Enhancing agroecosystem nitrogen management: microbial insights for improved nitrification inhibition.

Nitrification is a key microbial process in the nitrogen (N) cycle that converts ammonia to nitrate. Excessive nitrification, typically occurring in agroecosystems, has negative environmental impacts, including eutrophication and greenhouse gas emissions. Nitrification inhibitors (NIs) are widely used to manage N in agricultural systems by reducing nitrification rates and improving N use efficiency. However, the effectiveness of NIs can vary depending on the soil conditions, which, in turn, affect the microbial community and the balance between different functional groups of nitrifying microorganisms. Understanding the mechanisms underlying the effectiveness of NIs, and how this is affected by the soil microbial communities or abiotic factors, is crucial for promoting sustainable fertilizer practices. Therefore, this review examines the different types of NIs and how abiotic parameters can influence the nitrifying community, and, therefore, the efficacy of NIs. By discussing the latest research in this field, we provide insights that could facilitate the development of more targeted, efficient, or complementary NIs that improve the application of NIs for sustainable management practices in agroecosystems.

Drug discovery-based approach identifies new nitrification inhibitors.

Nitrogen (N) fertilization is crucial to sustain global food security, but fertilizer N production is energy-demanding and subsequent environmental N losses contribute to biodiversity loss and climate change. N losses can be mitigated be interfering with microbial nitrification, and therefore the use of nitrification inhibitors in enhanced efficiency fertilizers (EEFs) is an important N management strategy to increase N use efficiency and reduce N pollution. However, currently applied nitrification inhibitors have limitations and do not target all nitrifying microorganisms. Here, to identify broad-spectrum nitrification inhibitors, we adopted a drug discovery-based approach and screened 45,400 small molecules on different groups of nitrifying microorganisms. Although a high number of potential nitrification inhibitors were identified, none of them targeted all nitrifier groups. Moreover, a high number of new nitrification inhibitors were shown to be highly effective in culture but did not reduce ammonia consumption in soil. One archaea-targeting inhibitor was not only effective in soil, but even reduced - when co-applied with a bacteria-targeting inhibitor - ammonium consumption and greenhouse gas emissions beyond what is achieved with currently applied nitrification inhibitors. This advocates for combining different types of nitrification inhibitors in EEFs to optimize N management practices and make agriculture more sustainable.

Evaluation of a crop rotation with biological inhibition potential to avoid N2O emissions in comparison with synthetic nitrification inhibition.

Agriculture has increased the release of reactive nitrogen to the environment due to crops' low nitrogen-use efficiency (NUE) after the application of nitrogen-fertilisers. Practices like the use of stabilized-fertilisers with nitrification inhibitors such as DMPP (3,4-dimethylpyrazole phosphate) have been adopted to reduce nitrogen losses. Otherwise, cover crops can be used in crop-rotation-strategies to reduce soil nitrogen pollution and benefit the following culture. Sorghum (Sorghum bicolor) could be a good candidate as it is drought tolerant and its culture can reduce nitrogen losses derived from nitrification because it exudates biological nitrification inhibitors (BNIs). This work aimed to evaluate the effect of fallow-wheat and sorghum cover crop-wheat rotations on N2O emissions and the grain yield of winter wheat crop. In addition, the suitability of DMPP addition was also analyzed. The use of sorghum as a cover crop might not be a suitable option to mitigate nitrogen losses in the subsequent crop. Although sorghum-wheat rotation was able to reduce 22% the abundance of amoA, it presented an increment of 77% in cumulative N2O emissions compared to fallow-wheat rotation, which was probably related to a greater abundance of heterotrophic-denitrification genes. On the other hand, the application of DMPP avoided the growth of ammonia-oxidizing bacteria and maintained the N2O emissions at the levels of unfertilized-soils in both rotations. As a conclusion, the use of DMPP would be recommendable regardless of the rotation since it maintains NH4+ in the soil for longer and mitigates the impact of the crop residues on nitrogen soil dynamics.

Impact of dimethylpyrazole-based nitrification inhibitors on soil-borne bacteria.

Nitrogen (N) input from fertilizers modifies the properties of agricultural soils as well as bacterial community diversity, composition and relationships. This can lead to negative impacts such as the deterioration of system multifunctionality, whose maintenance is critical to normal nutrient cycling. Synthetic nitrification inhibitors (NIs) can be combined with fertilizers to improve the efficiency of N use by reducing N losses. However, analysis of their effects on non-target bacteria are scarce. This study aimed to analyze the effect of applying the NIs DMPP and DMPSA on the whole bacterial community. Through 16S rRNA amplicon sequencing we determined the differences between samples in terms of microbial diversity, composition and co-occurrence networks. The application of DMPP and DMPSA exerted little impact on the abundance of the dominant phyla. Nevertheless, several significant shifts were detected in bacterial diversity, co-occurrence networks, and the abundance of particular taxa, where soil water content played a key role. For instance, the application of NIs intensified the negative impact of N fertilization on bacterial diversity under high water-filled pore spaces (WFPS) (>64%), reducing community diversity, whereas alpha-diversity was not affected at low WFPS (<55%). Interestingly, despite NIs are known to inhibit ammonia monooxygenase (AMO) enzyme, both NIs almost exclusively inhibited Nitrosomonas genera among AMO holding nitrifiers. Thus, Nitrosomonas showed abundance reductions of up to 47% (DMPP) and 66% (DMPSA). Nonetheless, non-target bacterial abundances also shifted with NI application. Notably, DMPSA application partially alleviated the negative effect of fertilization on soil multifunctionality. A remarkable increase in populations related to system multifunctionality, such as Armatimonadetes (up to +21%), Cyanobacteria (up to +30%) and Fibrobacteres (up to +25%) was observed when DMPSA was applied. NI application substantially influenced microbial associations by decreasing the complexity of co-occurrence networks, decreasing the total edges and node connectivity, and increasing path distances.

Mitigation of yield-scaled nitrous oxide emissions and global warming potential in an oilseed rape crop through N source management.

Enhanced-efficiency nitrogen (N) fertilizers, such as those containing nitrification or urease inhibitors, can mitigate the carbon (C) footprint linked to the production of bioenergy crops through a reduction in direct nitrous oxide (N2O) emissions and indirect N2O losses. These indirect emissions are derived from ammonia (NH3) volatilization, which also have important environmental and health implications. The evaluation of the global warming potential (GWP) of different N sources using site-specific data of yield and direct and indirect emissions is needed for oilseed rape under rainfed semi-arid conditions, especially when meteorological variability is taken into account. Using urea as a N source, the N2O mitigation efficacy of the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) alone or combined with the nitrification inhibitor 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) was evaluated under field conditions in a rainfed oilseed rape (Brassica napus L.) crop. Two additional N sources from calcium ammonium nitrate (CAN), with and without DMPSA, were included. The GWP of the treatments was estimated considering the emissions from inputs, operations and other direct and indirect emissions of greenhouse gases (GHGs), such as methane (CH4) and the volatilization of NH3. We also measured the abundance of key genes involved in nitrification and denitrification to improve the understanding of N2O emissions on a biochemical basis under the conditions of our study. The results show that due to the intense rainfall after fertilization and a rewetting event, N2O losses from fertilizers without inhibitors were greater than those previously reported under Mediterranean conditions, while NH3 losses were low and not affected by the urease inhibitor. The cumulative N2O emissions (which were greatly influenced by a rewetting peak three months after fertilization) from the urea fertilization were significantly higher than those from CAN. The presence of NBPT significantly reduced N2O losses by an average of 71%, with respect to urea. The use of DMPSA with CAN resulted in an abatement of N2O emissions (by 57%) and a significant increase in oil yield in comparison with CAN alone. All inhibitor-based treatments were effective in abating N2O emissions during the rewetting peak. The abundances of the nitrifier and denitrifier communities, especially ammonia-oxidizing bacteria (AOB), significantly decreased relative to the urea or CAN treatments as inhibitors were applied. Under the conditions of our study, the sustainability of a bioenergy crop such as oilseed rape can be improved by using inhibitors because they mitigated N2O emissions and/or enhanced the oil yield.

Mechanism of action of nitrification inhibitors based on dimethylpyrazole: A matter of chelation.

In agriculture, the applied nitrogen (N) can be lost in the environment in different forms because of microbial transformations. It is of special concern the nitrate (NO3-) leaching and the nitrous oxide (N2O) emissions, due to their negative environmental impacts. Nitrification inhibitors (NIs) based on dimethylpyrazole (DMP) are applied worldwide in order to reduce N losses. These compounds delay ammonium (NH4+) oxidation by inhibiting ammonia-oxidizing bacteria (AOB) growth. However, their mechanism of action has not been demonstrated, which represent an important lack of knowledge to use them correctly. In this work, through chemical and biological analysis, we unveil the mechanism of action of the commonly applied 3,4-dimethyl-1H-pyrazole dihydrogen phosphate (DMPP) and the new DMP-based NI, 2-(3,4-dimethyl-1H-pyrazol-1-yl)-succinic acid (DMPSA). Our results show that DMP and DMPSA form complexes with copper (Cu2+) cations, an indispensable cofactor in the nitrification pathway. Three coordination compounds namely [Cu(DMP)4Cl2] (CuDMP1), [Cu(DMP)4SO4]n (CuDMP2) and [Cu(DMPSA)2]·H2O (CuDMPSA) have been synthesized and chemical and structurally characterized. The CuDMPSA complex is more stable than those containing DMP ligands; however, both NIs show the same nitrification inhibition efficiency in soils with different Cu contents, suggesting that the active specie in both cases is DMP. Our soil experiment reveals that the usual application dose is enough to inhibit nitrification within the range of Cu and Zn contents present in agricultural soils, although their effects vary depending on the content of these elements. As a result of AOB inhibition by these NIs, N2O-reducing bacteria seem to be beneficed in Cu-limited soils due to a reduction in the competence. This opens up the possibility to induce N2O reduction to N2 through Cu fertilization. On the other hand, when fertilizing with micronutrients such as Cu and Zn, the use of NIs could be beneficial to counteract the increase of nitrification derived from their application.

Relationship between tillage management and DMPSA nitrification inhibitor efficiency.

Agricultural sustainability is compromised by nitrogen (N) losses caused by soil microbial activity. Nitrous oxide (N2O) is a potent greenhouse gas (GHG) produced as consequence of nitrification and denitrification processes in soils. Nitrification inhibitors (NI) as 3,4-dimethylpyrazole-succinic acid (DMPSA) are useful tools to reduce these N losses from fertilization. The objective of this work was to test the efficiency of DMPSA in two different tillage management systems, conventional tillage (CT) and no-tillage (NT), in a winter wheat crop under Humid Mediterranean conditions. N fertilizer was applied as ammonium sulphate (AS) with or without DMPSA in a single or split application, including an unfertilized treatment. GHG fluxes (N2O, CO2 and CH4) were measured by the closed chamber method. amoA and nosZI genes were quantified by qPCR as indicators of nitrifying and denitrifying populations. Nitrification was inhibited by DMPSA in both CT and NT, while the higher water filled pore space (WFPS) in NT promoted a better efficiency of DMPSA in this system. This higher efficiency might be due to a greater N2O reduction to N2 as result of the nosZI gene induction. Consequently, DMPSA was able to reduce N2O emissions down to the unfertilized levels in NT. Provided that NT reduced CO2 emissions and maintained crop yield compared to CT, the application DMPSA under NT management is a promising strategy to increase agro-systems sustainability under Humid Mediterranean conditions.