Optimizing Carbon Sources to Promote Soil Denitrifiers: Lessons for Incubations, Enrichments, and Bioaugmentation

Oxygen concentrations fluctuate in soil across time and space. Under anoxic conditions, the three main microbial metabolic pathways - denitrification, fermentation, and DNRA - compete for the same carbon (C) sources. Denitrification studies in complex soil communities often rely on incubation experiments to determine how various factors affect the regulatory biology of denitrifying organisms and their N2O emissions. These experiments typically require an exogenous C source to stimulate measurable activity, and the choice of C source is critical as it should support denitrification while minimizing competition from fermentation and DNRA. This consideration is equally important for the enrichment/isolation of diverse denitrifying organisms and bioaugmentation strategies. Here, we compare twelve C sources, including glutamic acid, acetate, an artificial root exudate cocktail (eight compounds, individually and in combination), and a clover extract. By combining high-resolution denitrification gas kinetics, metagenomic sequencing, and 15N-labelling, we aimed to find a C source(s) that (1) supports a diverse soil-derived denitrifying community and (2) limits the competition for C from alternative anaerobic pathways.