Winter and summer nitrous oxide and nitrogen oxides fluxes from a seasonally snow-covered subalpine meadow at Niwot Ridge,Colorado

The soil emission rates (fluxes) of nitrous oxide (N₂O) and nitrogen oxides (NO + NO₂ = NO _x ) through a seasonal snowpack were determined by a flux gradient method from near-continuous 2-year measurements using an automated system for sampling interstitial air at various heights within the snowpack from a subalpine site at Niwot Ridge, Colorado. The winter seasonal-averaged N₂O fluxes of 0.047–0.069 nmol m⁻² s⁻¹ were ~15 times higher than observed NO _x fluxes of 0.0030–0.0067 nmol m⁻² s⁻¹. During spring N₂O emissions first peaked and then dropped sharply as the soil water content increased from the release of snowpack meltwater, while other gases, including NO _x and CO₂ did not show this behavior. To compare and contrast the winter fluxes with snow-free conditions, N₂O fluxes were also measured at the same site in the summers of 2006 and 2007 using a closed soil chamber method. Summer N₂O fluxes followed a decreasing trend during the dry-out period after snowmelt, interrupted by higher values related to precipitation events. These peaks were up to 2–3 times higher than the background summer levels. The integrated N₂O-N loss over the summer period was calculated to be 1.1–2.4 kg N ha⁻¹, compared to ~0.24–0.34 kg N ha⁻¹ for the winter season. These wintertime N₂O fluxes from subniveal soil are generally higher than the few previously published data. These results are of the same order of magnitude as data from more productive ecosystems such as fertilized grasslands and high-N-cycling forests, most likely because of a combination of the relatively well-developed soils and the fact that subnivean biogeochemical processes are promoted by the deep, insulating snowpack. Hence, microbially mediated oxidized nitrogen emissions occurring during the winter can be a significant part of the N-cycle in seasonally snow-covered subalpine ecosystems.     

Nitrous oxide in brackish Lake Nakaumi, Japan II: the role of nitrification and denitrification in N2O accumulation

In order to understand the role of nitrification and denitrification in the accumulation of nitrous oxide (N₂O) in the hypolimnetic water of brackish Lake Nakaumi, the effects of dissolved oxygen (DO) concentration on these activities were investigated by incubation experiments. N₂O was produced during the oxidation of NH₄ ⁺ to NO₂ ⁻ in nitrification and during the reduction of NO₃ ⁻ to N₂ in denitrification. N₂O-producing activity by nitrification (N₂O_N) increased markedly with decreasing concentrations of DO. Low DO (10%–30% saturation) induced high N₂O_N. In contrast to nitrification, N₂O-producing activity by denitrification (N₂O_D) decreased with decreasing concentrations of DO. Little N₂O was accumulated during denitrification under low-level conditions of DO (10%–30%), because of further reduction of N₂O to N₂. It can therefore be assumed that N₂O produced as the by-product of nitrification is concurrently reduced to N₂ by denitrification under low-DO conditions. This would result in no substantial accumulation of N₂O during active nitrification in the hypolimnetic water of Lake Nakaumi. Received: July 6, 2001 / Accepted: December 10, 2001     

Dynamics of gaseous nitrogen and carbon fluxes in riparian alder forests

We studied greenhouse gas (GHG) fluxes in two differently loaded riparian Alnus incana-dominated forests in agricultural landscapes of southern Estonia: a 33-year-old stand in Porijõgi, in which the uphill agricultural activities had been abandoned since the middle of the 1990s, and a 50-year-old stand in Viiratsi, which still receives polluted lateral flow from uphill fields fertilized with pig slurry. In Porijõgi, closed-chamber based sampling lasted from October 2001 to October 2009, whereas in Viiratsi the sampling period was from November 2003 to October 2009. Both temporal and spatial variations in all GHG gas fluxes were remarkable. Local differences in GHG fluxes between micro-sites (“Edge”, “Dry” and “Wet” in Porijõgi, and “Wet”, “Slope” and “Dry” in Viiratsi) were sometimes greater than those between sites. Median values of GHG fluxes from both sites over the whole study period and all microsites did not differ significantly, being 45 and 42 mg CO₂-C m⁻² h⁻¹, 8 and 0.5 μg CH₄-C m⁻² h⁻¹, 1.0 and 2.1 mg N₂-N m⁻² h⁻¹, and 5 and 9 μg N₂O-N m⁻² h⁻¹, in Porijõgi and Viiratsi, respectively. The N₂:N₂O ratio in Viiratsi (40-1200) was lower than in Porijõgi (10-7600). The median values-based estimation of the Global Warming Potential of CH₄ and N₂O was 19 and 185 kg CO₂ equivalents (eq) ha⁻¹ yr⁻¹ in Porijõgi and −14 and 336 kg CO₂ eq ha⁻¹ yr⁻¹ in Viiratsi, respectively. A significant Spearman rank correlation was found between the mean monthly air temperature and CO₂, CH₄ and N₂ fluxes in Porijõgi, and N₂O flux in Viiratsi, and between the monthly precipitation and CH₄ fluxes in both study sites. Higher groundwater level significantly increases CH₄ emission and decreases CO₂ and N₂O emission, whereas higher soil temperature significantly increases N₂O, CH₄ and N₂ emission values. In Porijõgi, GHG emissions did not display any discernable trend, whereas in Viiratsi a significant increase in CO₂, N₂, and N₂O emissions has been found. This may be a result of the age of the grey alder stand, but may also be caused by the long-term nutrient load of this riparian alder stand, which indicates a need for the management of similar heavily loaded riparian alder stands.     

Evaluation of pilot-scale modified A2O processes for the removal of nitrogen compounds from sewage

In this study, pilot-scale experiments for the removal of nitrogen from sewage obtained from a county Y sewer system were performed using modified A₂O processes. Using this approach, the total amount of nitrogen discharged during denitrification of the influent was average 38.6 mg/L and a level of average 10.8 mg/L was maintained throughout the denitrification process, which resulted in an average removal efficiency that was greater than 72%. The nitrogen components in the effluent water consisted of 22% ammonia nitrogen, 6% nitrite nitrogen and 72% nitrate nitrogen, reaching a nitrification efficiency of 94%. In conclusion, since these advanced treatment methods, which involve modified A₂O processes, were successfully employed to remove nitrogen from sewage discharge, they hold promise for wide spread use by treatment plants.     

The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: Cloning, sequence analysis, and expression

The nitrous oxide (N₂O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N₂O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N₂O reductase), and NosL is also reported. A model of the Cu_A site in N₂O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the Cu_A domain may be a ligand to the catalytic Cu_Z site. In addition, the origin of the spectroscopically-observed Cys coordination to Cu_Z is discussed in terms of the sequence alignment of seven N₂O reductases.     

Nitrous oxide reductase of Flexibacter canadensis: a unique membrane-bound enzyme

Abstract The subcellular distribution of nitrous oxide reductase was studied in the gliding soil bacterium Flexibacter canadensis . Nitrous oxide reductase activity, as measured by the methyl viologen-nitrous oxide oxidoreductase assay, was associated entirely with the membrane fraction of cell-free extracts. The enzyme was liberated from the membranes with use of detergents but not by high-salt concentrations, thus implying that nitrous oxide reductase is an integral membrane protein. The nitrous oxide reductase of F. canadensis is the first reported example of a membrane-bound form of this respiratory enzyme.     

Experimental determination of nitrogen kinetic isotope fractionation: Some principles; illustration for the denitrification and nitrification processes

Summary A few principles relative to the presentation and use of nitrogen stable isotopic data are briefly reviewed. Some classical relationships between the isotope composition of a substrate undergoing a single-step unidirectional reaction, are introduced. They are illustrated through controlled experiments on denitrification in a soil, and through nitrification by pure cultures ofNitrosomonas europaea. In the latter case, the isotope fractionation is calculated from the isotopic composition of the residual substrate, then of the product and the result is shown to be statistically the same for the two procedures. The isotopic enrichment factor for denitrification is −29.4±2.4‰ at 20°C, and −24.6±0.9‰ at 30°C; for nitrification this factor is −34.7±2.5‰ under the experimental conditions employed.     

Chironomus plumosus larvae increase fluxes of denitrification products and diversity of nitrate-reducing bacteria in freshwater sediment

Benthic invertebrates affect microbial processes and communities in freshwater sediment by enhancing sediment-water solute fluxes and by grazing on bacteria. Using microcosms, the effects of larvae of the widespread midge Chironomus plumosus on the efflux of denitrification products (N₂O and N₂ + N₂O) and the diversity and abundance of nitrate- and nitrous-oxide-reducing bacteria were investigated. Additionally, the diversity of actively nitrate- and nitrous-oxide-reducing bacteria was analyzed in the larval gut. The presence of larvae increased the total effluxes of N₂O and N₂ + N₂O up to 8.6- and 4.2-fold, respectively, which was mostly due to stimulation of sedimentary denitrification; incomplete denitrification in the guts accounted for up to 20% of the N₂O efflux. Phylotype richness of the nitrate reductase gene narG was significantly higher in sediment with than without larvae. In the gut, 47 narG phylotypes were found expressed, which may contribute to higher phylotype richness in colonized sediment. In contrast, phylotype richness of the nitrous oxide reductase gene nosZ was unaffected by the presence of larvae and very few nosZ phylotypes were expressed in the gut. Gene abundance of neither narG, nor nosZ was different in sediments with and without larvae. Hence, C. plumosus increases activity and diversity, but not overall abundance of nitrate-reducing bacteria, probably by providing additional ecological niches in its burrow and gut.     

Nitrogen processing in the hyporheic zone of a pastoral stream

The distribution of nitrogen-transforming processes, and factors controlling their rates, were determined within the hyporheic zone of a lowland stream draining agricultural land. In the field, physicochemical parameters were measured along a 10m-long hyporheic flow line between downwelling and upwelling zones. Sediment cores were retrieved from the stream bed surface, and from 20, 40 and 60cm deep in each zone, and in the laboratory, water from the corresponding depth was percolated through each core at the natural flow rate. Concentrations of nitrogen species and oxygen were measured before and after flow through each core. Denitrification was measured using a ¹⁵N-nitrate tracer. Shallow and downwelling zone samples were clearly distinct from deeper and upwelling zone samples in terms of physicochemical conditions, microbial processes and factors controlling nitrogen processing. Denitrification was highest in surface and downwelling zone cores, despite high oxygen levels, probably due to high pore-water nitrate concentrations in these cores and isolation of the denitrifying bacteria from oxygen in the bulk water by the hyporheic biofilms. Denitrification was limited by oxygen inhibition in the downwelling group, and by nitrate availability in the upwelling group. Strong evidence indicated that dissimilatory nitrate reduction to ammonium, occurred in almost all cores, and outcompeted denitrification for nitrate. In contrast, nitrification was undetectable in all but two cores, probably because of intense competition for oxygen. Field patterns and lab experiments indicated that the hyporheic zone at this moderately N-rich site is a strong sink for nitrate, fitting current theories that predict where hyporheic zones are nitrate sinks or nitrate sources.     

Anaerobic denitrification in fungi from the coastal marine sediments off Goa, India

Denitrification is a microbial process during which nitrate or nitrite is reduced under anaerobic condition to gaseous nitrogen. The Arabian Sea contains one of the major pelagic denitrification zones and in addition to this, denitrification also takes places along the continental shelf. Prokaryotic microorganisms were considered to be the only players in this process. However recent studies have shown that higher microeukaryotes such as fungi can also adapt to anaerobic mode of respiration and reduce nitrate to harmful green house gases such as NO and N₂O. In this study we examined the distribution and biomass of fungi in the sediments of the seasonal anoxic region off Goa from two stations. The sampling was carried out in five different periods from October 2005, when dissolved oxygen levels were near zero in bottom waters to March 2006. We isolated mycelial fungi, thraustochytrids and yeasts. Species of Aspergillus and thraustochytrids were dominant. Fungi were isolated under aerobic, as well as anaerobic conditions from different seasons. Four isolates were examined for their denitrification activity. Two cultures obtained from the anoxic sediments showed better growth under anaerobic condition than the other two cultures that were isolated from oxic sediments. Our preliminary results suggest that several species of fungi can grow under oxygen deficient conditions and participate in denitrification processes.     

Contribution of sediment fluxes and transformations to the summer nitrogen budget of an Upper Mississippi River backwater system

Routing nitrate through backwaters of regulated floodplain rivers to increase retention could decrease loading to nitrogen (N)-sensitive coastal regions. Sediment core determinations of N flux were combined with inflow–outflow fluxes to develop mass balance approximations of N uptake and transformations in a flow-controlled backwater of the Upper Mississippi River (USA). Inflow was the dominant nitrate source (>95%) versus nitrification and varied as a function of source water concentration since flow was constant. Nitrate uptake length increased linearly, while uptake velocity decreased linearly, with increasing inflow concentration to 2 mg l⁻¹, indicating limitation of N uptake by loading. N saturation at higher inflow concentration coincided with maximum uptake capacity, 40% uptake efficiency, and an uptake length 2 times greater than the length of the backwater. Nitrate diffusion and denitrification in sediment accounted for 27% of the backwater nitrate retention, indicating that assimilation by other biota or denitrification on other substrates were the dominant uptake mechanisms. Ammonium export from the backwater was driven by diffusive efflux from the sediment. Ammonium increased from near zero at the inflow to a maximum mid-lake, then declined slightly toward the outflow due to uptake during transport. Ammonium export was small compared to nitrate retention. Handling editor: J. Padisak     

Greenhouse gas fluxes from the eutrophic Temmesjoki River and its Estuary in the Liminganlahti Bay (the Baltic Sea)

We studied concentrations of carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) in the eutrophic Temmesjoki River and Estuary in the Liminganlahti Bay in 2003–2004 and evaluated the atmospheric fluxes of the gases based on measured concentrations, wind speeds and water current velocities. The Temmesjoki River was a source of CO₂, CH₄ and N₂O to the atmosphere, whereas the Liminganlahti Bay was a minor source of CH₄ and a minor source or a sink of CO₂ and N₂O. The results show that the fluxes of greenhouse gases in river ecosystems are highly related to the land use in its catchment areas. The most upstream river site, surrounded by forests and drained peatlands, released significant amounts of CO₂ and CH₄, with average fluxes of 5,400 mg CO₂–C m⁻² d⁻¹ and 66 mg CH₄–C m⁻² d⁻¹, and concentrations of 210 μM and 345 nM, respectively, but N₂O concentrations, at an average of 17 nM, were close to the atmospheric equilibrium concentration. The downstream river sites surrounded by agricultural soils released significant amounts of N₂O (with an average emission of 650 μg N₂O–N m⁻² d⁻¹ and concentration of 22 nM), whereas the CO₂ and CH₄ concentrations were low compared to the upstream site (55 μM and 350 nM). In boreal regions, rivers are partly ice-covered in wintertime (approximately 5 months). A large part of the gases, i.e. 58% of CO₂, 55% of CH₄ and 36% of N₂O emissions, were found to be released during wintertime from unfrozen parts of the river.     

Denitrification ability of rhizobial strains isolated from Lotus sp.

Ten rhizobial strains isolated from Lotus sp. have been characterized by their ability to denitrify. Out of the 10 strains, the five slow-growing isolates grew well under oxygen-limiting conditions with nitrate as a sole nitrogen source, and accumulated nitrous oxide in the growth medium when acetylene was used to inhibit nitrous oxide reductase activity. All five strains contained DNA homologous to the Bradyrhizobium japonicum nirK, norBDQ and nosZ genes. In contrast, fast-growing lotus rhizobia were incapable of growing under nitrate-respiring conditions, and did not accumulate nitrous oxide in the growth medium. DNA from each of the five fast-growing strains showed a hybridization band with the B. japonicum nirK gene but not with norBDQ and nosZ genes. Partial 16S rDNA gene sequencing revealed that fast-growing strains could be identified as Mesorhizobium loti species and the slow-growers as Bradyrhizobium sp.     

Effects of sulfide and low redox potential on the inhibition of nitrous oxide reduction by acetylene in Pseudomonas nautica

Membrane introduction mass spectrometry was used to investigate the inhibitory effect of acetylene on the nitrous oxide reductase activity of intact cells of Pseudomonas nautica. We studied the effects of the concentrations of nitrate and sulfide, and the redox potential, which have all been implicated in causing a decrease in the inhibitory effects of acetylene during measurements of denitrification in natural environments. There was no evidence that the concentration of nitrate influenced the effect of acetylene. Lowering the redox potential with the reductant Ti(III)-nitrilotriacetate caused a slight alleviation of acetylene inhibition. Much greater effects at the same redox potential were obtained with concentrations of sulfide in the range 1-10 microM.     

Reduction of nitrite to nitrous oxide by a cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus

A cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus reduced nitrite to nitrous oxide in a stoichiometric reaction without nitric oxide as free intermediate. The membrane system had a specific requirement for FMN with NAD(P)H as electron donors. Other electron donors were ascorbate-reduced cytochrome c-551 or phenazine methosulfate. The membrane fraction contained tightly bound cytochrome cd which represented only a small portion of the total cytochrome cd of the cell. As further terminal oxidase cytochrome o was identified. The membrane fraction produced also nitrous oxide from nitric oxide, however, at a substantially lower rate than from nitrite when using ascorbate-reduced phenazine methosulfate as electron donor.     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

White on green: under-snow microbial processes and trace gas fluxes through snow,Niwot Ridge,Colorado Front Range

The importance of snow and related cryospheric processes as an ecological factor has been recognized since at least the beginning of the twentieth century. Even today, however, many observations remain anecdotal. The research to date on cold-lands ecosystems results in scientists being unable to evaluate to what extent changes in the cryosphere will be characterized by abrupt changes in local and global biogeochemical cycles, and how these changes in seasonality may affect the rates and timing of key ecological processes. Studies of gas exchanges through snow have revealed that snow plays an important role in modulating wintertime soil biogeochemical processes, and that these can be the driving processes for gas exchange at the snow surface. Previous research has primarily focused on carbon dioxide, and resulted from episodic experiments at a number of snow-covered sites. Here we report new insights from several field sites on Niwot Ridge in the Colorado Rocky Mountains, including a dedicated snow gas flux research facility established at the 3340 m Soddie site. A novel in situ experimental system was developed at this site to continuously sample trace gases from above and within the snowpack for the duration of seasonal snow cover. The suite of chemical species investigated includes carbon dioxide, nitrous oxide, nitrogen oxides, ozone, and volatile inorganic and organic gases. Wintertime measurements have been supplemented by soil chamber experiments and eddy covariance measurements to allow assessment of the contribution of wintertime fluxes to annual biogeochemical budgets. This research has resulted in a plethora of new insight into the physics of gas transport through the snowpack, and the magnitude and the chemical and biogeochemical processes that control fluxes at the soil-snowpack and the snow-atmosphere interface. This article provides an overview of the history and evolution of this research, and highlights the findings from the ten articles that constitute this special issue.     

Nitrification and nitrous oxide production potentials in aerobic soil samples from the soil profile of a Finnish coniferous site receiving high ammonium deposition

Abstract Using aerobic soil slurry technique nitrification and nitrous oxide production were studied in samples from a pine site in Western Finland. The site received atmospheric ammonium deposition of 7–33 kg N ha⁻¹ a⁻¹ from a mink farm. The experiments with soil slurries showed that the nitrification potential in the litter layer was higher at pH 6 than at pH 4. However, the nitrification potentials in the samples from the organic and mineral horizons at pH 6 and 4 were almost equal. Also N₂O was produced at a higher rate at pH 6 than at pH 4 in slurries of the litter layer samples. The reverse was true for samples from the organic and mineral horizons. The highest N₂O production and nitrification rates were measured in the suspensions of litter layer samples. Nitrification activity in field-moist soil samples was lower than the activity in the slurries indicating that the availability of ammonium limited nitrification in these soils. Acetylene (2.5 kPa) retarded nitrification activity (70-–100%) and N₂O production (40 – 90%) in soil slurries. Acetylene inhibited the N₂O production by 40–60% during the first 3 days after its addition to field-moist samples incubated in aerobic atmosphere. After 3 days the inhibition became much lower (4–5%). The results indicate that, in soil profiles of boreal coniferous forests receiving ammonium deposition, chemolithotrophic nitrification may have importance in the N₂O production, and that changes in soil pH affect differently nitrification as well as N₂O production in litter and deeper soil layers.