Feasibility and limitations of using specific nitrification inhibitors to differentiate ammonia oxidizer activity

Abstract

The application of nitrification inhibitors targeting distinct ammonia-oxidizing guilds—complete ammonia oxidizers (comammox), ammonia-oxidizing archaea (AOA), and bacteria (AOB)—has facilitated elucidating their functional significance across diverse ecosystems. However, the specificity and reliability of these inhibitors remain controversial and have not been thoroughly evaluated. In this study, we first conducted a meta-analysis of several specific nitrification inhibitors: acetylene, 1-octyne, 3,4-dimethylpyrazole phosphate (DMPP), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide (PTIO), and chlorate, focusing on their effects on AOA, AOB and comammox Nitrospira. Acetylene broadly inhibited the growth of all ammonia oxidizers. Both 1-octyne and higher concentrations (≥1.5%) of DMPP exhibited dual regulatory effects: inhibiting the growth of AOB and comammox Nitrospira (clade B for 1-octyne; all clades for DMPP) while stimulating AOA growth. PTIO inhibited AOA and AOB but had no significant effect on comammox Nitrospira clade A growth. In contrast, chlorate specifically inhibited comammox Nitrospira growth. To assess the ecological relevance of these synthesized patterns, particularly the guild-specific effects, we conducted microcosm experiments. The results revealed that PTIO failed to inhibit AOA growth in wetland soil. The specificity of 1-octyne and chlorate is dose-dependent. Notably, chlorate at 10 mM completely inhibited the growth of all ammonia oxidizers, while its addition consistently stimulated soil nitrous oxide (N₂O) emissions, indicating it cannot reliably differentiate the contribution of different nitrifier guilds to N₂O emissions. These findings collectively challenge the conventional use of inhibitor-based assays, underscoring the necessity to evaluate the effectiveness of inhibitors in distinguishing the functional importance of ammonia oxidizers.