Search

The greenhouse gas nitrous oxide (N 2 O) has strong potential to drive climate change. Soils are a major source of N 2 O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC , nirK , norCBQD , and nosRZDYFLX genes, which sequentially reduce nitrate (NO 3 - ) to nitrite (NO 2 - ), nitric oxide (NO), N 2 O, and dinitrogen (N 2 ). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK 2 and NnrR transcriptional regulators. To understand the role of FixK 2 and NnrR proteins in N 2 O turnover, we monitored real-time kinetics of NO 3 - , NO 2 - , NO, N 2 O, N 2 , and oxygen (O 2 ) in a fixK 2 and nnrR mutant using a robotized incubation system. We confirmed that FixK 2 and NnrR are regulatory determinants essential for NO 3 - respiration and N 2 O reduction. Furthermore, we demonstrated that N 2 O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO 3 - , and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N 2 O turnover in B. diazoefficiens .

Legumes can contribute to emissions of the potent greenhouse gas nitrous oxide (N2O) directly by some rhizobia species that are able to denitrify under free-living conditions and in symbiotic association with the plant. In this study, the capacity of Phaseolus vulgaris-Rhizobium etli symbiosis to emit N2O in response to nitrate (NO3-) has been demonstrated for the first time. We found that bacteroidal assimilatory nitrate reductase (NarB) as well as nitrite reductase (NirK) and nitric oxide reductase (cNor) denitrifying enzymes contribute to nitric oxide (NO) and N2O formation in nodules. We also show that R. etli NarK is involved in NO2- extrusion and links NO3- reduction by NarB in the cytoplasm with NirK and cNor denitrification activities in the periplasm. The knowledge generated in this work will be instrumental for exploring strategies and sustainable practices in agricultural soil management to increase legume crop yield and mitigate greenhouse gas emissions. HIGHLIGHTS Common bean nodules are able to produce N2O in response to nitrate. Nitrate reduction to nitrite in R. etli bacteroids catalyzed by assimilatory nitrate reductase NarB is involved in N2O emissions from common bean nodules. R. etli respiratory nitrite reductase NirK and nitric oxide reductase cNor support N2O production in common bean nodules. R. etli NarB and cNor are involved in NO homeostasis in common bean nodules. R. etli nitrite exporter NarK links nitrate reduction in the cytoplasm with denitrification activities in the periplasm. [ABSTRACT FROM AUTHOR]

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c -type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD , and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ–FixK2–NnrR and RegSR–NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD , and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA , a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes. [ABSTRACT FROM AUTHOR]

Aims: To determine the effect of three N-fertilisers on N2O emission and abundance of nitrification and denitrification genes in bulk and rhizosphere soil of tomato and common bean, two vegetable crops representative of main horticultural crops in South Spain. Methods: Four consecutive harvests of tomato and common bean fertilised with urea, ammonium or nitrate were carried out. The total abundance of bacteria, archaea, nitrifiers and denitrifiers was estimated by quantitative PCR. Soil physicochemical properties and N2O emission were also determined. Results: Regardless of the plant species, the highest N2O emission was produced by the soil treated with urea, followed by ammonium and nitrate. Bacteria were more abundant than archaea in the bulk and rhizosphere soil. The abundance of the ammonia-oxidising archaea was greater than the ammonia-oxidising bacteria in the rhizosphere, but lower in the bulk soil. N-fertilisation increased the gene copy number of denitrifiers, which were more abundant in the bulk soil. Conclusions: N-fertilisation decreases N2O production due to increased abundance of the nosZ gene. The abundance of nitrification and denitrification genes in bulk and rhizosphere soils is dependent on the type of fertiliser. For both plant species, the ratio of the genes involved in production and reduction of N2O by bulk and rhizosphere was similar. [ABSTRACT FROM AUTHOR]

Irrigation and soil management can impact soil nitrous oxide (N2O) emissions. Flood and sprinkler irrigation systems together with conventional tillage are the main practices used in the high yielding maize systems in Mediterranean Spain. The objective of this field work was to quantify the effect of the irrigation system (i.e., flood, F; and sprinkler, S) and the soil tillage system (i.e., conventional tillage, CT; no‐tillage maintaining the maize stover, NTr; and no‐tillage removing the maize stover, NT) on the N2O emissions from the soil during 3 yr (2015, 2016, and 2017). S irrigation, with mean values of 1.35 kg N2O‐N ha−1 yr−1 throughout the 3 yr, obtained 42% lower N2O emissions than F irrigation. On average of the three growing seasons, yield‐scaled N2O emissions by grain yield and by grain N uptake in F irrigation were two‐fold higher than in S irrigation. Moreover, in one out of three growing seasons (2017), no‐tillage systems (i.e., NTr and NT) showed greater yield‐scaled N2O emissions compared with CT. The higher maize grain yield with the S irrigation compared to F irrigation, as well as the lower N2O emissions reported under S irrigation resulted in the reduction of the yield‐scaled N2O emissions. Our findings highlight the role of sprinkler irrigation decreasing N2O emissions in comparison to flood irrigation in Mediterranean agroecosystems. [ABSTRACT FROM AUTHOR]

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK2-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK2 and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC , nirK , norCBQD , and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens , we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c 550 soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc 1 complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA , nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK2 as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK2 and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c -staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation. [ABSTRACT FROM AUTHOR]

The nitrous oxide and molecular N emissions from 5-cm length subsamples taken from 20-cm length sample corers containing eutric Cambisol soil fertilised either with urea, ammonium or nitrate for 1 year have been examined using gas chromatography. At the beginning of the incubation, the same N rate (260 kg N/ha) was added to the soil and kept constant during the experiment. The total abundance of the soil Bacteria and Archaea and that of nitrifiers and denitrifiers was estimated by quantitative PCR of the corresponding biotic variables 16S rRNA, amoA and napA, narG, nirK, nirS, norB, nosZI and nosZII genes. The abiotic variables dissolved oxygen, pH, exchangeable NH4+-N and NO3−-N contents and total C and total N were also analysed. None of the three fertilisers affected the total abundance of Bacteria and Archaea and nitrification was the main driver of nitrous oxide production in the 0- to 5-cm and 5- to 10-cm soil layers while denitrification was in the 10- to 15-cm and 15- to 20-cm soil horizons. Parallel to the reduction in the content of dissolved oxygen along the soil profile, there was a decrease in the total and relative abundance of the bacterial and archaeal amoA gene and an increase in the abundances of the denitrification genes, mainly in the 10- to 15-cm and 15- to 20-cm soil layers. A non-metric multidimensional scaling plot comparing the biotic and abiotic variables examined in each of the four 5-cm soil subsamples and the whole 20-cm sample showed a disparate effect of N fertilisation on N gas emissions and abundance of nitrifiers and denitrifiers bacterial and archaeal communities. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants. [ABSTRACT FROM AUTHOR]

The alfalfa endosymbiont Ensifer meliloti strain1021 is known to be an incomplete denitrifier due to its inability to grow anoxically using nitrate as respiratory substrate to produce ATP and grow under anoxic conditions. Although this bacterium contains and expresses the complete set of denitrification genes napEFDABC, nirK, norECBQD and nosRZDFYLX encoding the periplasmic nitrate reductase (Nap), Cu-containing nitrite reductase (NirK), c-type nitric oxide (cNor) and nitrous oxide reductase (Nos), respectively, the reasons of its inability to grow under anoxic conditions are still very poorly understood. In the present study, we have constructed an E. meliloti strain overexpressing napEFDABC genes (Nap+) and demonstrated that this strain is able to grow through anaerobic nitrate respiration. Furthermore, Nap+ showed increased NapC levels as well as Nap, Nir and cNor activities and higher capacity to produce NO and N2O compared to wild-type cells. These results suggest that the inability of E. meliloti to grow under anaerobic conditions using nitrate as electron acceptor is attributable to a limitation in the expression of the periplasmic nitrate reductase. [ABSTRACT FROM AUTHOR]

Wet deposition of reactive nitrogen (Nr) species is considered a main factor contributing to N inputs, of which nitrate (NO3-) is usually the major component in high-mountain lakes. The microbial group of denitrifiers are largely responsible for reduction of nitrate to molecular dinitrogen (N2) in terrestrial and aquatic ecosystems, but the role of denitrification in removal of contaminant nitrates in high-mountain lakes is not well understood. We have used the oligotrophic, high-altitude La Caldera lake in the Sierra Nevada range (Spain) as a model to study the role of denitrification in nitrate removal. Dissolved inorganic Nr concentration in the water column of la Caldera, mainly nitrate, decreased over the ice-free season which was not associated with growth of microbial plankton or variations in the ultraviolet radiation. Denitrification activity, estimated as nitrous oxide (N2O) production, was measured in the water column and in sediments of the lake, and had maximal values in the month of August. Relative abundance of denitrifying bacteria in sediments was studied by quantitative polymerase chain reaction of the 16S rRNA and the two phylogenetically distinct clades nosZI and nosZII genes encoding nitrous oxide reductases. Diversity of denitrifiers in sediments was assessed using a culture-dependent approach and after the construction of clone libraries employing the nosZI gene as a molecular marker. In addition to genera Polymorphum, Paracoccus, Azospirillum, Pseudomonas, Hyphomicrobium, Thauera, and Methylophaga, which were present in the clone libraries, Arthrobacter, Burkholderia, and Rhizobium were also detected in culture media that were not found in the clone libraries. Analysis of biological activities involved in the C, N, P, and S cycles from sediments revealed that nitrate was not a limiting nutrient in the lake, allowed N2O production and determined denitrifiers' community structure. All these results indicate that denitrification could be a major biochemical process responsible for the N losses that occur in La Caldera lake. [ABSTRACT FROM AUTHOR]

The powerful greenhouse gas, nitrous oxide (N2O) has a strong potential to drive climate change. Soils are the major source of N2O and microbial nitrification and denitrification the main processes involved. The soybean endosymbiont Bradyrhizobium diazoefficiens is considered a model to study rhizobial denitrification, which depends on the napEDABC, nirK, norCBQD, and nosRZDYFLX genes. In this bacterium, the role of the regulatory cascade FixLJ-FixK2-NnrR in the expression of napEDABC, nirK, and norCBQD genes involved in N2O synthesis has been previously unraveled. However, much remains to be discovered regarding the regulation of the respiratory N2O reductase (N2OR), the key enzyme that mitigates N2O emissions. In this work, we have demonstrated that nosRZDYFLX genes constitute an operon which is transcribed from a major promoter located upstream of the nosR gene. Low oxygen was shown to be the main inducer of expression of nosRZDYFLX genes and N2OR activity, FixK2 being the regulatory protein involved in such control. Further, by using an in vitro transcription assay with purified FixK2 protein and B. diazoefficiens RNA polymerase we were able to show that the nosRZDYFLX genes are direct targets of FixK2. [ABSTRACT FROM AUTHOR]

In the last decades, intensive agriculture management and the application of nitrogen fertilizer have led to high nitrogen losses to the environment. Among these losses is the soil emission of the potent greenhouse gas nitrous oxide (N 2 O) which is mainly produced by the soil microbial processes of nitrification and denitrification. The use of nitrification inhibitors with ammonium based fertilizers can be an efficient mitigation strategy to decrease these nitrogen losses. Soil water content and temperature are major factors controlling N 2 O production and, so, also the efficiency of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) to reduce these emissions. In this work we study the effect of soil water content and temperature on the abundance of soil bacteria, ammonia-oxidizing bacteria and denitrifying bacteria, and on the performance of DMPP modulating these different bacterial populations responsible for N 2 O production/consumption. The effect of DMPP on the abundance of the different bacterial populations was dependent on soil water content. Under 40% of soil water filled pore space (WFPS), the application of DMPP caused an expected decrease of the target amoA gene abundance while narG , nirK and nosZ gene abundances also decreased probably due to an indirect effect induced by the lack of substrate for denitrifiers. Under 80% of WFPS, while the application of DMPP did not induce any change in the amoA gene abundance, an unexpected increase in non-target denitrifying gene abundances ( narG, nirK and nosZ ) was observed at either 10 °C or 20 °C incubation temperatures. This finding opens a new gate of research in the use of DMPP, where nitrous oxide reductase enzyme gene ( nosZ ) abundance and expression should be studied in the future in order to better understand the performance of DMPP not only inhibiting nitrification, but also affecting the non-target denitrification process. [ABSTRACT FROM AUTHOR]

Bacteria and fungi have been shown to produce nitrous oxide (N 2 O) during denitrification, but their contribution after nitrate (NO 3 −) application to soil is not clearly established. In a microcosm experiment, the relative contribution of bacteria and fungi to N 2 O and carbon dioxide (CO 2) production by four contrasting soils representing different land uses after KNO 3 addition was studied. The soils were daily wetted to 80% water-filled pore space (WFPS) and kept under greenhouse conditions for 10 days. The fungicide cycloheximide and the bactericide streptomycin were used to determine the possible microbial origin of the N 2 O and CO 2 emissions. Non-target effects of the antibiotics on the emission of N 2 O and CO 2 were evaluated using the inhibitor additivity ratio (IAR). The abundance of the bacterial and fungal communities was estimated by quantitative PCR (qPCR) of the bacterial 16S rRNA gene and the fungal internal transcribed spacer (ITS) region, respectively. The gene copy number of bacterial denitrifiers was calculated after quantification of the nirK , nirS , norB , nosZ I and nosZ II genes. After 10 d, regardless of the soil type, the cumulative N 2 O emission from the soils treated with cycloheximide or streptomycin were similar. In all the four soils, N 2 O fluxes were greater (on average 1.8 ± 0.3 times) in soils amended with the fungicide than with the bactericide during incubation for the first 48–96 h. Greater N 2 O emissions (on average 1.7 ± 0.2 times) were detected in soils where bacteria were inhibited in comparison to those treated with the fungicide from 96 to 240 h. On average, 68.5% of the total CO 2 emitted during the 10-d incubation period was produced in soils treated with the fungicide and 31.5% in those treated with the bactericide. The greater contribution of bacteria to the production of N 2 O than fungi during the first 48–96 h was possibly due to a faster used of nitrate. Variations in the abundance of bacterial 16S rRNA genes, the ITS region, and the nirK , nirS , norB and nosZ I bacterial denitrification genes indicated that the antibiotics used to prevent the growth of bacteria and fungi were effective during incubation. These results suggest that both bacteria and fungi should be considered when designing and applying greenhouse gas mitigation strategies in soils and that their relative contribution to produce N 2 O and CO 2 can vary with time and nitrate availability. • In a 10-d microcosm experiment, bacteria dominated fungi for N 2 O emissions during the first 3 d after N-fertilisation. • Fungi dominated bacteria for N 2 O emissions during the remaining 7-d of treatment. • After 10-d, bacteria and fungi similarly contributed to N 2 O production in the four soils analysed. • Bacteria were greater CO 2 producers than fungi after the 10-d treatment in the four soils analysed. • Bacteria and fungi should be considered when designing greenhouse gas mitigation strategies. [ABSTRACT FROM AUTHOR]

• Several Zn sources (with or without N) were tested on an irrigated maize crop under semi-arid climate. • Zn fertilization increased N 2 O and respiration fluxes in comparison to urea alone. • Zinc fertilization produced a detrimental effect on nitrifying and denitrifying communities. • Greater effect of Zn sources on NosZ communities compared to other denitrifying genes was found. • The zinc sulphate but not the synthetic chelate increased Zn concentration in grain. Previous studies have shown that the use of zinc (Zn) chelate fertilizers combined with a nitrogen (N) fertilizer (urea) can lead to both agronomic (i.e., yields and Zn and N biofortification due to the synergies between both nutrients) and environmental (i.e., by reducing the emissions of nitrous oxide, N 2 O, derived from N fertilization) benefits under rainfed semi-arid conditions. However, little is known about the effect of Zn-N co-fertilization on greenhouse gas (GHG) emissions or soil microbial processes involved in N 2 O fluxes under non-flooded irrigated conditions (during the dry season). Under these conditions, water-filled pore space continuously fluctuates following a periodic pattern and soil temperatures are in the optimum range for soil microorganisms. In this context, a field experiment was conducted using a maize (Zea mays L.) crop treated with two N levels (no N application and 120 kg N ha−1 as urea), and three Zn sources (no Zn application, Zn sulphate, and Zn applied with a mixture of chelating compounds, DTPA-HEDTA-EDTA). Nitrous oxide, methane (CH 4) and carbon dioxide (CO 2) fluxes were measured using opaque chambers, as well as the total abundances of soil bacteria, archaea and nitrifier and denitrifier communities. Zn-N co-fertilization increased cumulative N 2 O emissions from 0.36 kg N-N 2 O ha−1 (for urea combined with Zn chelates) to 0.76 kg N-N 2 O ha−1 (for urea combined with Zn sulphate), with respect to urea without Zn application. The N 2 O emission factors were lower (0.34%–0.72%) than the IPCC default value of 1%. Total abundances of the nosZ denitrification gene, which is involved in the reduction in N 2 O to dinitrogen (N 2), were reduced by 75% on average in the plots that received Zn fertilizers. This reduction may explain the higher N 2 O emissions in these treatments. In contrast with the case with non-irrigated crops, Zn-N co-fertilization cannot be recommended as a strategy to mitigate N 2 O emissions in irrigated maize under semi-arid conditions, despite of the enhancement of Zn availability in soil. [ABSTRACT FROM AUTHOR]

• Cu addition during growth influenced soybean- Bradyrhizobium diazoefficiens symbiosis. • 20 μM Cu2+ was the stress threshold for soybean plants. • The addition of Cu reduced significantly N 2 O emissions produced by soybean nodules. • Cu accumulation in nodules affected denitrifying enzymes activities of bacteroids. • Nitrate reductase of bacteroids is proposed as a target for mitigation strategies. Agriculture is an important source of the greenhouse gas nitrous oxide (N 2 O) due to the over- or non-synchronised application of nitrogen to crops. Symbiotic nitrogen fixation (SNF) by legume-rhizobia symbiosis can be an effective strategy for N 2 O mitigation, but several environmental factors such as copper (Cu) availability might affect N 2 O emissions derived from legume crops. The aim of this research was to study how Cu can modulate N 2 O emissions by soybean root nodules. Soybean plants inoculated with Bradyrhizobium diazoefficiens USDA110 were grown in the presence of 4 mM KNO 3 and a battery of Cu2+ concentrations added during growth (0, 5, 10, 20, 40, 60 or 100 μM). N 2 O emissions were measured by gas chromatography in both, nodulated roots and detached nodules, after root flooding. Also, 15N isotope dilution was assayed for SNF and N 2 O determinations. Results showed that an excess of added Cu during growth significantly affected plant physiology, nodulation and SNF of the soybean- B.diazoefficiens symbiosis, being 20 μM the threshold that soybean plants can tolerate without suffering Cu stress. Meanwhile, Cu addition reduced statistically N 2 O emissions by soybean nodules. This reduction was correlated with Cu accumulation in nodules, which affected the denitrifying enzymes activities. Cu excess produced a simultaneous decrease of nitrate and nitrite reductase activities, but an increase of nitrous oxide reductase activity. Finally, the modulation of bacteroidal nitrate reductase activity is proposed as an effective target for the strategies for mitigation of N 2 O emissions derived from soybean crops, probably more effective than nitrous oxide reductase activity. [ABSTRACT FROM AUTHOR]

Nitrification and denitrification are known to co-occur in soils, but the effect of fertilisation history on N 2 O fluxes and the relative source partitioning of the N 2 O has not been thoroughly investigated. In this study, we therefore combined a high-tech 15N stable isotope tracing technique with quantitative PCR (qPCR) to explore the relative contributions of nitrification and denitrification to N 2 O production by a sandy-loam Eutric Cambisol soil treated repeatedly with ammonium sulfate [(NH 4) 2 SO 4 ] or potassium nitrate (KNO 3) for 3 years prior. Both soils (historically (NH 4) 2 SO 4 and historically KNO 3 treated) were amended separately with (15NH 4) 2 SO 4 and K15NO 3 and incubated at 80% water filled pore space for 30 days. Soil N 2 O emissions, NH 4 + and NO 3 − concentrations and their corresponding 15N-enrichments were determined. The effect of N addition on N transformation rates was also calculated. The total abundance of nitrifiers was estimated by qPCR of the amoA gene from bacteria and archaea, and that of denitrifiers by using the nirK , nirS , norB and nosZ I genes as molecular targets. In the historically (NH 4) 2 SO 4 -treated soil, 49.0–58.0% of the N 2 O emitted originated from nitrification and 42.0–51.0% from denitrification during incubation. The production of N 2 O was accompanied by a decrease in soil NH 4 + concentrations and a parallel increase in the concentration of soil NO 3 −. In addition, the abundance of the bacterial and archaeal amoA gene increased during the incubation. Conversely, in the soil historically treated with KNO 3 , the 15N isotopic analyses showed that denitrification contributed 84.0–99.0% of the total N 2 O produced. Decreases in soil NO 3 − concentrations paralleled the increase in 15N enrichment of N 2 O and the abundance of the nirK , nirS , norB and nosZ I genes. The results also showed that values of 15N 2 enrichment were significantly higher in the KNO 3 -treated soil, which is in line with the higher abundance of the nosZ I gene. Calculation of the N transformation rates indicated that autotrophic nitrification and denitrification were responsible for N 2 O production in the historically (NH 4) 2 SO 4 -treated soil and that denitrification was the most important N 2 O source in the soil treated with KNO 3. We conclude that N-fertilisation history, and not simply soil oxygen availability, affect the relative contributions of nitrification and denitrification to soil N 2 O emissions. Indeed, here we have shown that nitrification can be an important N 2 O source process even in soils maintained at high moisture contents. • There exist simultaneous nitrification and denitrification in suboxic N-fertilised soils. • Rates of 15N 2 O enrichment from each process were similar in NH 4 + treated soils. • Denitrification exceeded nitrification when NO 3 − was the applied fertiliser. • In that case, the highest 15N 2 O enrichment was due to 15NO 3 −, not to 15NH 4 +. • The targeted nitrification and denitrification genes increased during soil incubation. [ABSTRACT FROM AUTHOR]

• Urea-treated soils emitted more N 2 O than those amended with ammonium and then nitrate. • Nitrification occurs at moisture contents around 80% WFPS. • The ratio between the genes involved in N 2 O production and reduction decreased on a yearly basis. • Nitrate fertiliser increases the abundance of the N 2 O reducers over the N 2 O producers. • N-fertilisation reduces bacterial richness and alters the bacterial community composition. In a 3-year microcosm study the nitrous oxide (N 2 O) emission from an agricultural soil amended with urea, ammonium sulphate or potassium nitrate were examined. Soil samples were taken every year to determine the soil physicochemical properties, the total abundance of bacteria, archaea, nitrifier and denitrifier communities and the changes in the structure and composition of the bacterial community. Gene abundance and biodiversity were estimated using quantitative PCR and pyrosequencing, respectively. The soils were watered weekly to reach 80% water filled pore space and varied from 80 to 62% during that time. N-fertilisation increased the abundance of bacteria and decreased that of archaea. Soils treated with ammonium or urea emitted more N 2 O than that amended with potassium nitrate and produced yearly increases in the abundance of nitrification genes. Also, the abundance of the denitrification genes gradually increased during the experimental period. N 2 O emission decreased on a yearly basis and so did the ratio of the genes involved in N 2 O production and reduction. A non-metric multidimensional scaling plot showed that N 2 O emission was positively related with the norB gene and negatively with the nosZ. The Shannon and Simpson diversity indices indicated that N-fertilisation reduced the number of operational taxonomic units (OTUs) and that the bacterial community became dominated by a small group of OTUs, respectively. N-fertilisation reduced the number of OTUs whose relative abundance was lower than 1%. We conclude that potassium nitrate reduces N 2 O emissions and increases the abundance of the N 2 O reducers. This work also shows that N-fertilisation decreases soil biodiversity and that its response depends on the type of the N-fertiliser. [ABSTRACT FROM AUTHOR]