High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

In-depth analysis of N2O fluxes in tropical forest soils of the Congo Basin combining isotope and functional gene analysis

Primary tropical forests generally exhibit large gaseous nitrogen (N) losses, occurring as nitric oxide (NO), nitrous oxide (N₂O) or elemental nitrogen (N₂). The release of N₂O is of particular concern due to its high global warming potential and destruction of stratospheric ozone. Tropical forest soils are predicted to be among the largest natural sources of N₂O; however, despite being the world’s second-largest rainforest, measurements of gaseous N-losses from forest soils of the Congo Basin are scarce. In addition, long-term studies investigating N₂O fluxes from different forest ecosystem types (lowland and montane forests) are scarce. In this study we show that fluxes measured in the Congo Basin were lower than fluxes measured in the Neotropics, and in the tropical forests of Australia and South East Asia. In addition, we show that despite different climatic conditions, average annual N₂O fluxes in the Congo Basin’s lowland forests (0.97 ± 0.53 kg N ha⁻¹ year⁻¹) were comparable to those in its montane forest (0.88 ± 0.97 kg N ha⁻¹ year⁻¹). Measurements of soil pore air N₂O isotope data at multiple depths suggests that a microbial reduction of N₂O to N₂ within the soil may account for the observed low surface N₂O fluxes and low soil pore N₂O concentrations. The potential for microbial reduction is corroborated by a significant abundance and expression of the gene nosZ in soil samples from both study sites. Although isotopic and functional gene analyses indicate an enzymatic potential for complete denitrification, combined gaseous N-losses (N₂O, N₂) are unlikely to account for the missing N-sink in these forests. Other N-losses such as NO, N₂ via Feammox or hydrological particulate organic nitrogen export could play an important role in soils of the Congo Basin and should be the focus of future research.Subject terms: Microbiology, Biogeochemistry     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Genetic and Functional Variation in Denitrifier Populations along a Short-Term Restoration Chronosequence

Complete removal of plants and soil to exposed bedrock, in order to eradicate the Hole-in-the-Donut (HID) region of the Everglades National Park, FL, of exotic invasive plants, presented the opportunity to monitor the redevelopment of soil and the associated microbial communities along a short-term restoration chronosequence. Sampling plots were established for sites restored in 1989, 1997, 2000, 2001, and 2003. The goal of this study was to characterize the activity and diversity of denitrifying bacterial populations in developing HID soils in an effort to understand changes in nitrogen (N) cycling during short-term primary succession. Denitrifying enzyme activity (DEA) was detected in soils from all sites, indicating a potential for N loss via denitrification. However, no correlation between DEA and time since disturbance was observed. Diversity of bacterial denitrifiers in soils was characterized by sequence analysis of nitrite reductase genes (nirK and nirS) in DNA extracts from soils ranging in nitrate concentrations from 1.8 to 7.8 mg kg⁻¹. High levels of diversity were observed in both nirK and nirS clone libraries. Statistical analyses of clone libraries suggest a different response of nirS- and nirK-type denitrifiers to factors associated with soil redevelopment. nirS populations demonstrated a linear pattern of succession, with individual lineages represented at each site, while multiple levels of analysis suggest nirK populations respond in a grouped pattern. These findings suggest that nirK communities are more sensitive than nirS communities to environmental gradients in these soils.     

Aerobic nitrous oxide production through N-nitrosating hybrid formation in ammonia-oxidizing archaea

Soil emissions are largely responsible for the increase of the potent greenhouse gas nitrous oxide (N₂O) in the atmosphere and are generally attributed to the activity of nitrifying and denitrifying bacteria. However, the contribution of the recently discovered ammonia-oxidizing archaea (AOA) to N₂O production from soil is unclear as is the mechanism by which they produce it. Here we investigate the potential of Nitrososphaera viennensis, the first pure culture of AOA from soil, to produce N₂O and compare its activity with that of a marine AOA and an ammonia-oxidizing bacterium (AOB) from soil. N. viennensis produced N₂O at a maximum yield of 0.09% N₂O per molecule of nitrite under oxic growth conditions. N₂O production rates of 4.6±0.6 amol N₂O cell⁻¹ h⁻¹ and nitrification rates of 2.6±0.5 fmol NO₂⁻ cell⁻¹ h⁻¹ were in the same range as those of the AOB Nitrosospira multiformis and the marine AOA Nitrosopumilus maritimus grown under comparable conditions. In contrast to AOB, however, N₂O production of the two archaeal strains did not increase when the oxygen concentration was reduced, suggesting that they are not capable of denitrification. In ¹⁵N-labeling experiments we provide evidence that both ammonium and nitrite contribute equally via hybrid N₂O formation to the N₂O produced by N. viennensis under all conditions tested. Our results suggest that archaea may contribute to N₂O production in terrestrial ecosystems, however, they are not capable of nitrifier-denitrification and thus do not produce increasing amounts of the greenhouse gas when oxygen becomes limiting.     

Denitrification Activity of a Remarkably Diverse Fen Denitrifier Community in Finnish Lapland Is N-Oxide Limited

Peatlands cover more than 30% of the Finnish land area and impact N₂O fluxes. Denitrifiers release N₂O as an intermediate or end product. In situ N₂O emissions of a near pH neutral pristine fen soil in Finnish Lapland were marginal during gas chamber measurements. However, nitrate and ammonium fertilization significantly stimulated in situ N₂O emissions. Stimulation with nitrate was stronger than with ammonium. N₂O was produced and subsequently consumed in gas chambers. In unsupplemented anoxic microcosms, fen soil produced N₂O only when acetylene was added to block nitrous oxide reductase, suggesting complete denitrification. Nitrate and nitrite stimulated denitrification in fen soil, and maximal reaction velocities (v_max) of nitrate or nitrite dependent denitrification where 18 and 52 nmol N₂O h^-1 g_DW ^-1, respectively. N₂O was below 30% of total produced N gases in fen soil when concentrations of nitrate and nitrite were <500 μM. v_max for N₂O consumption was up to 36 nmol N₂O h^-1 g_DW ^-1. Denitrifier diversity was assessed by analyses of narG, nirK/nirS, and nosZ (encoding nitrate-, nitrite-, and nitrous oxide reductases, respectively) by barcoded amplicon pyrosequencing. Analyses of ~14,000 quality filtered sequences indicated up to 25 species-level operational taxonomic units (OTUs), and up to 359 OTUs at 97% sequence similarity, suggesting diverse denitrifiers. Phylogenetic analyses revealed clusters distantly related to publicly available sequences, suggesting hitherto unknown denitrifiers. Representatives of species-level OTUs were affiliated with sequences of unknown soil bacteria and Actinobacterial, Alpha-, Beta-, Gamma-, and Delta-Proteobacterial sequences. Comparison of the 4 gene markers at 97% similarity indicated a higher diversity of narG than for the other gene markers based on Shannon indices and observed number of OTUs. The collective data indicate (i) a high denitrification and N₂O consumption potential, and (ii) a highly diverse, nitrate limited denitrifier community associated with potential N₂O fluxes in a pH-neutral fen soil.     

Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

Nitrous oxide (N₂O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N₂O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10¹ and 10² target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10⁵ to 10⁷ target copies g⁻¹ of dry soil, whereas genes for 16S rRNA were found at 10⁸ to 10⁹ target copies g⁻¹ of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.     

Isolation,genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO₃⁻ and NO₂⁻) to N₂ via the intermediates NO and N₂O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.     

Insights into the Effect of Soil pH on N2O and N2 Emissions and Denitrifier Community Size and Activity

The objective of this study was to investigate how changes in soil pH affect the N₂O and N₂ emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H₂SO₄ solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N₂O and N₂ emissions using the ¹⁵N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N₂O/(N₂O + N₂) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N₂O production were observed. Denitrification activity and N₂O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between nirS, napA, and narG gene copy numbers and the N₂O/(N₂O + N₂) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.Denitrification is the microbial reduction of NO₃⁻ via NO₂⁻ to gaseous NO, N₂O, and N₂, which are then lost into the atmosphere (36). It therefore results in considerable loss of nitrogen, one of the most limiting nutrients for crop production in agriculture (20). Denitrification is also of environmental concern since, together with nitrification, it is the main biological process responsible for N₂O emissions (7). N₂O is a potent greenhouse gas which has a global warming potential about 320 times greater than that of CO₂ and has a lifetime of approximately 120 years (32). In the stratosphere, N₂O can also react with O₂ to produce NO, which induces the destruction of stratospheric ozone (8). N₂O can be released into the atmosphere by incomplete denitrification due to the effect of environmental conditions on the regulation of the different denitrification reductases (14, 41, 51), but it has recently been suggested that it could also be due to lack of nitrous oxide reductase in some denitrifiers (19, 41). Since N₂O is an intermediate in the denitrification pathway, both the amount of N₂O produced and the N₂O/(N₂O + N₂) ratio are important in understanding and predicting N₂O fluxes from soils.The main environmental factors known to influence the N₂O/(N₂O + N₂) ratio are pH, organic carbon and NO₃⁻ availability, water content, and O₂ partial pressure (50). Soil pH is one of the most important factors influencing both denitrification and N₂O production (43). In general, the denitrification rate increases with increasing pH values (up to the optimum pH) while, in contrast, the N₂O/(N₂O + N₂) ratio decreases (50). This relationship has been characterized in laboratory experiments (9, 45), but it is not clear whether the same relationships exist in the field because of methodological limitations of in situ measurement of N₂ emissions (16). Nevertheless, ¹⁵N tracing experiments based on the addition of a labeled denitrification substrate to soil offer a useful tool to quantify emissions of both N₂O and N₂ in situ (47, 49). Soil pH is also an important factor influencing denitrifier community composition (35, 39), which can be an important driver of denitrification activity and N₂O emissions (5, 21). A recent study reported a negative relationship between the proportion of bacteria genetically capable of reducing N₂O within the total bacterial community and the N₂O/(N₂O + N₂) ratio, with both being strongly correlated with soil pH (38).The objective of the present study was to explore the effect of changes in soil pH on in situ N₂O and N₂ emissions, denitrifying enzyme activity (DEA), and potential N₂O production. In addition, we also investigated whether differences in N fluxes could be related to changes in the size of the microbial community possessing the different denitrification genes. A field experiment was conducted using replicated grassland plots in which the soil pH was modified by addition of either acid or hydroxide to the soil. A ¹⁵N tracer method was used to provide information on N emissions. In addition to measuring potential denitrification activity, the size of the denitrifier community was determined by real-time PCR quantification of the denitrification genes.     

Quantification of Nitrogen Reductase and Nitrite Reductase Genes in Soil of Thinned and Clear-Cut Douglas-Fir Stands by Using Real-Time PCR

The abundance of nifH, nirS, and nirK gene fragments involved in nitrogen (N) fixation and denitrification in thinned second-growth Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest soil was investigated by using quantitative real-time PCR. Prokaryotic N cycling is an important aspect of N availability in forest soil. The abundance of universal nifH, Azotobacter sp.-specific nifH (nifH-g1), nirS, and nirK gene fragments in unthinned control and 30, 90, and 100% thinning treatments were compared at two long-term research sites on Vancouver Island, Canada. The soil was analyzed for organic matter (OM), total carbon (C), total N, NH₄-N, NO₃-N, and phosphorus (P). The soil horizon accounted for the greatest variation in nutrient status, followed by the site location. The 30% thinning treatment was associated with significantly greater nifH-g1 abundance than the control treatment in one site; at the same site, nirS in the mineral soil horizon was significantly reduced by thinning. The abundance of nirS genes significantly correlated with the abundance of nirK genes. In addition, significant correlations were observed between nifH-g1 abundance and C and N in the organic horizon and between nirS and nirK and N in the mineral horizon. Overall, no clear influence of tree thinning on nifH, nirS, and nirK was observed. However, soil OM, C, and N were found to significantly influence N-cycling gene abundance.Nitrogen (N) is a limiting nutrient in most Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest ecosystems. Understanding the links between forest management and forest ecosystem function, including the cycling of N, is of paramount importance to researchers and forest managers. Management practices such as thinning and clear-cutting can alter the soil microbial community, potentially altering the rate and amount of net N addition or loss to the forest floor. Clear-cutting alters the functional diversity of soil microorganisms and alters soil characteristics (temperature, pH, moisture, and nutrient status). Thinning and clear-cutting can increase nitrification, denitrification, and leaching of N in soil, all of which can reduce the available N (2, 13, 22, 41, 47). Clear-cutting in Douglas-fir forests can also remove associated gene pools of diazotrophic microorganisms (46). It is not yet well understood how clear-cutting or thinning affects the abundance of N-cycling microorganisms. We focus on two populations of N-cycling microorganisms: diazotrophs, which biologically fix N₂ gas to ammonia, and denitrifiers, which reduce N oxides and result in the release of N-containing gasses.Fixation of N by diazotrophic microorganisms is the primary source of N addition to undisturbed, unfertilized forest soil ecosystems (9, 39). The diazotrophic community is most often studied in situ using the marker gene for nitrogenase reductase (nifH); the diversity and abundance of diazotrophic microorganisms as determined by nifH characterization may be used as an indicator of overall soil ecological health. Diazotrophs can be symbiotic, associated (e.g., with a specific plant or fungal biomass), or free-living in the soil. Endophytic diazotrophs fix ∼100 times more N than free-living strains (9). Free-living diazotrophs such as Azotobacter vinelandii and A. chroococcum may fix between 0 and 60 kg of N ha⁻¹ year⁻¹ (9) and, because of a relative dearth of endophytic interactions in coniferous forests, free-living diazotrophs can be an important source of N in these soils. Cultural studies have shown that free-living diazotrophs improve the establishment of mycorrhizae and conifer seedlings, with relative activity fluctuating according to season, site aspect, and moisture conditions (11). Fixed-N inputs act as a catalyst for interlinked N-cycling events, e.g., fungal decomposition of woody debris and organic material (28). Nitrogen fixation in temperate forest soil is directly related to the amounts of soil organic matter (17). However, it is unclear how nifH gene abundance relates to the amount of total carbon (C) and organic matter (OM) and N in forest soil. It is also unknown how common silvicultural practices (e.g., clear-cutting and thinning) affect diazotrophic abundance or how diazotrophic abundance may in turn affect cycling of soil nutrients.The reduction of inorganic N oxides by denitrifying microorganisms can cause N loss from forest soil ecosystems, as well as the release of greenhouse gases into the atmosphere. The loss of N from temperate forest soil as N₂O has been reported as ranging from 0.2 to 7.0 kg ha⁻¹ year⁻¹, depending largely on soil nitrogen status, soil moisture, and temperature (57). Robertson and Tiedje (44) state that soil N loss in coniferous ecosystems due to denitrification is regulated by nitrification potential (e.g., nitrate levels) in the soil, and while not considered a major N loss component following clear-cutting, this loss is generally of the same magnitude as the N loss due to leaching. Denitrification is primarily studied using molecular approaches by monitoring several genes in the denitrification pathway: cytochrome cd₁-containing nitrite reductase (nirS), Cu-containing nitrite reductase (nirK), nitrous oxide reductase (nosZ), and membrane-bound nitrate reductase A (narG). The nirS and nirK genes were the denitrification genes used in the present study. Studies demonstrating (i) that the nirS gene is more diverse than nirK in soil and (ii) the domination of the nirK population by a relatively reduced number of clones have been published (42, 45). However, recent meta-analysis of studies involving nirK and nirS has shown that both communities tend to be phylogenetically clustered in undisturbed soils (23).To compare the effects of silvicultural practices on the abundance of diazotrophs and denitrifiers, we used quantitative real-time PCR (qPCR) assays to quantify nifH, nirS, and nirK genes in soil. This method can be used to quantify target sequences in environmental samples. Several qPCR protocols for the analysis of functional gene abundance in soil have been developed for N-cycling genes, including nifH, ammonia monooxygenase (amoA), nirK, nirS, nosZ, and narG (21, 24, 31, 38, 43, 54, 55). The objectives of the present study were (i) to quantify nifH, nirS, and nirK; (ii) to compare the effects of thinning and clear-cutting in Douglas-fir stands on the abundance of total diazotrophs, free-living diazotrophs, and denitrifiers; and (iii) to elucidate the relationships between N-cycling genes and nutrient abundance in forest soils.     

Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ _I and nosZ _II) in lake sediments. Summertime N₂O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS, nirK, nosZ _I, and nosZ _II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O reductase genes, i.e. the (nirS+nirK)/nosZ _I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ _I at the inter-lake scale and (nirS+nirK)/nosZ _I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.     

Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen

Wetlands are sources of denitrification-derived nitrous oxide (N₂O). Thus, the denitrifier community of an N₂O-emitting fen (pH 4.7 to 5.2) was investigated. N₂O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10¹¹ cells·g_DW⁻¹ (where DW is dry weight) and 10⁸ cells·g_DW⁻¹, respectively, in both 0- to 10-cm and 30- to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (v_max) values for denitrification in anoxic microcosms ranged from 1 to 24 and −19 to −105 nmol N₂O h⁻¹· g_DW⁻¹, with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N₂O approximated 40% of total nitrogenous gases produced at in situ pH, which was likewise the optimal pH for denitrification. Gene libraries of narG and nosZ (encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of narG and nosZ sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of narG and nosZ revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N₂O at in situ pH.Nitrous oxide (N₂O) is a potent greenhouse gas with a global warming potential that is 300-fold higher than that of CO₂, and its concentration increased from 270 ppb in 1750 to 319 ppb in 2005 (17). N₂O can be produced in soils during denitrification, nitrification, the dissimilatory reduction of nitrate to nitrite and/or ammonium (hereafter referred to as dissimilatory nitrate reduction), or the chemical transformation of nitrite or hydroxylamine (5, 7, 49). The percentage of N₂O produced in any of these processes is variable, depending mainly on the redox potential, pH, and C/N ratio (49). In anoxic ecosystems such as waterlogged soils, most of the N₂O is considered to be denitrification derived (7, 9). Complete denitrification is the sequential reduction of nitrate to dinitrogen (N₂) via nitrite, nitric oxide (NO), and N₂O (75). The main product of denitrification varies with the organism and in situ conditions and is usually either N₂O or N₂ (68). N₂O can occur as a by-product during dissimilatory nitrate reduction when accumulated nitrite interacts with nitrate reductase to form N₂O (59). The production of N₂O by dissimilatory nitrate reducers is favored in environments with large amounts of readily available organic carbon (65). Thus, their contribution to nitrate-dependent production of N₂O in soils is likely insignificant compared to that of denitrifiers.The oxidoreductases involved in denitrification are termed dissimilatory nitrate reductase (Nar, encoded by narGHJI, or Nap, encoded by napEDABC), nitrite reductase (Nir, encoded by nirK and nirS), NO reductase (cNor and qNor, encoded by norBC and norB, respectively), and N₂O reductase (Nos, encoded by nosZ) (75). Nitrate reductase is also found in dissimilatory nitrate reducers (60). narG can therefore be used as a molecular marker to assess both denitrifiers and dissimilatory nitrate reducers, whereas nosZ is specific for the assessment of denitrifiers (25, 43, 48).Denitrification in soils is regulated by temperature, pH, substrate (i.e., carbon) availability, and water content (10, 24, 66). Although denitrification increases with increasing temperature, it can still occur at temperatures below 0°C (10, 24). Low temperatures appear to limit the activity of N₂O reductase more severely than other enzymes involved in denitrification and thus yield higher relative amounts of denitrification-derived N₂O (24). Although denitrification activity usually decreases under acidic conditions, the relative percentage of N₂O to total denitrification-derived nitrogenous gases increases with increasing acidity, a result attributed to the sensitivity of N₂O reductase to low pH (27, 70). However, denitrifier communities can be adapted to the in situ pH of the system (40, 58, 73).Wetlands are ecosystems in which denitrification is likely a dominant source of emitted N₂O (7, 44, 45). The identification and analysis of main drivers for N₂O production (i.e., the microbiota catalyzing N₂O production and consumption) is thus of major concern in such environments. Fens are specialized wetlands characterized by soil acidity (67). However, information on acid-tolerant denitrifier communities of such wetlands is scarce. It is hypothesized that fens harbor a diverse, hitherto unknown, denitrifier community that is adapted to in situ conditions and associated with N₂O fluxes (i.e., fen denitrifiers are acid tolerant and have a high affinity for nitrate and N₂O). Thus, the main objectives of the present study were to evaluate the capacities of denitrifier communities of an N₂O-emitting fen (20) to produce or consume N₂O and to determine if a novel and diverse denitrifier community was associated with these capacities.     

Nitrogen loss from grassland on peat soils through nitrous oxide production

Nitrous oxide (N₂O) in soils is produced through nitrification and denitrification. The N₂O produced is considered as a nitrogen (N) loss because it will most likely escape from the soil to the atmosphere as N₂O or N₂. Aim of the study was to quantify N₂O production in grassland on peat soils in relation to N input and to determine the relative contribution of nitrification and denitrification to N₂O production. Measurements were carried out on a weekly basis in 2 grasslands on peat soil (Peat I and Peat II) for 2 years (1993 and 1994) using intact soil core incubations. In additional experiments distinction between N₂O from nitrification and denitrification was made by use of the gaseous nitrification inhibitor methyl fluoride (CH₃F).Nitrous oxide production over the 2 year period was on average 34 kg N ha^-1 yr^-1 for mown treatments that received no N fertiliser and 44 kg N ha^-1 yr^-1 for mown and N fertilised treatments. Grazing by dairy cattle on Peat I caused additional N₂O production to reach 81 kg N ha^-1 yr^-1. The sub soil (20–40 cm) contributed 25 to 40% of the total N₂O production in the 0–40 cm layer. The N₂O production:denitrification ratio was on average about 1 in the top soil and 2 in the sub soil indicating that N₂O production through nitrification was important. Experiments showed that when ratios were larger than l, nitrification was the major source of N₂O. In conclusion, N₂O production is a significant N loss mechanism in grassland on peat soil with nitrification as an important N₂O producing process.     

Intergenomic Comparisons Highlight Modularity of the Denitrification Pathway and Underpin the Importance of Community Structure for N2O Emissions

Nitrous oxide (N₂O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N₂O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N₂O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N₂O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N₂O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N₂O emissions than nirK type denitrifiers under favorable environmental conditions_. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N₂O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N₂O emissions.     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.     

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N₂ production with activities of 0.33–0.64 nmol N₂ g⁻¹ soil h⁻¹, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N₂ production with lower activities of 0.08–0.26 nmol N₂ g⁻¹ soil h⁻¹. Higher anammox bacterial cells were observed (0.75–1.4 × 10⁷ copies g⁻¹ soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 10⁶ copies g⁻¹ soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia'' and ‘Candidatus Brocadia'' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.     

Co-Occurring Anammox,Denitrification, and Codenitrification in Agricultural Soils

Anammox and denitrification mediated by bacteria are known to be the major microbial processes converting fixed N to N₂ gas in various ecosystems. Codenitrification and denitrification by fungi are additional pathways producing N₂ in soils. However, fungal codenitrification and denitrification have not been well investigated in agricultural soils. To evaluate bacterial and fungal processes contributing to N₂ production, molecular and ¹⁵N isotope analyses were conducted with soil samples collected at six different agricultural fields in the United States. Denitrifying and anammox bacterial abundances were measured based on quantitative PCR (qPCR) of nitrous oxide reductase (nosZ) and hydrazine oxidase (hzo) genes, respectively, while the internal transcribed spacer (ITS) of Fusarium oxysporum was quantified to estimate the abundance of codenitrifying and denitrifying fungi. ¹⁵N tracer incubation experiments with ¹⁵NO₃⁻ or ¹⁵NH₄⁺ addition were conducted to measure the N₂ production rates from anammox, denitrification, and codenitrification. Soil incubation experiments with antibiotic treatments were also used to differentiate between fungal and bacterial N₂ production rates in soil samples. Denitrifying bacteria were found to be the most abundant, followed by F. oxysporum based on the qPCR assays. The potential denitrification rates by bacteria and fungi ranged from 4.118 to 42.121 nmol N₂-N g⁻¹ day⁻¹, while the combined potential rates of anammox and codenitrification ranged from 2.796 to 147.711 nmol N₂-N g⁻¹ day⁻¹. Soil incubation experiments with antibiotics indicated that fungal codenitrification was the primary process contributing to N₂ production in the North Carolina soil. This study clearly demonstrates the importance of fungal processes in the agricultural N cycle.