Quantitative evaluation of inhibitory effect of various substances on anaerobic ammonia oxidation (anammox)

The inhibitory effect of 20 substances of various chemical species on the anaerobic ammonia oxidation (anammox) activity of an enrichment culture, predominated by Candidatus Brocadia, was determined systematically by using a ¹⁵N tracer technique. The initial anammox rate was determined during first 25 min with a small-scale anaerobic batch incubation supplemented with possible inhibitors. Although Cu²⁺ and Mn²⁺ did not inhibit anammox, the remaining 18 substances [Ni²⁺, Zn²⁺, Co²⁺, [Formula: see text] , Fe²⁺, 4 amines, ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N,N'-bis (2-hydroxyphenylacetic acid) (EDDHA), citric acid, nitrilotriacetic acid (NTA), N,N-dimethylacetamide (DMA), 1,4-dioxane, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and tetrahydrofuran (THF)] were inhibitory. Inhibitory effect of NTA, EDDHA, THF, DMF, DMA and amines on anammox was first determined in this study. Inhibitory effects of metals were re-evaluated because chelators, which may interfere inhibitory effect, have been used to dissolve metal salts into assay solution. The relative anammox activities as a function of concentration of each substance were described successfully (R² > 0.91) either with a linear inhibition model or with a Michaelis-Menten-based inhibition model. IC₅₀ values were estimated based on either model, and were compared. The IC₅₀ values of the 4 chelators (0.06-2.7 mM) and 5 metal ions (0.02-1.09 mM) were significantly lower than those of the 4 amines (10.6-29.1 mM) and 5 organic solvents (3.5-82 mM). Although it did not show any inhibition within 25 min, 0.1 mM Cu²⁺ completely inhibited anammox activity in 240 min, suggesting that the inhibitory effect caused by Cu²⁺ is time-dependent.