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

Wetland soils, despite covering only 8% of the terrestrial Earth's surface, store a substantial amount of organic carbon (C). Microbial decomposition of C and nitrogen (N) in these soils leads to significant releases of greenhouse gases (GHGs), including nitrous oxide (N₂O). N₂O is a potent GHG with a global warming potential 265 times that of CO₂ and is a major ozone-depleting substance. The sources of N₂O are susceptible to change with environmental shifts, and wetlands are increasingly affected by land-use changes like drainage and afforestation. To mitigate N₂O emissions, a comprehensive understanding of the biogeochemical pathways and environmental factors influencing microbial N cycling and N₂O dynamics is crucial. Several microbial processes contribute to N₂O production, including denitrification, nitrifier denitrification, and DNRA, primarily under anoxic conditions. In contrast, ammonia oxidation (nitrification) is an aerobic process carried out by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and comammox *Nitrosopira*. AOA not only directly produce N₂O but also supply substrates for denitrification. However, the environmental conditions favoring these processes and N₂O production/consumption are not fully understood. AOA may play a crucial, yet understudied, role in terrestrial N₂O emissions. This study aimed to analyze 645 wetland soils to determine how microbial community structure and function contribute to N₂O emissions. The researchers hypothesized that high N₂O production is linked to the diversity and abundance of nitrifying microbes, particularly archaeal nitrifiers, and that their relative abundance to denitrifiers is the most significant factor explaining global N₂O emissions from wetland soils.

Existing literature highlights the importance of wetland soils as a major source of N2O, a potent greenhouse gas and ozone depleter. Studies have implicated various microbial processes, including denitrification, nitrifier denitrification, and dissimilatory nitrate reduction to ammonium (DNRA), in N2O production, predominantly under anoxic conditions. However, the relative contribution of these processes and the influence of environmental factors remain unclear. The role of ammonia-oxidizing archaea (AOA) in N2O production has received increasing attention, with some studies suggesting their significant contribution to N2O emissions in various ecosystems. Prior research has investigated the relationship between microbial diversity and N2O emissions, with varying results. This study builds on these previous works by conducting a global-scale analysis of microbial communities and their functional roles in N2O emissions from wetland soils, considering various environmental factors.

This study involved the collection of soil and gas samples from 645 wetland sites across six continents, encompassing various climate types and land-use intensities. N₂O fluxes were measured in situ using the static chamber method, while potential N₂ production was measured ex situ using a He-O₂ method. Soil samples were analyzed for various physicochemical parameters, including pH, nutrient levels, carbon and nitrogen content, and water content. Microbial community composition was analyzed using metabarcoding of bacterial 16S, archaeal 16S, and fungal 18S-ITS rRNA genes (Illumina and PacBio sequencing). Functional metagenomes were analyzed to estimate the relative abundance of N-cycle genes. Absolute quantification of N-cycle gene abundances was performed using qPCR. Statistical analyses, including Spearman correlation, partial least squares regression (PLS), random forest modeling, structural equation modeling (SEM), and generalized additive models (GAM), were used to identify the key microbial groups and environmental factors influencing N₂O emissions. Comparative genomics analysis of archaeal OTUs was conducted to investigate genetic mechanisms underlying N₂O production. The data was processed using various bioinformatics pipelines (Lotus, PipeCraft, MATAFILER) and statistical software (R).

The study revealed that warmer soils and more intensive land use led to increased N₂O emissions. N₂O emissions showed an exponential relationship with temperature, and land-use type significantly influenced emissions. Archaeal diversity increased towards lower latitudes, contrasting with bacterial diversity which peaked at mid-latitudes. Fungal diversity showed no clear latitudinal trend. The relative abundance of AOA from the phylum Thaumarchaeota was the most strongly correlated microbial group with N₂O emissions. The Soil Crenarchaeotic Group (SCG) showed the strongest positive correlation with N₂O emissions among all genera. Specific OTUs closely associated with 'Candidatus Nitrosotenuis chungbukensis MY2' and 'Candidatus Nitrosocosmicus oleophilus MY3' showed the strongest correlations with N₂O fluxes. Metagenomic analysis showed that the relative abundance of the archaeal *amoA* gene was strongly correlated with N₂O emissions, followed by an OG with unknown function. Archaea were more enriched in aerobic ammonia-oxidizing pathways compared to bacteria. qPCR analysis revealed strong positive correlations between the abundance of archaeal and bacterial *amoA* genes and N₂O emissions. Archaeal *amoA* abundance was slightly higher than bacterial *amoA* abundance, supporting the importance of archaea in nitrification. Other N-cycle genes showed weaker or no correlations with N₂O emissions. N₂O emissions increased with the diversity of N-cycle functional genes. Archaeal *amoA* abundance had a unimodal relationship with mean annual air temperature, peaking around 20°C. The AOA/AOB ratio showed a strong positive correlation with mean annual air temperature and soil temperature.

The findings demonstrate the significant role of archaeal nitrifiers in N₂O emissions from wetland soils globally. The strong correlation between archaeal *amoA* abundance and N₂O emissions, along with the comparative genomics analysis showing enrichment of aerobic ammonia-oxidizing pathways in archaea, supports the importance of archaeal nitrification in driving N₂O production. The increased diversity of N-cycle functional genes correlated with higher N₂O emissions suggests functional complementarity between nitrification and denitrification processes, particularly in drained soils. The influence of temperature and soil moisture on archaeal *amoA* abundance highlights the impact of climatic conditions on N₂O emissions. The results highlight the need to consider both the structure and function of the soil microbiome when predicting and mitigating N₂O emissions from wetlands. The decoupling of taxonomic and functional diversity emphasizes the importance of functional redundancy in N cycling microbes. These results complement previous studies on the role of archaea in N2O emissions in various environments.

This study provides compelling evidence for the crucial role of archaeal nitrifiers in global N₂O emissions from wetland soils. The findings highlight the need to consider both the functional diversity of the soil microbiome and environmental factors like temperature and drainage when predicting future N₂O emissions from these ecosystems. Future research should focus on elucidating the mechanisms underlying archaeal N₂O production and investigating the complex interplay between nitrification and denitrification in shaping N₂O fluxes under various environmental conditions. The study underscores the importance of considering microbial community structure and function in assessing and mitigating greenhouse gas emissions from wetlands.

While this study provides a comprehensive global-scale analysis, it is important to note some limitations. The cross-sectional nature of the study design does not allow for definitive causal inferences. The study relied on correlations and statistical modeling, which may not fully capture the complex interactions between microbial communities and environmental factors. Further research using manipulative experiments and more detailed mechanistic studies is needed to better understand the causal relationships between microbial processes and N2O emissions. The sampling strategy, while aiming for broad representation, may still have biases in representing the full spectrum of global wetland environments.