Revisiting the involvement of ammonia oxidizers and denitrifiers in nitrous oxide emission from cropland soils

Nitrous oxide (N₂O), an ozone-depleting greenhouse gas, is generally produced by soil microbes, particularly NH₃ oxidizers and denitrifiers, and emitted in large quantities after N fertilizer application in croplands. N₂O can be produced via multiple processes, and reduced, with the involvement of more diverse microbes with different physiological constraints than previously thought; therefore, there is a lack of consensus on the production processes and microbes involved under different agricultural practices. In this study, multiple approaches were applied, including N₂O isotopocule analyses, microbial gene transcript measurements, and selective inhibition assays, to revisit the involvement of NH₃ oxidizers and denitrifiers, including the previously-overlooked taxa, in N₂O emission from a cropland, and address the biological and environmental factors controlling the N₂O production processes. Then, we synthesized the results from those approaches and revealed that the overlooked denitrifying bacteria and fungi were more involved in N₂O production than the long-studied ones. We also demonstrated that the N₂O production processes and soil microbes involved were different based on fertilization practices (plowing or surface application) and fertilization types (manure or urea). In particular, we identified the following intensified activities: (1) N₂O production by overlooked denitrifying fungi after manure fertilization onto soil surface; (2) N₂O production by overlooked denitrifying bacteria and N₂O reduction by long-studied N₂O-reducing bacteria after manure fertilization into the plowed layer; and (3) N₂O production by NH₃-oxidizing bacteria and overlooked denitrifying bacteria and fungi when urea fertilization was applied into the plowed layer. We finally propose the conceptual scheme of N flow after fertilization based on distinct physiological constraints among the diverse NH₃ oxidizers and denitrifiers, which will help us understand the environmental context-dependent N₂O emission processes.