The role of riverbed substrates in N2O and CH4 emission: Insights from metagenomic analysis of epilithic biofilms

Riverbed substrates are critical in N₂O and CH₄ emission with functional microbes adhering to them. However, the role of substrates remains to be fully understood. This study monitors N₂O and CH₄ emission and collects epilithic biofilms on riverbed substrates with various diameters and size heterogeneity from 10 sections along a mountain river. Compared with the global range, moderate water-air exchange rates of N₂O (−2.34–29.2 μg/m²/h) and rapid CH₄ emission (−2.58–35.2 mg/m²/h) are observed. Based on metagenomic analysis, the abundances of nirS and fmdA genes, which encode catalysts in the denitrification and the hydrogenotrophic methanogenesis process, are found to be significantly higher in the medium diameter group (2–100 mm), implying higher N₂O and CH₄ emission. Meanwhile, the abundance of nirS and nirK genes, which are key to N₂O production, is significantly higher in the low size heterogeneity group, promoting N₂O release. In contrast, the abundance of ftr, pta,ackA and ACS genes critical in the methanogenesis processes are significantly lower in the low size heterogeneity group, inhibiting CH₄ emission. For N₂O production, the nitrification process is found to be dominated by species of Nocardioides and Planctomycetales, denitrification process by species of Tabrizicola and Rhodobacteraceae, and dissimilatory nitrate reduction to ammonium process by Leptospiraceae species. In contrast, CH₄ is mainly generated by species of Pirellula and Proteobacteria through hydrogenotrophic, acetoclastic and methylotrophic methanogenesis respectively. A structural equation model indicated that substrate physical properties are equally or even more important as/than the aquatic nutrients concentration for N₂O or CH₄ emission in mountain rivers.