Off-target effects of biological nitrification inhibitors on soil microbial substrate use and enzyme activity in an agricultural soil

High nitrification rates and substantial nitrogen (N) losses through nitrate leaching and N₂O emissions make current agricultural practices unsustainable, contributing to greenhouse gas emissions and environmental pollution. Synthetic nitrification inhibitors (SNIs) can be amended with N-fertilizers to reduce the conversion of ammonia to nitrate by soil nitrifiers. SNIs aim to increase agricultural nitrogen use efficiency (NUE), but they have several disadvantages (e.g., costs, ineffectiveness in the field, possible accumulation in the food chain). The use of biological nitrification inhibitors (BNIs), naturally occurring in plant root exudates, could become an alternative to SNIs. Potential BNIs should be highly specifically targeting nitrification, but for most known BNIs it is unclear if and how they affect other soil microorganisms and biogeochemical processes.

This study aimed to investigate possible off-target effects of BNIs in agricultural soils. We tested the effect of two candidate BNIs (Methyl 3-(4-hydroxyphenyl)propionate and DL-limonene) in slurry assays on soil microbial communities from a typical Austrian agricultural field (Linz, pH 6.89±0.12, fertilized with 120 kg N ha^-1 yr^-1), and compared them to a known SNI (nitrapyrin), and two further nitrification inhibitors (phenylacetylene and octyne). The slurries were incubated for eight days and CO₂ production, pH, as well as nitrate- and N₂O accumulation were measured. At the end of the incubation, we analyzed fluorescence-based enzyme activity, as well as microbial substrate use efficiency using ‘Biolog©’ assays to test the influence on general microbial activity, selected microbial soil processes, and the effectiveness of nitrification inhibition, respectively.

Our results showed that both tested BNIs significantly reduced net nitrification rates, but also affected other biogeochemical processes, even though limonene lost some effectiveness during the incubation. MHPP was heavily respired by heterotrophic microorganisms, leading to a drop in pH and heterotrophic competition for the remaining ammonium, therefore likely acting as an indirect nitrification inhibitor. Extracellular enzymes were also affected: MHPP led to increased potential β-glucosidase activity, while nitrapyrin led to a decrease in potential phosphatase activity. General soil microbial substrate use diversity seemed to be unaffected by the input of either BNIs or SNIs. Whether or not the observed off-target effects are positive and what they mean for the large-scale application of BNIs in the agricultural industry remains to be further investigated.