as a complementary marker gene [21]. Environmental surveys of pmoA and mmoX sequences have revealed a large diversity of potential methanotrophs outside the cultivated lineages [18]. This novel diversity includes the alphaproteobacterial pmoA group, upland soil cluster alpha (USCα), which is found predominantly in aerobic soils [18], and has recently been identified in permafrost-associated systems [22, 23]. Members of this group have a high affinity for methane and are capable of oxidising methane from the atmosphere [24,25,26]. Atmospheric methane oxidisers are responsible for the uptake of ~30 Tg of methane (~5% of the total sink) per year, and are the only known biological sink [27,28,29].

Our understanding of permafrost methanotrophs has focused on intact permafrost where activity is low [10, 22, 30, 31], or artificially thawed incubations [22, 32, 33], with no naturally thawing gradient sites studied to date. Here, the methanotroph communities at Stordalen Mire are examined through metagenomics, metatranscriptomics and paired biogeochemical data, across an environmental thaw gradient, peat depths (surface, mid, deep; spanning the top ~50 cm), and time (September 2010–August 2012). Characterising the presence, genomic potential and activity of methanotrophs across the Stordalen Mire thaw gradient is a significant step towards elucidating the wider role of methanotrophs in the changing Arctic and subarctic environments, and their impact on the global carbon cycle.

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