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Microbial nitrogen (N) transformations in soil, notably denitrification, result in the production of the potent greenhouse and ozone depleting gas nitrous oxide (N2O). Soil chemistry and microbiome composition impact N2O emission potential but the relative importance of these factors as determinants of N2O emission in denitrifying systems is rarely tested. In addition, previous linkages between microbiome composition and N2O emission potential rarely demonstrate causality. Here, we determined the relative impact of microbiome composition (i.e. soil extracted cells) and chemistry (i.e. water extractable chemicals) on N2O emission potential utilizing an anoxic cell based assay system. Cells and chemistry for assays were sourced from soils with contrasting N2O/N2O+N2ratios, combined in various combinations and denitrification gas production was measured in response to nitrate addition. Average directionless effects of cell and chemical extract on N2O/N2O+N2(Cell: Δ0.16, Chemical extract: Δ0.22) and total N2O hypothetically emitted (Cell: Δ2.62 μmol-N, Chemical extract: Δ4.14 μmol-N) indicated chemistry is the most important determinant of N2O emissions. Independent pH differences of just 0.6 points impacted N2O/N2O+N2on par with independent chemical extract differences, supporting the dominance of this variable in previous studies. However, impacts on overall N2O hypothetically emitted were smaller suggesting that soil pH manipulation may not necessarily be a successful approach to mitigate emissions over a fixed time period. In addition, we observed increased N2O accumulation and emission potential at the end of incubations concomitant with predicted decreases in carbon availability suggesting that carbon limitation increases N2O emission transiently with the magnitude of emission dependent on the both chemical and microbiome controls.

Inoculating agricultural soils with N2O-respiring bacteria (NRB) can reduce N2O-emissions, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB's, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB's were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

Soil denitrification produces the potent greenhouse gas nitrous oxide (N2O) and by further reduction of N2O, the harmless inert gas N2. N2O emission is determined by rate and timing of the N2O producing and reducing steps which are sensitive to a series of proximal and distal regulators such as pH and microbial community composition. Microbial community associations to N2O emission potential (N2O/(N2O+N2)) are commonly entangled with pH leaving the true role of community composition unclear. Here, we leverage a set of soil microbiomes strongly linked to rainfall above pH to test the hypothesis that microbiome vs. N2O emission potential (N2O/(N2O+N2)) correlations will be maintained across alternative distal drivers. N2O emission potential (N2O/(N2O+N2)) and denitrification gas (NO, N2O, N2) kinetics were assessed by automated gas chromatography while community composition was assessed by 16S rRNA gene sequencing and qPCR of nosZI and II genes. Analyses revealed a sustained correlation between microbiome and N2O emission potential (N2O/(N2O+N2)) in the absence of a pH effect. Further, a continuum of gas accumulation phenotypes linked to NO accumulation and sensitive to carbon addition are identified. Separate phenotypes carried out N2O production and reduction steps more concurrently or sequentially and thus determined N2O accumulation and emission potential (N2O/(N2O+N2)). Concurrent N2O producing/reducing soils typically contained NO accumulation to a low steady state, while carbon addition manipulations which increased NO accumulation also increased sequentiality of N2O production/reduction and thus emission potential (N2O/(N2O+N2)). These features may indicate a conserved NO inhibitory mechanism across multiple effectors (rainfall, community composition, carbon availability).HighlightsN2O emission potential is linked to microbiome changes associated with rainfall, but not to pH.Sequential vs. concurrent denitrification phenotypes differing in NO and N2O accumulation are identified.High N2O accumulation is associated with increased NO accumulation.Sequentiality of N2O production/reduction determines soil N2O emission potential.Sequentiality of N2O reduction was susceptible to manipulation via carbon addition.