Using BONCAT-FACS to probe the active soil microbial community during nitrous oxide production

Nitrous oxide (N ₂ O) is a potent greenhouse gas and is largely produced by incomplete denitrification. Although we know many of the microbial species that denitrify, we are still unable to reliably predict N ₂ O production from soils. Recent work in microbial ecology has shown that when key microbes are considered as members of functional ensembles rather than isolated, the predictive power linking their activity to emergent properties increases dramatically. We hypothesized that the active microbial community during high N ₂ O production would be taxonomically distinct from the inactive portion and increases in N₂O production rates would correlate more strongly with increased abundance across multiple active taxa than with dominance by a single active species. We conducted a microcosm experiment where agricultural soil was incubated in anaerobic vials for up to 15 hours while tracking N ₂ O production. Using bioorthogonal non-canonical amino acid tagging paired with fluorescence-activated cell sorting and 16S rRNA amplicon sequencing (BONCAT-FACS-Seq), we probed the active subset of the microbial community throughout the incubation period. Analysis of 16S rRNA gene amplicons revealed that the active and inactive fractions contained distinct taxa, and the taxonomic composition of the active fraction shifted over time. We found that less than 1% of the microbial community was responsible for N ₂ O flux rates as high as 3.84 µg N ₂ O-N g dry soil-1 hr-1. The level of activity (median fluorescent intensity of active cells) correlated well with N ₂ O production rates. The Ensemble Quotient Optimization for Microbiomes (mEQO) tool was used to identify an ensemble of eight organisms whose combined abundance best correlated with N₂O fluxes. Overall, our results reveal that N₂O fluxes are driven not by changes in a single taxon but by shifting ensembles of active microorganisms whose combined functional potential supports consistent emissions. This study applied a novel conceptual and methodological framework with a distinct focus on the active microbial community, rather than the entire community; if our observation that N ₂ O flux rates are correlated with an ensemble of organisms is broadly confirmed, then framing denitrification as a community trait may increase predictability of this key process.