Optimizing carbon sources for detailed denitrification studies in soil bacterial communities: supporting diverse denitrifiers while limiting competing processes
Studies examining how denitrification and nitrous oxide (N2O) emissions respond to environmental factors are commonly performed in soil microcosms to improve experimental control relative to field conditions. To achieve even better control of the conditions experienced by individual cells, bacteria can first be extracted from the soil and incubated in a liquid medium. Such systems require an exogenous carbon (C) source to stimulate measurable microbial activity, and the choice of substrate is critical. It should support a diverse denitrifying community representative of unsaturated soils while minimizing competing anaerobic pathways (e.g., fermentation and DNRA) to ensure ecologically meaningful results with minimized interference from parallel processes. 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. Using high-resolution denitrification gas kinetics, metagenomic sequencing, and 15N isotope labelling, we sought to identify C sources that (i) support a diverse soil-derived denitrifying community and (ii) limit the competition for C from alternative anaerobic pathways. Among the tested substrates, only the clover extract supported denitrification while maintaining a complex denitrifying community. However, it also promoted fermentation and DNRA, revealing a trade-off must exist between fostering denitrifier diversity and limiting competing anaerobic processes in controlled incubations. More broadly, our results highlight that the C source is a methodological fulcrum in controlled microbiome studies, shaping community composition and determining which metabolic processes dominate. This work provides a foundation for designing ecologically relevant experiments to better understand, predict, and mitigate N2O emissions from denitrification.
Studies examining how denitrification and nitrous oxide (N2O) emissions respond to environmental factors are commonly performed in soil microcosms to improve experimental control relative to field conditions. To achieve even better control of the conditions experienced by individual cells, bacteria can first be extracted from the soil and incubated in a liquid medium. Such systems require an exogenous carbon (C) source to stimulate measurable microbial activity, and the choice of substrate is critical. It should support a diverse denitrifying community representative of unsaturated soils while minimizing competing anaerobic pathways (e.g., fermentation and DNRA) to ensure ecologically meaningful results with minimized interference from parallel processes. 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. Using high-resolution denitrification gas kinetics, metagenomic sequencing, and 15N isotope labelling, we sought to identify C sources that (i) support a diverse soil-derived denitrifying community and (ii) limit the competition for C from alternative anaerobic pathways. Among the tested substrates, only the clover extract supported denitrification while maintaining a complex denitrifying community. However, it also promoted fermentation and DNRA, revealing a trade-off must exist between fostering denitrifier diversity and limiting competing anaerobic processes in controlled incubations. More broadly, our results highlight that the C source is a methodological fulcrum in controlled microbiome studies, shaping community composition and determining which metabolic processes dominate. This work provides a foundation for designing ecologically relevant experiments to better understand, predict, and mitigate N2O emissions from denitrification.