Genomic evidence that microbial carbon degradation is dominated by iron redox metabolism in thawing permafrost

Permafrost soils contain ~1600 Pg of organic carbon (OC), nearly 60% of the global belowground OC pool, most stored in a perennially frozen state. Arctic warming is increasing soil temperatures, thaw depth, and thaw duration, likely enhancing microbial activity, OC oxidation, and CO₂ and CH₄ emissions. The fate of this OC depends on how the permafrost microbiome responds to thaw, yet there are uncertainties regarding both short- and long-term changes in composition and metabolism. Short-term laboratory warming often shows rapid shifts in composition, gene abundance, and expression linked to CO₂ and CH₄ production, while long-term in situ warming experiments frequently show little change, potentially due to substrate depletion. A recent field study found microbiome composition correlates strongly with annual thaw duration along soil depth profiles, suggesting thaw duration is a key driver of microbial responses. Redox conditions are also critical; much OC degradation in thawing permafrost occurs under reducing conditions that favor anaerobic and fermentative processes. Anaerobic respiration in thawed soils is tightly coupled to iron (Fe) cycling, with Fe(III) serving as a terminal electron acceptor for OC oxidation to CO₂. Fe-rich tundra soils can account for 40–60% of ecosystem respiration under reducing conditions, and Fe(III)-reducing bacteria can suppress methanogenesis by outcompeting methanogens for substrates. To clarify how OC degradation pathways are influenced by Fe-mediated processes over different thaw durations, the authors simulated thaw along a soil depth profile of Fe-rich wet sedge tundra and quantified relative and absolute microbiome composition and functional gene changes during short-term (7-day) and extended (30-day) thaw under reducing conditions.

Prior work indicates: (1) Short-term thaw incubations (days) can cause rapid shifts in permafrost microbiome composition and functional potential or expression toward active-layer-like communities and OC-degrading pathways. (2) Long-term field warming often shows minimal microbiome compositional change, potentially due to sustained substrate depletion, though thaw duration along depth profiles strongly correlates with microbiome structure. (3) Redox conditions strongly mediate microbial processes during thaw; Fe(III) and humic substance reduction contribute substantially (up to 40–60%) to ecosystem respiration in Fe-rich tundra and can suppress methane production when Fe(III)-reducing bacteria outcompete methanogens for substrates. These contrasting findings motivate controlled tests of thaw duration effects and Fe-mediated metabolism on OC degradation in permafrost soils.

Study site and sampling: Wet sedge tundra at Imnavait Creek, North Slope, Brooks Range, Alaska (68°36'35.36"N, 149°18′29.80°W). One frozen soil core (10 cm diameter, 1 m length) collected 19 July 2019 from a valley-bottom location, cleaned aseptically, sectioned into 30-cm increments, frozen at −80 °C. Soil layers defined by long-term surveys: active layer (AL, 0–50 cm), transition zone (TZ, 50–70 cm), permafrost (PF, 70+ cm). Incubations: For each layer, ~50 g wet soil (homogenized while frozen) placed in triplicate 250 mL amber jars (N=9 total). Each jar received 50 mL sterile deionized water; soils mixed to slurries. Jars sealed with airtight lids and septa, headspace purged with N₂ to simulate reducing conditions, incubated 4 °C in dark. Time points: subsamples (~2 g) collected frozen at T0 (pre-incubation), T7 (7 days), T30 (30 days), immediately frozen at −80 °C (N=27 DNA samples total). DNA extraction and sequencing: DNA extracted with Qiagen RNeasy PowerSoil DNA Elution Accessory kit. 16S rRNA gene V4 region amplified with 515f-806r dual-barcoded primers; pooled library sequenced on Illumina MiSeq (UM Microbiome Core). Metagenomes prepared with Nextera XT libraries and sequenced on Illumina NextSeq 2000 (150 bp PE; UM Advanced Genomics Core). 16S analysis: QIIME2 v2020.11; reads quality-filtered with DADA2; taxonomy assigned via scikit-learn naïve Bayes classifier against SILVA v138. Metagenome analysis: Reads trimmed/filtered with BBDuk (BBMap suite) with dereplication; co-assembly with MEGAHIT; contigs indexed with Bowtie2; reads mapped with BBMap; contigs database built in Anvi’o; coding sequences predicted with Prodigal; single-copy marker genes identified with HMMER (two marker gene collections). Functional annotation: KEGG via GhostKOALA; Fe-related genes via FeGenie (HMMs for Fe metabolism, acquisition, and cycling). Counts normalized as genes per million (GPM) to account for library size and CDS length. Internal standard and absolute quantification: 5.5 ng Thermus thermophilus HB-8 DNA spiked into each soil prior to extraction to enable calculation of absolute abundance of 16S rRNA gene copies and KEGG orthologs per gram soil relative to recovered internal standard reads. Statistics: R v4.1.2. Alpha diversity (Shannon H′) tested by two-way ANOVA with Tukey HSD for soil layer and time effects. 16S reads rarefied to 71,245 reads per sample; beta diversity (Bray–Curtis) tested by PERMANOVA; visualized via PCOA (ggplot2). Pairwise comparisons of dominant phyla by two-way ANOVA with Tukey HSD. Functional genes: beta diversity via PERMANOVA on GPM-normalized counts; KEGG tiers II–IV and FeGenie categories compared across time points within each soil layer by two-way ANOVA with Tukey HSD. Absolute abundance changes expressed as Log10 fold-change relative to T0 using internal standard–based normalization.

• Short-term thaw (7 days at 4 °C) produced no significant changes in taxonomic composition, Fe-associated functional genes, or OC degradation pathways across soil layers compared to T0. - Extended thaw (30 days) caused strong microbiome shifts, especially in TZ and PF: taxonomic Shannon diversity decreased by −15.4% (AL), −66.5% (TZ), −64.6% (PF); functional gene diversity decreased in TZ (−8.7%) and PF (−6.9%). - Gammaproteobacteria increased markedly in relative abundance by T30: +25.8% (AL), +81.5% (TZ), +87.0% (PF). - Fe-cycling taxa dominated after extended thaw: Rhodoferax sp. (Fe(III)-reducing) rose to 10.4% (AL), 62.3% (TZ), 62.6% (PF) relative abundance; Gallionella sp. (Fe(II)-oxidizing) increased to 8.0% (AL), 1.6% (TZ), 3.1% (PF). Geobacter sp. remained rare (<0.8% AL; <0.1% TZ/PF). Fe-cycling taxa constituted ~20% of AL and ~65% of TZ and PF communities by T30. - Absolute abundances (16S rRNA gene copies) increased substantially by T30 (internal standard–based): Rhodoferax sp. Log10 fold-change +2.1 (AL), +4.4 (TZ), +4.6 (PF); Gallionella sp. +2.2 (AL), +3.6 (TZ), +4.1 (PF). Total non–T. thermophilus 16S copies increased by >2 orders of magnitude in TZ and PF; AL showed proportional shifts without large total increase. - Functional gene shifts mirrored taxonomy: KEGG orthologs attributed to Rhodoferax sp. increased from 0.1–0.3% at T0 to 9.3% (AL), 49.5% (TZ), 48.4% (PF) at T30; absolute abundance Log10 fold-change +1.2 to +4.0. Gallionella-attributed KOs rose to 2.5% (AL), 1.1% (TZ), 1.6% (PF); Log10 fold-change +1.4 to +2.9. - Fe-associated gene categories (FeGenie): siderophore production genes increased by +7.5% (AL), +22.4% (TZ), +25.5% (PF) relative abundance; Fe(III) reduction genes increased in TZ (+3.9%) and PF (+2.6%); Fe(II) oxidation genes increased in TZ (+4.8%) and PF (+4.3%). - OC degradation pathways increased with extended thaw: aromatic compound degradation genes increased in absolute abundance (Log10 fold-change) by +0.3 (AL), +1.6 (TZ), +1.4 (PF); benzoate/aminobenzoate/fluorobenzoate degradation increased by +0.3–0.4 (AL), +1.7–2.2 (TZ), +1.6–2.0 (PF). Pyruvate metabolism increased in TZ and PF (+0.9 each). - Methane metabolism genes decreased in TZ (−0.5) and PF (−0.3) Log10 fold-change, suggesting suppression of acetoclastic methanogenesis. - Abstract-level markers of Fe cycling rose: genes for Fe(III) reduction (e.g., mtrE) and Fe(II) oxidation (e.g., cyc1) increased concurrently with benzoate and pyruvate metabolism genes. Overall, microbial carbon degradation during extended thaw was dominated by iron redox metabolism, enhancing CO₂-generating pathways while suppressing CH₄-related pathways in Fe-rich wet sedge tundra.

Extended thaw under reducing, likely sub-oxic conditions led to coordinated growth of Fe-cycling Gammaproteobacteria and increases in Fe(III) reduction and Fe(II) oxidation genes, tightly coupled with genes for degradation of aromatic compounds and central carbon metabolism (benzoate, aminobenzoate, fluorobenzoate, pyruvate). The dominance of Rhodoferax sp. (facultative Fe(III)-reducing anaerobe) and the rise of Gallionella sp. (microaerophilic Fe(II) oxidizer) indicate an Fe redox cycle that fuels microbial respiration and carbon transformations following thaw. Suppression of methane metabolism genes, particularly associated with acetoclastic methanogens (e.g., Methanothrix, Methanosarcina), is consistent with Fe(III) reducers outcompeting methanogens under reducing conditions, shifting carbon flux toward CO₂ rather than CH₄. No significant changes after 7 days suggest that short-term thaw is insufficient to drive community-wide reorganization in wet sedge tundra, contrasting with some other tundra types and highlighting the role of thaw duration, substrate availability, vegetation, and initial community structure. The observed pattern points to Fe-mediated microbial respiration as a dominant control on early post-thaw OC degradation in Fe-rich tundra, with important implications for greenhouse gas balance as thaw duration increases with climate warming.

A 30-day thaw at 4 °C under reducing conditions drove large increases in Gammaproteobacteria, especially Fe(III)-reducing Rhodoferax sp. and Fe(II)-oxidizing Gallionella sp., most strongly in transition-zone and permafrost layers. Fe-cycling taxa reached ~65% relative abundance in TZ and PF and ~20% in AL, with absolute abundances rising by 2–5 orders of magnitude. Fe(III) reduction and Fe(II) oxidation gene abundances increased alongside genes for aromatic compound and benzoate degradation and pyruvate metabolism, while methane metabolism genes declined, indicating enhanced CO₂ production and suppression of CH₄ pathways. As thaw duration at depth increases with climate warming, Fe-mediated microbial respiration is likely to play a dominant role in permafrost carbon cycling across wet sedge tundra, a major Arctic tundra type.

The incubation conditions simulated reducing, likely sub-oxic environments; oxygen concentrations were not directly measured, and the inference of sub-oxic conditions is based on taxon responses (growth of Gallionella sp., lack of Geobacter sp.). Experiments were conducted on soils from a single core/site and under laboratory slurry conditions at 4 °C with N₂-purged headspace, which may not capture all in situ spatial and temporal variability across tundra types or redox dynamics.