Unique high Arctic methane metabolizing community revealed through in situ 13CH4-DNA-SIP enrichment in concert with genome binning

Ianina Altshuler,Isabelle Raymond-Bouchard,Elisse Magnuson,Lyle G Whyte,Charles W Greer,Julien Tremblay

doi:10.1038/s41598-021-04486-z

Abstract

Greenhouse gas (GHG) emissions from Arctic permafrost soils create a positive feedback loop of climate warming and further GHG emissions. Active methane uptake in these soils can reduce the impact of GHG on future Arctic warming potential. Aerobic methane oxidizers are thought to be responsible for this apparent methane sink, though Arctic representatives of these organisms have resisted culturing efforts. Here, we first used in situ gas flux measurements and qPCR to identify relative methane sink hotspots at a high Arctic cytosol site, we then labeled the active microbiome in situ using DNA Stable Isotope Probing (SIP) with heavy 13CH4 (at 100 ppm and 1000 ppm). This was followed by amplicon and metagenome sequencing to identify active organisms involved in CH4 metabolism in these high Arctic cryosols. Sequencing of 13C-labeled pmoA genes demonstrated that type II methanotrophs (Methylocapsa) were overall the dominant active methane oxidizers in these mineral cryosols, while type I methanotrophs (Methylomarinovum) were only detected in the 100 ppm SIP treatment. From the SIP-13C-labeled DNA, we retrieved nine high to intermediate quality metagenome-assembled genomes (MAGs) belonging to the Proteobacteria, Gemmatimonadetes, and Chloroflexi, with three of these MAGs containing genes associated with methanotrophy. A novel Chloroflexi MAG contained a mmoX gene along with other methane oxidation pathway genes, identifying it as a potential uncultured methane oxidizer. This MAG also contained genes for copper import, synthesis of biopolymers, mercury detoxification, and ammonia uptake, indicating that this bacterium is strongly adapted to conditions in active layer permafrost and providing new insights into methane biogeochemical cycling. In addition, Betaproteobacterial MAGs were also identified as potential cross-feeders with methanotrophs in these Arctic cryosols. Overall, in situ SIP labeling combined with metagenomics and genome binning demonstrated to be a useful tool for discovering and characterizing novel organisms related to specific microbial functions or biogeochemical cycles of interest. Our findings reveal a unique and active Arctic cryosol microbial community potentially involved in CH4 cycling.

Highlights

Greenhouse gas (GHG) emissions from Arctic permafrost soils create a positive feedback loop of climate warming and further GHG emissions
While the soluble methane monooxygenase is only found in some methanotrophs, the particulate membrane bound form (pMMO) is ubiquitous in all known methanotrophs, with the notable exception of Methylocella and Methyloferula species, which only contain a soluble form of the enzyme[18]
Our objective focused on identifying active in situ organisms involved in methane cycling in remote high Arctic ice wedge polygon mineral cryosols that were previously shown to act as methane sinks, this is crucial for understanding future GHG emissions from widespread Arctic permafrost soils that are highly impacted by climate change[23,24]

Summary

Introduction

Greenhouse gas (GHG) emissions from Arctic permafrost soils create a positive feedback loop of climate warming and further GHG emissions. We first used in situ gas flux measurements and qPCR to identify relative methane sink hotspots at a high Arctic cytosol site, we labeled the active microbiome in situ using DNA Stable Isotope Probing (SIP) with heavy 13CH4 (at 100 ppm and 1000 ppm) This was followed by amplicon and metagenome sequencing to identify active organisms involved in CH4 metabolism in these high Arctic cryosols. Previous studies have shown that pmoA genes detected in high Arctic mineral cryosols acting as methane sinks at atmospheric methane consentrations (~ 2 ppm) are phylogenetically related to high affinity methane oxidizers (USCα and USCΥ) from upland forest s oils[21,23] To date, these particular Arctic atmospheric methane oxidizers have not been cultured, limiting our understanding of their physiology and metabolic potential and their role in the global methane cycle

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