ABSTRACT

Nitrification is the rate-limiting step of N cycling. It is performed in two successive steps by ammonia oxidizing bacteria (ΑΟΒ) or archaea (ΑΟΑ) (oxidation of ΝΗ3 to ΝΟ2-), and nitrite oxidizing bacteria (ΝΟΒ) (oxidation of NO2- to NO3-). 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 demonstrates 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 (i) participating in the modular (NOB) or the non-modular (Comammox) nitrification process or (ii) modulating downstream microbial processes in N cycling like denitrification. Furthermore, little information is available regarding the impact of NI on the diversity of AOA and AOB. 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. This project aims to provide a holistic assessment, at in vitro and in soil level, of the inhibitory effects of EQ, QI and EQNL on nitrification. This will be achieved via (i) the in vitro assessment of the effects of EQ, its derivatives and other known NI on the growth (q-PCR) and the activity (RT-q-PCR, NH3, NO2-, NO3-) of selected terrestrial strains of AOA/AOB and NOB, and (ii) the in situ study of the impact of all these compounds  on the abundance (q-PCR) and activity (RT-q-PCR, NH3, NO3-) of ΑΟΑ/ΑΟΒ and Comammox, and on the diversity of AOA/AOB (using high throughput amplicon sequencing approaches), in soil microcosms under various operating conditions (pH, temperature). In addition, the impact of all NI on NOB and denitrifying bacteria will be assessed using advanced molecular tools. The dissipation and metabolism of the investigated NI in soil will be also determined to facilitate results interpretation and enable correlations between effects and persistence. Our findings are expected to advance our understanding of the interaction of the microbial groups participating in N cycling and NI and might potentially result in the development of a novel, universal NI attractive for industrial exploitation.