Structure and function of the soil microbiome underlying N₂O emissions from global wetlands

Wetland soils, despite covering only about 8% of terrestrial surfaces, store large stocks of organic carbon and nitrogen whose microbial degradation releases greenhouse gases, notably nitrous oxide (N₂O), a potent greenhouse gas and major ozone-depleting substance. Land-use change (e.g., drainage for afforestation or agriculture) and warming threaten to increase N₂O emissions from wetlands. Multiple microbial pathways contribute to N₂O production under largely anoxic conditions (classical denitrification, nitrifier denitrification, DNRA), while ammonia oxidation (first step of nitrification) is aerobic and performed by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and comammox Nitrospira. AOA can both directly produce N₂O and supply oxidized nitrogen substrates for denitrification, but the environmental controls favoring different processes remain unclear. The authors assembled a global dataset of wetland soils to test how microbial community structure and functions relate to N₂O emissions, hypothesizing that high N₂O production is mainly related to the diversity and abundance of nitrifiers—especially archaeal nitrifiers—whose absolute and relative abundance to denitrifiers would best explain global wetland N₂O emissions.

Study design and sites: Gas and soil were sampled during the growing season (2011–2018) across 29 regions on six continents spanning Köppen climate types A (tropical rainy), C (temperate), and D (boreal). Within 76 wetland soil sites encompassing vegetation types (mosses, sedges, grasses, herbs, trees, bare soil) and land uses (natural bog, fen, forest; agricultural arable, hay field, pasture; peat extraction), 196 stations (1–4 per site) were established. In situ gas flux measurements: At each station, 3–5 opaque 65 L PVC static chambers (total 645 chambers) with collars (0.5 m diameter, inserted 0.1 m) were used to measure N₂O fluxes. After 3–12 h stabilization, headspace gas was sampled every 20 min over 1 h, with at least three sessions per location in 3 days. Water-table height was recorded and soil temperature measured at 10 and 20 cm. N₂O concentrations were analyzed within 2 weeks using Shimadzu GC-2014 with ECD/TCD and autosampler; fluxes were calculated from linear regressions, applying quality thresholds. Potential N₂ measurements: Intact soil cores (0–10 cm) from 252 chambers at 26 sites were incubated under helium–oxygen atmosphere at climate-appropriate temperatures to determine potential N₂ production using GC-TCD; fluxes were calculated from linear regression with r² ≥ 0.81 (p < 0.1). Soil physicochemical analyses: From 0–10 cm soils, plant-available P (NH₄-lactate), K (flame photometry), Mg (flow injection with titanium-yellow), Ca (flame photometry), pH (1 N KCl), NH₄⁺-N and NO₃⁻-N (2 M KCl extract, flow injection), total C and N (dry combustion), organic matter (loss on ignition), and soil water content were determined; bulk density estimates were used to derive SWC. DNA extraction and sequencing: DNA was extracted from 0.2 g frozen soil (Qiagen DNeasy PowerSoil). Metabarcoding targeted bacteria (16S V4; primers 515F/806RB; Illumina NovaSeq 2×250 bp), archaea (long 16S with SSULArF/SSU1000ArR; PacBio Sequel II), and eukaryotes (18S V9 + full ITS; ITS9mun/ITS4ngsUni; PacBio). Replicate PCRs (optimized cycles) were pooled; low-depth libraries were re-run. Shotgun metagenomics: For each station, three replicate soils were pooled equimolarly (196 pooled samples). Libraries (Nextera XT) were sequenced on Illumina NovaSeq (2×150 bp) targeting ~5 million reads per sample; low-yield samples were resequenced. Quantitative PCR: Absolute abundances (gene copies g⁻¹ dry soil) were quantified for bacterial/archaeal 16S rRNA and N-cycle genes: denitrification (nirS, nirK, nosZ clades I/II), nitrogen fixation (nifH), DNRA (nrfA), ammonia oxidation (bacterial amoA, archaeal amoA, comammox amoA), anammox- and n-damo-related 16S rRNA. qPCR used Rotor-Gene Q with SYBR Green chemistry; quantification followed LinRegPCR recommendations. Bioinformatics: Illumina 16S reads were processed with LotuS (quality filtering, chimera removal via UCHIME, LCA taxonomy against SILVA). PacBio amplicons were processed with PipeCraft (mothur demultiplexing, vsearch filtering/uchime_denovo, ITSx for ITS, UPARSE clustering at 98% similarity, UNITE-based taxonomy + LCA). Shotgun metagenomes were processed with MATAFILER: quality filtering/trimming (sdm), merging (FLASH), DIAMOND blastx against eggNOG v4 to derive orthologous groups (OGs), and SSU miTags via SortMeRNA against SILVA with LCA assignment. Comparative genomics: 385 complete archaeal genomes (NCBI, Oct 7, 2020) were used to map archaeal OTUs to nearest genomes and retrieve functional annotations (IMG/JGI) to infer pathway distributions (e.g., ammonia oxidation in Thaumarchaeota genera). Statistical analyses: Abundances were normalized (Hellinger), diversity indices computed on rarefied data (vegan). Associations were tested via Spearman correlations (Benjamini–Hochberg correction), partial least squares (PLS) with VIP-based variable selection, Random Forest for predictor importance, structural equation modeling (piecewiseSEM) for direct/indirect effects, and generalized additive models (mgcv) for non-linear responses. Environmental predictors included latitude, climate (MAT, MAP, soil temperature), hydrology (water table, water content), soil chemistry (pH, C/N, C, N, NH₄⁺, NO₃⁻, Ca, K, Mg, P), organic matter, and decomposition grade (Von Post).

- N₂O emissions increased with temperature and land-use intensity. Temperature of the warmest month showed an exponential relationship with N₂O; land-use type explained emissions strongly (adjusted r = 0.364, p < 0.001), highest in bare soils and lowest in forests. Emissions declined toward higher latitudes (latitudinal model examples: N₂O vs latitude r² = 0.23, p = 2.927e-05). - Potential N₂ production peaked in temperate climates and was negatively related to land-use intensity. - Microbial diversity patterns: Archaeal diversity increased toward low latitudes; bacterial diversity peaked at mid-latitudes; fungal diversity peaked at MAT 10–15 °C. Archaeal diversity best explained by soil C/N; bacterial diversity by pH. - Taxonomic associations: Thaumarchaeota (ammonia-oxidizing archaea, AOA) relative abundance most strongly correlated with N₂O flux; major bacterial phyla (Proteobacteria, Acidobacteriota, Chloroflexi) were abundant but not significantly associated with N₂O. - Specific archaeal taxa: Soil Crenarchaeotic Group (SCG) had the strongest positive correlation with N₂O among genera (metagenomes). From 620 archaeal OTUs (PacBio; 5 sites), 11 OTUs (5 Nitrososphaerales) correlated positively with N₂O (r > 0.35, q < 0.2). Strongest OTU-level correlations included ‘Ca. Nitrosotenuis chungbukensis MY2’ (r = 0.488, p < 0.001) and ‘Ca. Nitrosocosmicus oleophilus MY3’ (r = 0.477, p < 0.001), both known N₂O producers. - Metagenomic functional signals: Archaeal amoA (ENOG411114F) relative abundance showed the strongest correlation with N₂O emission (r = 0.625, p < 0.001). Soil NO₃⁻ content correlated with archaeal amoA (r = 0.551, p < 0.001). Comparative genomics indicated greater enrichment of aerobic ammonia-oxidizing pathways in archaea versus bacteria (5.3% vs 0.3% of genomes carrying the pathway). - qPCR gene abundances: Absolute archaeal amoA correlated best with N₂O (r = 0.458, p < 0.001), followed by bacterial amoA (r = 0.313, p < 0.001). Archaeal amoA copy numbers were slightly higher than bacterial amoA (F = 6.00, p = 0.015). Comammox amoA abundance and its correlation with N₂O were weak and lower than archaeal amoA. - Denitrification genes: nirK/nirS had weak or no correlation with N₂O; nosZ (N₂O reductase) correlated positively with N₂O and was strongly correlated with nir genes, potentially indicating enhanced N₂O reduction capacity where denitrification potential is high. Potential N₂ production did not correlate with nosZ abundance. - Functional diversity: N₂O emissions increased with the diversity of N-cycle functional genes measured by qPCR (GAM; positive relationship), supporting functional complementarity (e.g., nitrification supplying substrates for denitrification) in drained soils with mixed redox conditions. - Environmental controls on AOA: Archaeal amoA displayed a unimodal relationship with MAT, peaking around 20 °C (r²adj = 0.255, p < 0.001). AOA/AOB ratio correlated positively with air and soil temperatures, consistent with higher temperature optima of AOA. - Latitudinal contrasts: Archaeal amoA abundance increased toward low latitudes (r² = 0.18, p = 0.0004), while nir gene abundances showed no significant latitudinal trend (p = 0.785).

The study shows that global wetland N₂O emissions are shaped by both climate/land-use and the functional composition of soil microbiomes. Warming and drainage enhance conditions favorable to ammonia-oxidizing archaea (AOA), which are closely linked to N₂O emissions across diverse wetland types. Despite lower overall abundance than bacteria, archaeal nitrifiers—particularly Thaumarchaeota (e.g., Nitrososphaera, Nitrosocosmicus, Nitrosotenuis, Nitrosarchaeum)—emerge as key correlates of N₂O flux. Multiple lines of evidence converge: metagenomic orthologs (archaeal amoA), taxonomic signals (Thaumarchaeota/SCG), qPCR-based absolute gene abundances (archaeal amoA), and comparative genomics showing a higher prevalence of ammonia-oxidation pathways in archaea than bacteria. Denitrification potential alone did not consistently predict N₂O emissions, likely due to metabolic versatility, gene modularity, and environmental modulation (pH, C content) of N₂O:N₂ end products. Positive covariation of nir and nosZ suggests concurrent potential for N₂O production and reduction; where nosZ is abundant, N₂O consumption may temper emissions. In contrast, the strong relationships between archaeal amoA and N₂O across climates and land uses point to nitrification (and nitrifier-associated pathways) as a widespread driver, especially under warmer, moderately oxic conditions in drained wetlands. The increase of N₂O with N-cycle functional gene diversity further implies that complementary processes (nitrification supplying NO₂⁻/NO₃⁻ for denitrification) amplify emissions under fluctuating redox regimes common in wetlands. Collectively, these findings refine understanding of microbial controls over wetland N₂O fluxes, highlighting AOA as pivotal contributors whose global distribution and adaptation to low oxygen and ammonia align with wetland conditions. This has implications for predicting responses to climate warming and land-use change, which are expected to shift microbiome structure toward higher AOA dominance and elevate N₂O emissions.

This global analysis demonstrates that nitrifying microbes—particularly ammonia-oxidizing archaea—are key microbial associates of wetland soil N₂O emissions. N₂O fluxes rise with warming and drainage, track increases in archaeal amoA abundance, and are higher where N-cycle functional gene diversity is greater, consistent with complementary nitrification–denitrification interactions. Comparative genomics supports a broader distribution of ammonia-oxidation capacity in archaea than in bacteria, reinforcing the centrality of AOA in wetland nitrogen cycling. Future work should (1) mechanistically resolve thaumarchaeal N₂O production pathways (e.g., roles of cytochrome P450 NO reductases versus hybrid nitrosation), (2) quantify the relative contributions of nitrification versus denitrification to N₂O across wetland types and hydrological regimes, (3) integrate vegetation, climate, and land-use dynamics into predictive models, and (4) assess how management of drainage and nutrient regimes can mitigate AOA-fueled N₂O emissions.

- Observational, correlative design limits causal inference; the authors note they could not distinguish cause and effect. - Mechanisms of thaumarchaeal N₂O production remain unresolved; gene presence (e.g., cytochrome P450 NO reductase homologs) varies across taxa, and AOA lacking these homologs can still produce N₂O. - Gene abundance and diversity metrics (metagenomes/qPCR) may not reflect in situ activity or flux partitioning among processes; metatranscriptomic or isotopic approaches were not applied globally. - PacBio long-read archaeal community data were available from a subset of sites (5), potentially underrepresenting archaeal taxonomic diversity globally. - Denitrification pathway modularity and environmental factors (e.g., pH, organic C) complicate linking denitrifier gene abundances to N₂O fluxes, contributing to weak correlations. - Potential N₂ measurements were ex situ under controlled atmospheres and available for fewer sites, which may not fully capture field variability.