N-cycling and nitrification inhibitors: In vitro and in soil assessment of their spectrum of activity and mode of action

Nitrification is the rate-limiting step of N cycling. It is performed in two successive steps by ammonia oxidizing bacteria (ΑΟΒ) or archaea (ΑΟΑ), and nitrite oxidizing bacteria (ΝΟΒ). Recently, bacteria of the genus Nitrospira sp. (formerly known as ΝΟΒ)  were found to be able to perform the complete oxidation of ammonia to nitrate, and they were named Comammox (Complete-ammonia-oxidation). Nitrification could have undesirable environmental effects leading to the production of NO3 which could contaminate groundwater and air (via the process of denitrification). Nitrification inhibitors (NI) are used as supplements in slow-release fertilizers (nitrapyrin and DCD) to reduce the rate of nitrification and hence to improve N use efficiency and reduce N losses (ΝΟ3-, ΝxO). The majority of currently available NI demonstrate a selective activity towards ΑΟΒ, while their impact on AOA is either limited or unknown. In addition, we know nothing about the effects of NI on other functional microbial groups of N cycling and on the non-target soil microbiota. Ethoxyquin (EQ), an antioxidant commonly used in fruit packaging plants, and its main oxidative derivatives quinone imine (QI) (major metabolite) and 2,4-dimethyl-6-ethoxyquinoline (EQNL) (minor metabolite) showed a clear inhibitory effect on nitrification appearing as a temporary and equivalent inhibition of both AOA and AOB. Our main focus is to develop novel NIs like EQ and its derivative, evaluate their spectrum of activity against target microbial groups comparatively to established NIs, and identify their biochemical mode of action through proteomic approaches.  In this frame our lab in collaboration with Dr G. Nicol (Ecole Central del Lyon) has developed in vitro assays with selected AOB and AOA strains and is currently investigating the effects of novel NIs in soil and at the biochemical level.

Figure Nitrification Inhibitors







Call of ELIDEK for the support of postdoctoral fellows, Title: Looking up for novel nitrification inhibitors: New stories with old compounds. Funding Body: Hellenic Foundation for Research and Innovation (ELIDEK), Greece. Total Funding 200000  €. Duration: 2018-2020





  1. Papadopoulou E.S., Tsachidou P., Sulowic S., Menkissoglu-Spiroudi U., Karpouzas D.G., (2016) Land spreading of wastewaters from the fruit packaging industry and potential effects on soil microbes: Effects of the antioxidant ethoxyquin and its metabolites on ammonia oxidizers. Applied and Environmental Microbiology 82: 747-755
  2. Vasileiadis S., Puglisi E., Papadopoulou E.S., Pertile G., Suciu N., Papolla, A., Tourna M., Karas P.A., Papadimitriou F., Kasiotakis A, Ipsilanti N., Ferrarini A., Sulowic S., Fornasier F., Nicol G.W, Trevisan M., Karpouzas D.G (2018) Blame it on the metabolite: 3,5-dichloraniline rather than the parent compound iprodione induces strong effects on the diversity and function of soil microorganisms. Applied and Environmental Microbiology 84(22): e01536-18


- Dr Evangelia Papadopoulou

- Eleftheria Bachtsevani, MSc student