Microbial and chemical predictors of methane release from a stratified thermokarst permafrost hotspot

Abstract

Soils are dynamic interfaces that can act as both sources and sinks of methane (CH₄), yet the microbial processes underlying these fluxes remain poorly constrained in current Earth system models-particularly in thawing permafrost regions. Accurately quantifying subsurface microbial activity and its response to environmental variation is essential for improving predictions of CH₄ emissions under shifting temperature regimes. Here, we explore the potential of volatile organic compounds (VOCs) as early chemical indicators of microbial processes driving CH₄ production within a thermokarst-associated CH₄ hotspot. Field surveys at Big Trail Lake, a young thermokarst feature in central Alaska, identified localized CH₄ emission zones. Anaerobic soil laboratory microcosms from 50, 200, and 400 cm depths were incubated at -4 °C, 5 °C, and 12 °C to simulate freeze-thaw transitions. Methane flux increased markedly with temperature, and microbial community shifts revealed Methanosarcina spp. as the dominant methanogen, particularly at 200 cm. VOC profiling showed strong depth- and temperature-dependent patterns, with the 50 cm layer exhibiting the greatest chemical diversity. Notably, 200 cm soils produced VOC signatures overlapping with those from pure Methanosarcina acetivorans C2A cultures, supporting the identification of shared metabolites linked to active methanogenesis. Extended 60-day incubations confirmed temperature-sensitive CH₄ production. Carbon isotopic enrichment in CH₄ was unexpectedly strong with warming, and metagenomic detection of ANME-associated markers-including multiheme cytochromes and formate dehydrogenases-supports temperature-sensitive anaerobic oxidation of methane as a significant control on isotopic signatures. Calculated Q₁₀ values for methanogenesis exceeded typical values for boreal soils, highlighting an underappreciated temperature responsiveness of Arctic methanogens. Together, these results demonstrate that VOCs can serve as informative biomarkers of subsurface microbial activation and offer a novel diagnostic tool for detecting early-stage CH₄ hotspot formation. Incorporating such chemically and biologically resolved metrics into process-based models will be critical for improving forecasts of CH₄ release from thawing permafrost landscapes.

Keywords: biogeochemical modeling; carbon cycling; methane hotspot; methane isotopes; methanotrophy; microbial VOCs; permafrost thaw; thermokarst soils.

Figure 1

Overview of experimental design for investigating VOC dynamics in thawing permafrost soils. (a) Aerial view of the thermokarst lake system in Alaska with outlined sampling transect and soil core collection site. (b) Anaerobic microcosm setup for VOC measurement showing triplicate incubations for each depth, housed in bioreactors with VOC-trapping thin films. (c) Anaerobic microcosm setup for methane carbon isotope mole fraction measurement across different temperatures at 100 cm depth, housed in bioreactors and slurred with added Milli-Q water.

Figure 2

Genus-level microbial community composition in thermokarst soil from Big Trail Lake (BTL) across depth and incubation temperature, based on shotgun metagenomic sequencing. Bar plots represent the average relative abundance of annotated genera from triplicate samples at each depth (50 cm, 200 cm, 400 cm) and temperature condition (−5 °C, 4 °C, and 12 °C). Prominent genera are labeled, and “Others” denotes the combined abundance of taxa contributing <2% relative abundance each. Depth- and temperature-driven shifts are evident, including increased representation of methanogens (e.g., Methanosarcina*), iron-cycling bacteria (e.g.,* Sideroxydans*, Gallionella), and diverse heterotrophs, indicating microbial reorganization during simulated thaw*.

Figure 3

Genus-level distribution of methane-cycling microbial communities across soil depths and incubation temperatures. Stacked bar plot showing the relative abundance of methanogenic and methanotrophic genera in anaerobic incubations of thermokarst lake soil from 50 cm, 200 cm, and 400 cm depths, incubated at 5 °C, 12 °C, and −4 °C. Methanosarcina (green) dominates across all depths, with pronounced enrichment at 200 cm under warming (12 °C) and freeze–thaw (−4 °C) conditions, suggesting elevated methanogenic potential. Methylotrophic genera such as Methylobacterium, Methylotenera, and Methylocystis also show depth- and temperature-specific shifts, reflecting dynamic microbial responses to thaw and freeze cycles. Relative abundance data presented here may be influenced by relic DNA, particularly in permafrost soils. As such, these patterns should be interpreted as potential shifts in community composition, not direct measures of viable cell abundance.

Figure 4

Mesocosm methane incubation results. (a) Methane carbon-isotope composition as a function of incubation temperature. Methane carbon-isotopes enrich linearly with response to temperature at the end of a 60-day anaerobic incubation. Error bars report first standard deviation of calibration uncertainty at injected methane concentration. (b) Temperature sensitivity of CH₄ flux from thermokarst soil incubations expressed as Q₁₀ values over time. Curves represent the ratio of fluxes (R₂/R₁) across temperature differentials (T₂–T₁) for incubation days 7 (dark blue), 15 (orange), 22 (green), and 60 (light blue). The isoTEM Boreal Forest baseline (purple) represents the average Q₁₀ value typically used in ecosystem models for boreal regions. Lower Q₁₀ values observed at Day 60 indicate elevated methanotrophic communities.

Figure 5

Heatmap showing temperature-dependent differential expression of volatile organic compounds (VOCs) across soil depths (50 cm, 200 cm, 400 cm). VOC intensities were standardized and clustered to reveal patterns of chemical response under warming (5 °C, 12 °C) and re-freezing (−4 °C) conditions. Warmer colors indicate higher relative abundance of specific VOCs. Distinct enrichment patterns at 50 cm and 200 cm under thawed conditions highlight depth- and temperature-specific chemical signatures associated with microbial activity and potential methane hotspot emergence.

Figure 6

Chemical classification of volatile organic compounds (VOCs) across incubation conditions and temperatures. Bar plots represent the number of VOCs detected per superclass under each experimental condition (50, 200, 400 mg soil or methanogen inoculum; 12 °C, 5 °C, or freeze–thaw “m4C” treatments). High temperatures (12 °C) yielded greater diversity in organometallics, benzenoids, and lipids, particularly in methanogen-enriched incubations (200_12C). Cold or frozen conditions (e.g., 200_m4C, 50_m4C) showed shifts toward organic nitrogen compounds, salts, and stress-associated metabolites such as phenylpropanoids. Minimal background VOCs were observed in blank controls. These profiles suggest distinct VOC fingerprints related to microbial community structure, temperature response, and methanogenic potential.

Figure 7

Numerical modeling of methane dynamics during 60-day mesoscale anaerobic incubations of thermokarst soils from Big Trail Lake. (Left panel) Simulated subsurface temperature (solid lines) and CH₄ concentration (dashed lines) at 50 cm (green) and 200 cm (red) depths. Temperatures reflect initial transient responses followed by stabilization, while CH₄ concentrations increase over time, with higher accumulation at 200 cm. (Right panel) Modeled CH₄ fluxes at the soil surface under three temperature regimes—Cold (0 °C, blue), Mid (4 °C, green), and Warm (10 °C, red)—highlight the exponential temperature sensitivity of CH₄ efflux. Warmer conditions lead to accelerated and sustained increases in surface CH₄ emissions, suggesting enhanced microbial methanogenesis and vertical transport. These simulations support empirical findings of temperature-amplified CH₄ release in thawing permafrost.

Figure 8

Temperature-dependent methanogenesis pathway activity inferred from metagenomic functional potential. Arrows indicate the relative abundance of key methanogenesis-associated genes across three temperatures: −4 °C (green), 5 °C (red), and 12 °C (purple). Line thickness corresponds to quartile ranges of relative abundance, with dotted lines representing <10.04% (25th percentile), thin solid lines between 10.04–17.38% (50th percentile), medium lines between 17.38–21.48% (75th percentile), and thick solid lines >21.48%. Pathways include hydrogenotrophic (right), acetoclastic (bottom), and methylotrophic (left) methanogenesis routes. Note: This diagram reflects the aggregate functional potential of the microbial community and does not imply that all pathways co-occur within a single organism. Gene presence was inferred from metagenomic annotations of unbinned, read-based data and should be interpreted as community-scale metabolic potential.