Impacts of substrate properties and aquatic nutrient concentrations on the relative abundance of nitrifying/denitrifying genes and the associated microbes in epilithic biofilms.

Substrates like sand or gravels and aquatic nutrient concentrations of rivers are highly heterogeneous, influencing the abundance of functional genes in epilithic biofilms where nitrification-denitrification processes take place. To analyze how the relative abundance of nitrifying/denitrifying genes and the associated microbes changes with the physical properties of substrates and aquatic concentrations of nutrients, this paper utilized metagenomics to comprehensively characterize these functional genes (i.e., amoA, hao, and nxrB involved in nitrification, and napA, narG, nirS, norB, and nosZ associated with denitrification) from epilithic biofilms collected along the Shitingjiang River in Southwest China and further obtained the relative abundance of major nitrifiers and denitrifiers. The results show that substrate size most significantly affects the relative abundance of hao and norB by altering the hydrodynamic conditions. In sampling sites with high heterogeneity in substrate size distribution, the relative abundance of most denitrifying genes is also higher. The carbon-nitrogen ratio negatively correlates with the relative abundance of all the nitrifying genes, while ammonium, total inorganic carbon, and total organic carbon concentrations positively affect the relative abundance of amoA and nxrB. As to the relative abundance of nitrifiers and denitrifiers, mainly belonging to phyla Proteobacteria and Actinobacteria, substrate heterogeneity and the aquatic concentrations of nutrients have greater influences than substrate size. Also, the substrate heterogeneity exerted positive influence on functional species of Pseudogemmobacter bohemicus and Paracoccus zhejiangensis. Considering the genes' functions and the dominant species linked to denitrification, nitrous oxide is more likely to occur in rivers with higher heterogeneity and larger substrates.