Nitrification inhibitors impose distinct effects on comammox bacteria and canonical ammonia oxidizers under high N fertilization regimes

The recent discovery of complete ammonia oxidizing (comammox) bacteria expanded the diversity of nitrifiers and raised fundamental questions regarding their relative contribution to nitrification in agricultural soils. One such open question remains their response to N inputs and nitrification inhibitors (NIs). We employed two microcosm experiments, in a soil where both clade A and clade B comammox Nitrospira were present, to (i) identify the type (ammonium sulphate vs. urea) and the level of N fertilization (0 to 500 mg N kg-1 dwt soil) that is conducive to the growth of comammox bacteria, and subsequently (ii) to assess the impact of three commercial NIs (DCD, nitrapyrin (NP), DMPP) and a new potent NI (ethoxyquin (EQ)) on the function, diversity and dynamics of comammox bacteria comparatively to the canonical ammonia-oxidizing microorganisms (AOM). The highest concentration of ammonium sulphate induced a more consistent increase of comammox Nitrospira compared to AOA and AOB which showed a dose-dependent increase by both N types. DCD and DMPP were more effective than NP and EQ in inhibiting nitrification, an effect mostly driven by their selective inhibitory activity on AOB that functionally prevailed in the studied soil. Interestingly, the inhibition of AOB by DCD and DMPP was complemented by proliferation of comammox Nitrospira and AOA, suggesting that competition with AOB is a key factor restraining comammox bacteria and AOA in N-fertilized agricultural soils. NIs induced significant structural changes on the AOB (except for EQ) and comammox communities, but not on AOA. We noted a clade-dependent response of comammox bacteria to NIs; clade B was neriched by DCD and DMPP but was depleted by NP and EQ, whereas clade A showed a weak but complementary response to DCD, DMPP and NP. Our findings provide pioneering evidence that (i) releasing comammox Nitrospira and AOA from the competition of AOB allows them to proliferate efficiently, even under high inorganic ammonium conditions and NI presence; (ii) comammox Nitrospira clade B are more responsive than clade A to high inorganic N input and NI treatments.