Quantitative evaluation of inhibitory effect of various substances on anaerobic ammonia oxidation (anammox) Tomotaka Nakamura, 1 Yuhki Harigaya, 1 Yuya Kimura, 2 Megumi Kuroiwa, 1 Yuhri Kurata, 1 Kazuichi Isaka, 2 and Yuichi Suwa 1 , * Department of Biological Sciences, Chuo University,1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan 1 and Matsudo Research Center, Infrastructure Systems Company, Hitachi Ltd., Kami-Hongo, Matsudo, Chiba 271-0064, Japan 2 Received 31 January 2017; accepted 14 April 2017 Available online xxx 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 15 N tracer technique. The initial anammox rate was determined during first 25 min with a small-scale anaerobic batch incubation supplemented with possible inhibitors. Although Cu 2D and Mn 2D did not inhibit anammox, the remaining 18 substances [Ni 2D , Zn 2D , Co 2D , MoO 4 2L , Fe 2D , 4 amines, ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N,N 0 - 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 (R 2 > 0.91) either with a linear inhibition model or with a MichaeliseMenten-based inhibition model. IC 50 values were estimated based on either model, and were compared. The IC 50 values of the 4 chelators (0.06e2.7 mM) and 5 metal ions (0.02e1.09 mM) were significantly lower than those of the 4 amines (10.6e29.1 mM) and 5 organic solvents (3.5e82 mM). Although it did not show any inhibition within 25 min, 0.1 mM Cu 2D completely inhibited anammox activity in 240 min, suggesting that the inhibitory effect caused by Cu 2D is time-dependent. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Anaerobic ammonia oxidation; 15 N-tracer; IC 50 ; Inhibition models; Amines; Metals; Organic solvents; Chelators] Anaerobic ammonia oxidation (anammox) is an anoxic reaction carried out by chemolithoautotrophic bacteria, in which the elec- tron donor is ammonium and the electron acceptor is nitrite (1e4). The anammox process has been considered a novel cost-effective wastewater treatment process (5) because it requires neither external electron donor nor aeration, which are major expenses in conventional nitrificationedenitrification processes. It has been applied to various wastewater sources, including various industrial wastewaters (6e8), landfill leachates, sludge concentrates, semi- conductor effluents, and livestock wastewaters, among others. When applying the anammox process to treat various types of wastewaters, it is necessary to consider the influence of substances coexisting with NH 4 þ on anammox activity. Wastewater may oc- casionally contain substances that could adversely affect to wastewater treatment performance. For example, it has been re- ported that heavy metals are inhibitory to anammox, and, in fact, Cu 2þ , Zn 2þ and other heavy metals have been detected in the effluent of an anaerobic digester, in anaerobic piggery wastewater, and in slurries from the dairy products industry (9,10); similarly, Cu 2þ has been detected in industrial wastewater from the semi- conductor industry (11,12). The adverse effects of heavy metals have been previously studied (8,13e20). Various metal species are necessary nutrients for anammox microorganisms. Copper and molybdenum are key cofactors in nitrite reductase (21) and nitrite oxidoreductase (22), which are responsible for anammox catabolism, and nickel and zinc are required for hydrogenase and dehydrogenase reactions, respectively (23). Thus, while some metal species are necessary nutrients for anammox microorganisms, they may also inhibit anammox activity when present in excess. The effects of other substances, such as monoethanolamine (MEA) (24), flocculants, allylthiourea, chloramphenicol (25), and anions (chloride, acetate, phosphate and sulfide), on the anammox process have also been examined (2,25). Information on the inhibitory effects of various substances on the anammox process has obtained recently, but systematic and quantitative data for a variety of chemical sub- stances are still lacking. In previous studies, inhibitory effects have been examined by monitoring the nitrogen removal performance of a bench- or pilot- scale anammox reactor, or by using a batch test where the rates of overall substrate consumption, N 2 gas emission, or increases in headspace gas pressure were determined as an index of anammox activity (26). These procedures are all indirect, in terms of * Corresponding author. Tel.: þ81 3 3817 7132; fax: þ81 3 3817 7102. E-mail address: y-suwa@bio.chuo-u.ac.jp (Y. Suwa). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2017 1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.04.010 Please cite this article in press as: Nakamura, T., et al., Quantitative evaluation of inhibitory effect of various substances on anaerobic ammonia oxidation (anammox), J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.04.010