First microscale data on depth profiles of microbial N₂O reduction, O2 availability, and pore networks inside contrasting single soil aggregates

A major greenhouse gas, nitrous oxide (N 2 O) significantly emitted from agricultural soils, is reduced to innocuous N 2 gas by the activity of two groups of N 2 O-reducing microbes (typical clade I and more recently discovered atypical clade II) having different enzymatic efficiency. Yet, basic information such as the locations of N₂O reduction hotspots and soil factors regulating their formations is still lacking. In addition, oxygen availability, which is strongly constrained by soil pore property, likely dictates their ecology in soil as N 2 O reductase enzyme (coded by nosZ genes) is inhibited by O 2 . Accordingly, the aim of this study was to assess the mechanistic linkage among soil pore networks, chemical microenvironments (pH, Eh, and O 2 and N 2 O abundances), ecology of N₂O-reducing microbes, and the occurrence of N₂O reduction hotspots in single soil aggregates. Using water-stable macroaggregates from two contrasting soil types (highly porous Andosol and less porous clay-rich Acrisol), we determined microscale depth profiles of N 2 O and O 2 dynamics, three-dimensional pore properties, and the two N 2 O reducer populations in the single aggregates after 48-hour lab incubation under a water-saturated condition. The N 2 O and O 2 depth profiles showed the increase in N 2 O production with O 2 depletion towards deeper part of the incubated aggregates, indicating denitrification N 2 O production especially in Andosol aggregate where O 2 availability was higher. The gene distribution with depth clearly showed higher abundance of nosZ harboring microbes (including both clades I and II) in the Acrisol aggregate than Andosol aggregate especially towards the aggregate interior. In the Acrisol aggregate, the abundance of nosZ clade I harboring microbes was maximum at the middle depth corresponding to N 2 O maxima, whereas the nosZ clade II harboring microbes had slightly different niche as their population monotonically increased towards the aggregate core, which were consistent with theoretical O 2 availability and pore connectivity. The current findings underscore the intimate connection between soil physical complexity and microbial ecology, which merits further investigation. • Distribution of N 2 O-reducing microbes varies within a single soil aggregate. • Microbial distribution correlates with O 2 /N 2 O dynamics regulated by pore properties. • The pore property strongly influences the microbial N 2 O reduction potential in soil.