Comparative Analysis of Denitrification and Other Nitrogen Cycle Genes in Diverse Environmental Microbiomes

The nitrogen cycle includes the microbial transformation of reactive nitrogenous molecules within a given ecosystem. Shotgun metagenomics, the unbiased sequencing of a microbial community, can reveal the unique gene profile and community composition of diverse microbiomes. We used NCycDB, an assembly-free, alignment-based pipeline, to determine and compare the potential nitrogen cycle processes in seven distinct microbiomes: wetland soils, agricultural soils, forest soils, compost, river water, and pond water from Knox County, Ohio, and lake water from the McMurdo Dry Valley Region in Antarctica. Soil metagenomes showed high levels of bacterial denitrifier genes, whereas freshwater metagenomes showed fewer denitrifiers. In wetland and agricultural soil cores, the relative abundance of norB, the marker gene for nitric oxide reductase, was a predictor of ambient N2O flux as measured by gas chromatography. Denitrifiers were predicted using the Kraken2/Bracken pipeline. In Ohio wetland soils, the relative abundance of Bradyrhizobium species were strongly associated with denitrification genes. We also explored how freshwater environmental factors select for or against nitrogen cycle genes. Denitrifier genes were positively correlated with phosphate concentration. Denitrifiers and denitrification genes were detected under microaerobic conditions, demonstrating that denitrification activity diminishes proportionally to oxygen concentration. An improved understanding of the denitrifier community in diverse microbiomes is necessary to mitigate excess N2O emissions from anthropogenic activities such as wetland drainage and crop production. IMPORTANCETerrestrial nitrogen limitations have historically reduced crop output. To fertilize crops, humans have developed synthetic N fixation methods, such as the Haber-Bosch process, which now account for half of global nitrogen uptake in biomass. The resulting nitrogen surplus in soils leads to eutrophication, harmful algal blooms in lakes and other aquatic ecosystems, and increased N2O emissions from denitrification via anaerobic respiration by soil bacteria. Considering that the nitrogen cycle is controlled by microbial communities, we need a better understanding of how nitrogen-cycling microbes control N runoff and N2O flux in each unique microbiome.