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

Abstract 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. Studies on denitrification 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 and isolation of diverse denitrifying organisms and for bioaugmentation-based N2O-mitigation strategies. Here, we compared 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 isotope 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. Among the tested substrates, only the clover extract supported denitrification and maintained a complex denitrifying community. Yet, it also promoted fermentation and DNRA, revealing that a trade-off must exist between fostering denitrifier diversity and limiting growth of organisms using competing anaerobic pathways. More broadly, our results highlight that C source is a methodological fulcrum in controlled soil microbiome studies. It shapes community composition, drives metabolic processes, and ultimately determines the ecological relevance of experimental outcomes and the success of enrichments and soil bioaugmentation approaches. Competing Interest Statement The authors have declared no competing interest.