nosZ Clade Research Articles

Short- and long-term temperature responses of soil denitrifier net N<sub>2</sub>O efflux rates, inter-profile N<sub>2</sub>O dynamics, and microbial genetic potentials

Abstract. Production and reduction of nitrous oxide (N2O) by soil denitrifiers influence atmospheric concentrations of this potent greenhouse gas. Accurate projections of the net N2O flux have three key uncertainties: (1) short- vs. long-term responses to warming, (2) interactions among soil horizons, and (3) temperature responses of different steps in the denitrification pathway. We addressed these uncertainties by sampling soil from a boreal forest climate transect encompassing a 5.2 ∘C difference in the mean annual temperature and incubating the soil horizons in isolation and together at three ecologically relevant temperatures in conditions that promote denitrification. Both short-term exposure to warmer temperatures and long-term exposure to a warmer climate increased N2O emissions from organic and mineral soils; an isotopic tracer suggested that an increase in N2O production was more important than a decline in N2O reduction. Short-term warming promoted the reduction of organic horizon-derived N2O by mineral soil when these horizons were incubated together. The abundance of nirS (a precursor gene for N2O production) was not sensitive to temperature, whereas that of nosZ clade I (a gene for N2O reduction) decreased with short-term warming in both horizons and was higher from a warmer climate. These results suggest a decoupling of gene abundance and process rates in these soils that differs across horizons and timescales. In spite of these variations, our results suggest a consistent, positive response of denitrifier-mediated net N2O efflux rates to temperature across timescales in these boreal forests. Our work also highlights the importance of understanding cross-horizon N2O fluxes for developing a predictive understanding of net N2O efflux from soils.

Shifting abundances of communities associated with nitrogen cycling in soils promoted by sugarcane harvest systems

Sugarcane cultivation supports Brazil as one of the largest world sugar and ethanol producer. In order to understand the impact of changing sugarcane harvest from manual to mechanized harvest, we studied the effect of machinery traffic on soil and consequently soil compaction upon soil microbial communities involved in nitrogen cycling. The impact of sugarcane harvest was dependent on soil depth and texture. At deeper soil layers, mechanized harvesting increases the abundance of nitrogen fixers and denitrifying communities (specifically nosZ clade I and II) while manual harvesting increases the abundance of ammonia oxidizers (specifically AOA) and increases denitrifying communities (nosZ clade I and II) on top and at intermediate depth. The effect of change on the harvest system is more evident on sandy soil than on clay soil, where soil indicators of compaction (bulk density and penetration resistance) were negatively correlated with soil microorganisms associated with the nitrogen cycle. Our results point to connections between soil compaction and N transformations in sugarcane fields, besides naming biological variables to be used as proxies for alterations in soil structure.

Dinitrogen (N2) pulse emissions during freeze-thaw cycles from montane grassland soil

Short-lived pulses of soil nitrous oxide (N2O) emissions during freeze-thaw periods can dominate annual cumulative N2O fluxes from temperate managed and natural soils. However, the effects of freeze thaw cycles (FTCs) on dinitrogen (N2) emissions, i.e., the dominant terminal product of the denitrification process, and ratios of N2/N2O emissions have remained largely unknown because methodological difficulties were so far hampering detailed studies. Here, we quantified both N2 and N2O emissions of montane grassland soils exposed to three subsequent FTCs under two different soil moisture levels (40 and 80% WFPS) and under manure addition at 80% WFPS. In addition, we also quantified abundance and expression of functional genes involved in nitrification and denitrification to better understand microbial drivers of gaseous N losses. Our study shows that each freeze thaw cycle was associated with pulse emissions of both N2O and N2, with soil N2 emissions exceeding N2O emissions by a factor of 5–30. Increasing soil moisture from 40 to 80% WFPS and addition of cow slurry increased the cumulative FTC N2 emissions by 102% and 77%, respectively. For N2O, increasing soil moisture from 40 to 80% WFPS and addition of slurry increased the cumulative emissions by 147% and 42%, respectively. Denitrification gene cnorB and nosZ clade I transcript levels showed high explanatory power for N2O and N2 emissions, thereby reflecting both N gas flux dynamics due to FTC and effects of different water availability and fertilizer addition. In agreement with several other studies for various ecosystems, we show here for mountainous grassland soils that pulse emissions of N2O were observed during freeze-thaw. More importantly, this study shows that the freeze-thaw N2 pulse emissions strongly exceeded those of N2O in magnitude, which indicates that N2 emissions during FTCs could represent an important N loss pathway within the grassland N mass balances. However, their actual significance needs to be assessed under field conditions using intact plant-soil systems.

Wheat rhizodeposition stimulates soil nitrous oxide emission and denitrifiers harboring the nosZ clade I gene

Stimulatory effects of growing plants on nitrous oxide (N2O) emissions have been widely reported in terrestrial ecosystems, but the potential mechanisms responsible for these effects remain unclear. This study revealed that wheat can induce a 3.5–9.2-fold increase in N2O emissions under different soil fertility levels, and that this “plant” source of N2O occurs in the rhizosphere. Moreover, plants induced soil niche differentiation between denitrifiers harboring the nitrous oxide reductase genes nosZI and nosZII. Pulse labeling of wheat demonstrated that 67% of 13C-labeled nosZI-type denitrifiers, but no nosZII-type denitrifiers, were more abundant in the rhizosphere than in bulk soil. Furthermore, a higher percentage of bacterial genomes containing nitrite reductase genes was found within plant-associated nosZI-type denitrifiers than nosZII-type denitrifiers, favoring NO2− to N2O conversion. Overall, this study revealed a strong selective stimulating effect of wheat on soil denitrifiers through root-derived carbon and a key role of the nosZI-type community in rhizosphere denitrification.

Lucerne (Medicago sativa) alters N2O-reducing communities associated with cocksfoot (Dactylis glomerata) roots and promotes N2O production in intercropping in a greenhouse experiment

Lower emissions of the greenhouse gas nitrous oxide (N2O) are generally observed from intercropped compared to sole cropped systems. This could be due to better N-use efficiency, but differences in microbial communities establishing in the rhizosphere may also play a role as the only known biological sink for N2O is its reduction to nitrogen gas (N2) by bacteria and archaea that possess the nosZ gene encoding the N2O reductase. Nitrous oxide reducing communities can be divided into two clades, I and II, and their relative abundance and diversity may have important consequences for N2O emissions. Here, we examine how intercropping with a legume (Medicago sativa, “lucerne”) and a grass (Dactylis glomerata, “cocksfoot”) species, compared to sole cropping of each species, affects the N2O emission potential, and the structure and abundance of root-associated N2O-reducing microbial communities. In a rhizobox experiment, we show that intercropping resulted in higher total shoot biomass compared to sole cropping. Further, N2O production rates were significantly higher in intercropped cocksfoot roots compared to sole cropping of either species. This coincided with lower abundances of nosZ clade II communities in intercropped compared to sole cropped cocksfoot roots, suggesting that these organisms likely act as a N2O sink. Phylogenetic placement of sequencing reads placed root-associated nosZ clade II reads close to Ignavibacteria and Opitutaceae, which harbour non-denitrifying N2O reducers with the genetic capacity to also perform dissimilatory nitrate reduction to ammonium (DNRA). We observed a shift in the composition of the cocksfoot root-associated nosZI communities towards incomplete denitrifiers terminating with N2O in intercropped roots. Overall, we hypothesize that such alterations of plant-microbe and/or microbe-microbe interactions contributed to the higher potential N2O emission rate observed in intercropped cocksfoot roots. Understanding the nature of these interactions would represent an important step forward for the design of management practices that minimize N2O emissions.

Conversion of monoculture cropland and open grassland to agroforestry alters the abundance of soil bacteria, fungi and soil-N-cycling genes.

Integration of trees in agroforestry systems can increase the system sustainability compared to monocultures. The resulting increase in system complexity is likely to affect soil-N cycling by altering soil microbial community structure and functions. Our study aimed to assess the abundance of genes encoding enzymes involved in soil-N cycling in paired monoculture and agroforestry cropland in a Phaeozem soil, and paired open grassland and agroforestry grassland in Histosol and Anthrosol soils. The soil fungi-to-bacteria ratio was greater in the tree row than in the crop or grass rows of the monoculture cropland and open grassland in all soil types, possibly due to increased input of tree residues and the absence of tillage in the Phaeozem (cropland) soil. In the Phaeozem (cropland) soil, gene abundances of amoA indicated a niche differentiation between archaeal and bacterial ammonia oxidizers that distinctly separated the influence of the tree row from the crop row and monoculture system. Abundances of nitrate (napA and narG), nitrite (nirK and nirS) and nitrous oxide reductase genes (nosZ clade I) were largely influenced by soil type rather than management system. The soil types’ effects were associated with their differences in soil organic C, total N and pH. Our findings show that in temperate regions, conversion of monoculture cropland and open grassland to agroforestry systems can alter the abundance of soil bacteria and fungi and soil-N-cycling genes, particularly genes involved in ammonium oxidation.

Catch Crop Residues Stimulate N2O Emissions During Spring, Without Affecting the Genetic Potential for Nitrite and N2O Reduction.

Agricultural soils are a significant source of anthropogenic nitrous oxide (N2O) emissions, because of fertilizer application and decomposition of crop residues. We studied interactions between nitrogen (N) amendments and soil conditions in a 2-year field experiment with or without catch crop incorporation before seeding of spring barley, and with or without application of N in the form of digested liquid manure or mineral N fertilizer. Weather conditions, soil inorganic N dynamics, and N2O emissions were monitored during spring, and soil samples were analyzed for abundances of nitrite reduction (nirK and nirS) and N2O reduction genes (nosZ clade I and II), and structure of nitrite- and N2O-reducing communities. Fertilization significantly enhanced soil mineral N accumulation compared to treatments with catch crop residues as the only N source. Nitrous oxide emissions, in contrast, were stimulated in rotations with catch crop residue incorporation, probably as a result of concurrent net N mineralization, and O2 depletion associated with residue degradation in organic hotspots. Emissions of N2O from digested manure were low in both years, while emissions from mineral N fertilizer were nearly absent in the first year, but comparable to emissions from catch crop residues in the second year with higher precipitation and delayed plant N uptake. Higher gene abundances, as well as shifts in community structure, were also observed in the second year, which were significantly correlated to availability. Both the size and structure of the nitrite- and N2O-reducing communities correlated to the difference in N2O emissions between years, while there were no consistent effects of management as represented by catch crops or fertilization. It is concluded that N2O emissions were constrained by environmental, rather than the genetic potential for nitrite and N2O reduction.

The link between nitrous oxide emissions, microbial community profile and function from three full-scale WWTPs

Few attempts have been made in previous studies to link the microbial community structure and function with nitrous oxide (N2O) emissions at full-scale wastewater treatment plants (WWTPs). In this work, high-throughput sequencing and reverse transcriptase-qPCR (RT-qPCR) was applied to activated sludge samples from three WWTPs for two seasonal periods (winter and summer) and linked with the N2O emissions and wastewater characteristics. The total N2O emissions ranged from 7.2 to 937.0 g N-N2O/day, which corresponds to an emission factor of 0.001 to 0.280% of the influent NH4-N being emitted as N2O. Those emissions were related to the abundance of Nitrotoga, Candidatus Microthrix and Rhodobacter genera, which were favored by higher dissolved oxygen (DO) and nitrate (NO3−) concentrations in the activated sludge tanks. Furthermore, a relationship between the nirK gene expression and N2O emissions was verified. Detected N2O emission peaks were associated with different process events, related to aeration transition periods, that occurred during the regular operation of the plants, which could be potentially associated to increased emissions of the WWTP. The design of mitigation strategies, such as optimizing the aeration regime, is therefore important to avoid process events that lead to those N2O emissions peaks. Furthermore, this study also demonstrates the importance of assessing the gene expression of nosZ clade II, since its high abundance in WWTPs could be an important key to reduce the N2O emissions.

The Abundance of Fungi, Bacteria and Denitrification Genes during Insect Outbreaks in Scots Pine Forests

Outbreaks of defoliating insects may affect microbial populations in forests and thereby mass balances and ecosystem functioning. Here, we investigated the microbial dynamics in Scots pine (Pinus sylvestris L.) forests during outbreaks of the nun moth (Lymantria monacha L.) and the pine-tree lappet (Dendrolimus pini L.). We used real-time PCR (polymerase chain reaction) to quantify genes that characterize bacterial and fungal abundance and the denitrification processes (nirK, nirS, nosZ clades I and II) in different forest compartments and we analyzed the C and N content of pine needles, insect feces, larvae, vegetation layers, organic layers, and mineral soil horizons. The infestation of the nun moth increased the bacterial abundance on pine needles, in the vegetation layer, and in the upper organic layer, while fungal populations were increased in the vegetation layer and upper organic layer during both outbreaks. In soil, the abundance of nirK increased after insect defoliation, while the C/N ratios decreased. nosZ clades I and II showed variable responses in different soil layers and to different defoliating insects. Our results illustrate changes in the microbial populations in pine forests that were infested by defoliating insects and changes in the chemical soil properties that foster these populations, indicating a genetic potential for increased soil N2O emissions during the defoliation peak of insect outbreak events.

Immobilization of Azospira sp. strain I13 by gel entrapment for mitigation of N2O from biological wastewater treatment plants: Biokinetic characterization and modeling

Development of a strategy to mitigate nitrous oxide (N2O) emitted from biological sources is important in the nexus of wastewater treatment and greenhouse gas emission. To this end, immobilization of N2O-reducing bacteria as a biofilm has the potential to ameliorate oxygen (O2) inhibition of the metabolic activity of the bacteria. We demonstrated the effectiveness of calcium alginate gel entrapment of the nosZ clade II type N2O-reducing bacterium, Azospira sp. strain I13, in reducing levels of N2O, irrespective of the presence of O2. Azospira sp. strain I13cells in the gel exhibited N2O reduction up to a maximum dissolved oxygen concentration of 100μM in the bulk liquid. The maximum apparent N2O uptake rate, [Formula: see text] , by gel immobilization did not appreciably decrease, retaining 72% of the N2O reduction rate of the cell suspension of Azospira sp. strain I13. Whereas gel immobilization increased the apparent half-saturation constant for N2O, [Formula: see text] , and the apparent O2 inhibition constant, [Formula: see text] , representing the degree of O2 resistance, correspondingly increased. A mechanistic model introducing diffusion and the reactions of N2O consumption was used to describe the experimental observations. Incorporating Thieles modulus into the model determined an appropriate gel size to achieve N2O reduction even under aerobic conditions.

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2O respiring bacteria

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by μmax /Ks , in organisms with nosZ clade II.

Differences in microbial community structure and nitrogen cycling in natural and drained tropical peatland soils

Tropical peatlands, which play a crucial role in the maintenance of different ecosystem services, are increasingly drained for agriculture, forestry, peat extraction and human settlement purposes. The present study investigated the differences between natural and drained sites of a tropical peatland in the community structure of soil bacteria and archaea and their potential to perform nitrogen transformation processes. The results indicate significant dissimilarities in the structure of soil bacterial and archaeal communities as well as nirK, nirS, nosZ, nifH and archaeal amoA gene-possessing microbial communities. The reduced denitrification and N2-fixing potential was detected in the drained tropical peatland soil. In undisturbed peatland soil, the N2O emission was primarily related to nirS-type denitrifiers and dissimilatory nitrate reduction to ammonium, while the conversion of N2O to N2 was controlled by microbes possessing nosZ clade I genes. The denitrifying microbial community of the drained site differed significantly from the natural site community. The main reducers of N2O were microbes harbouring nosZ clade II genes in the drained site. Additionally, the importance of DNRA process as one of the controlling mechanisms of N2O fluxes in the natural peatlands of the tropics revealed from the results of the study.

Life on N2O: deciphering the ecophysiology of N2O respiring bacterial communities in a continuous culture

Reduction of the greenhouse gas N2O to N2 is a trait among denitrifying and non-denitrifying microorganisms having an N2O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N2O emissions from ecosystems. To increase our understanding of the ecophysiology of N2O reducers, we determined the thermodynamic growth efficiency of N2O reduction and the selection of N2O reducers under N2O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N2O as electron acceptor and acetate as electron donor. The biomass yields were higher during N2O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N2O. Denitrifiers were favored over non-denitrifying N2O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N2O, as defined by μmax/Ks. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N2O respiration under growth-limited conditions.

Resolving N2 O respiration pathways: a tale of two NosZ clades.

Resolving N2 O respiration pathways: a tale of two NosZ clades.

Peaks of in situ N2 O emissions are influenced by N2 O-producing and reducing microbial communities across arable soils.

Agriculture is the main source of terrestrial N2 O emissions, a potent greenhouse gas and the main cause of ozone depletion. The reduction of N2 O into N2 by microorganisms carrying the nitrous oxide reductase gene (nosZ) is the only known biological process eliminating this greenhouse gas. Recent studies showed that a previously unknown clade of N2 O-reducers (nosZII) was related to the potential capacity of the soil to act as a N2 O sink. However, little is known about how this group responds to different agricultural practices. Here, we investigated how N2 O-producers and N2 O-reducers were affected by agricultural practices across a range of cropping systems in order to evaluate the consequences for N2 O emissions. The abundance of both ammonia-oxidizers and denitrifiers was quantified by real-time qPCR, and the diversity of nosZ clades was determined by 454 pyrosequencing. Denitrification and nitrification potential activities as well as insitu N2 O emissions were also assessed. Overall, greatest differences in microbial activity, diversity, and abundance were observed between sites rather than between agricultural practices at each site. To better understand the contribution of abiotic and biotic factors to the insitu N2 O emissions, we subdivided more than 59,000 field measurements into fractions from low to high rates. We found that the low N2 O emission rates were mainly explained by variation in soil properties (up to 59%), while the high rates were explained by variation in abundance and diversity of microbial communities (up to 68%). Notably, the diversity of the nosZII clade but not of the nosZI clade was important to explain the variation of insitu N2 O emissions. Altogether, these results lay the foundation for a better understanding of the response of N2 O-reducing bacteria to agricultural practices and how it may ultimately affect N2 O emissions.

Spatial and phyloecological analyses of nosZ genes underscore niche differentiation amongst terrestrial N2O reducing communities

The only known biological sink for the greenhouse gas nitrous oxide (N2O) is its reduction to nitrogen gas (N2) by bacteria and archaea that possess the nosZ gene conferring this trait. Phylogenetic and genomic evidence indicates that N2O reducing communities can be divided into two genetically distinct groups, termed clade I and II. Differing abundance or diversity of each clade may have significant consequences for N2O emissions from soil. However, whether the two groups respond similarly to gradients of environmental or biotic factors in soils remains unclear. Here, we explore spatial patterns of nosZ clade I and II abundance, phylogenetic diversity and community structure across a 44-ha farm, and compare with edaphic factors and abundances of ammonia oxidizing and denitrifying communities, which are the main N2O producers in soil. Contrasting spatial distributions of the total abundance and phylogenetic diversity of each clade, as well as disparate associations with various edaphic and biotic factors indicated potential niche differentiation between the two clades. This is supported by the greater occurrence of significant phylogenetic clustering or overdispersion in clade I communities compared to clade II, indicating differences in the underlying mechanisms of community assembly for each group. Variance partitioning of phylogenetic community structure further showed that biotic factors, particularly the abundance of denitrifiers, played a more substantial role in explaining clade II community structure compared to that of clade I. Finally, identification of nosZ lineages driving differences in community structure and spatial distribution patterns of gene abundances suggests a shift in the genetic potential for N2O production and consumption dynamics across the farm.

N2O production by denitrification in an urban river: evidence from isotopes, functional genes, and dissolved organic matter

Rivers are important sources of N2O emissions into the atmosphere. Nevertheless, N2O production processes in rivers are not well identified. We measured concentrations and isotopic ratios of N2O, NH4 +, NO2 −, and NO3 − in surface water to identify the microbial processes of N2O production along the Tama River in Japan. We also measured the functional gene abundance of nitrifiers and denitrifiers (amoA-bacteria, nirK, nirS, nosZ clade I, nosZ clade II) together with concentrations of dissolved organic carbon (DOC) and fluorescence intensities of protein and humic components of dissolved organic matter (DOM) to support the elucidation of N2O production processes. The observed nitrogen (δ15N) and oxygen (δ18O) of N2O were within the expected isotopic range of N2O produced by nitrate reduction, indicating that N2O was dominantly produced by denitrification. The positive significant correlation between N2ONet concentration and nirK gene abundance implied that nitrifiers and denitrifiers are contributors to N2O production. Fluorescence intensities of protein and humic components of DOM and concentrations of DOC did not show significant correlations with N2O concentrations, which suggests that DOC and abundance of DOM components do not control dissolved N2O. Measurement of isotope ratios of N2O and its substrates was found to be a useful tool to obtain evidence of denitrification as the main source of N2O production along the Tama River.

Mitigating N2O emissions from clover residues by 3,4-dimethylpyrazole phosphate (DMPP) without adverse effects on the earthworm Lumbricus terrestris

In EU-28, temporary grasslands constitute more than 10% of the total arable land. Grassland tillage will return up to 400 kg N ha−1 in residues that can lead to a pulse of N2O emissions. Here a novel application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) was evaluated in a 28-d mesocosm experiment, where DMPP spraying prior to tillage was simulated. Above-ground parts of 15N-labelled clover residues were treated with DMPP and either placed at 10 cm depth to simulate ploughing (PL), or mixed with soil at 0–10 cm depth to simulate rotovation (RO). Earthworms (Lumbricus terrestris) were introduced to study their role in residue decomposition and N2O emissions. Fluxes and isotopic composition of N2O were determined with dynamic chambers using laser spectroscopy. A gradual increase in 15N-enrichment of N2O indicated that denitrification was the main source. DMPP reduced cumulative N2O emissions in PL from 241 to 146 mg N m−2; the reduction in RO was smaller, from 103 to 94 mg N m−2, and not significant, possibly due to higher oxygen and soil NO3− availability. After 28 d incubation, on average > 90% of the earthworms were recovered, and in vivo N2O production from L. terrestris accounted for only 1–6% of residue-induced emissions. The guts were removed from worms to quantify DNA and mRNA of denitrifying genes (nirK, nirS, nosZ clade I and II). It was found that earthworm in vivo N2O production rates were negatively correlated to mRNA of nosZ-I, but positively to nir/nos ratio, suggesting the active involvement of denitrifiers associated with ingested feed in these emissions. Earthworm body tissue was enriched with 15N, with no effect of DMPP. This showed that the worms had fed on the clover residues, and that DMPP did not interfere with feeding behavior or nitrogen assimilation. The observed effects show that DMPP treatment of clover residue before tillage has the potential to modify N2O emissions without adverse effects on residue decomposition or soil fauna.

Simulated animal trampling of a free‐draining stony soil stimulated denitrifier growth and increased nitrous oxide emissions

AbstractWinter forage grazing systems in New Zealand cause compaction of soil by grazing animals, especially when the soil is wet. However, there is little information on the effects of animal trampling on denitrifiers in soil, despite their importance for N2O production. Here, we report a field study of the abundance of the denitrifying genes nirS, nirK, and nosZ and N2O emissions following the application of dairy cow urine in a free‐draining stony soil. Importantly, we found that simulated animal trampling altered some of the denitrifying microbial communities, thus leading to increased N2O emissions. Over the 111 day measurement period, the abundance of nitrite (NO2−)‐reducing nirS gene copy numbers increased significantly by 87% in the trampled soil with urine (P < 0.01) and increased by 40% in the trampled soil without urine (P < 0.05), but the nirS gene abundance did not change significantly in the nontrampled soil. The abundance of NO2− reducing nirK gene copy numbers was not affected by trampling, but increased significantly following urine application. The abundance of N2O‐reducing nosZ clade I and nosZ clade II gene copy numbers increased significantly in the trampled soil, but did not change significantly in the nontrampled soil. N2O emissions from the trampled soil were about twice that from the nontrampled soil without urine (1.20 and 0.62 kg N2O‐N per ha, respectively) and about eight times greater (6.24 kg N2O‐N per ha) than from nontrampled soil (0.80 kg N2O‐N per ha) when urine was applied. These results strongly suggest that animal trampling during winter forage grazing can have a major impact on denitrifying communities in soil, which in turn stimulate greater denitrification with increased N2O emissions.

Reconstructing the Genetic Potential of the Microbially-Mediated Nitrogen Cycle in a Salt Marsh Ecosystem.

Coastal ecosystems are considered buffer zones for the discharge of land-derived nutrients without accounting for potential negative side effects. Hence, there is an urgent need to better understand the ecological assembly and dynamics of the microorganisms that are involved in nitrogen (N) cycling in such systems. Here, we employed two complementary methodological approaches (i.e., shotgun metagenomics and quantitative PCR) to examine the distribution and abundance of selected microbial genes involved in N transformations. We used soil samples collected along a well-established pristine salt marsh soil chronosequence that spans over a century of ecosystem development at the island of Schiermonnikoog, The Netherlands. Across the examined soil successional stages, the structure of the populations of genes involved in N cycling processes was strongly related to (shifts in the) soil nitrogen levels (i.e., , ), salinity and pH (explaining 73.8% of the total variation, R2 = 0.71). Quantification of the genes used as proxies for N fixation, nitrification and denitrification revealed clear successional signatures that corroborated the taxonomic assignments obtained by metagenomics. Notably, we found strong evidence for niche partitioning, as revealed by the abundance and distribution of marker genes for nitrification (ammonia-oxidizing bacteria and archaea) and denitrification (nitrite reductase nirK, nirS and nitrous oxide reductase nosZ clades I and II). This was supported by a distinct correlation between these genes and soil physico-chemical properties, such as soil physical structure, pH, salinity, organic matter, total N, , and , across four seasonal samplings. Overall, this study sheds light on the successional trajectories of microbial N cycle genes along a naturally developing salt marsh ecosystem. The data obtained serve as a foundation to guide the formulation of ecological models that aim to effectively monitor and manage pristine and impacted salt marsh areas. Such models should account for the ecology as well as the historical contingency of N cycling communities.