N Kinetic Analysis of N(2)O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification

A series of N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the "denitrifying enzyme" nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% N) and 0.4 mM nitrite (99% N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.     

Revision of N2O-Producing Pathways in the Ammonia-Oxidizing Bacterium Nitrosomonas europaea ATCC 19718

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N₂O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N₂O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O₂ tensions. Anoxic resting-cell assays and instantaneous nitrite (NO₂⁻) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N₂O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N₂O production during growth at both atmospheric and reduced O₂ tensions. Anoxic resting-cell assays and measurements of instantaneous NO₂⁻ reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N₂O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.     

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K_m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K_m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from ¹³NO₃⁻ by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.     

Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor

Cells of the obligately lithotrophic species Nitrosomonas europaea and Nitrosomonas eutropha were able to nitrify and denitrify at the same time when grown under oxygen limitation. In addition to oxygen, nitrite was used as an electron acceptor. The simultaneous nitrification and denitrification resulted in significant formation of the gaseous N-compounds nitrous oxide and dinitrogen, causing significant nitrogen loss. In mixed cultures of N. europaea and various chemoorganotrophic bacteria, the nitrogen loss was strongly influenced by the partners growing under oxygen limitation. Under anoxic conditions, pure cultures of N. eutropha were able to denitrify with molecular hydrogen as electron donor and nitrite as the only electron acceptor in a sulfide-reduced complex medium. The increase of cell numbers was directly coupled to nitrite reduction. Nitrous oxide and dinitrogen were the only detectable end products. In pure cultures of N. eutropha and mixed cultures of N. eutropha and Enterobacter aerogenes, ammonium and nitrite disappeared slowly at a molar ratio of about one when oxygen was absent. However, under these conditions cell growth was not measurable.     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

Characterization of a Nitrite Reductase Involved in Nitrifier Denitrification

Nitrifier denitrification is the conversion of nitrite to nitrous oxide by ammonia-oxidizing organisms. This process, which is distinct from denitrification, is active under aerobic conditions in the model nitrifier Nitrosomonas europaea. The central enzyme of the nitrifier dentrification pathway is a copper nitrite reductase (CuNIR). To understand how a CuNIR, typically inactivated by oxygen, functions in this pathway, the enzyme isolated directly from N. europaea (NeNIR) was biochemically and structurally characterized. NeNIR reduces nitrite at a similar rate to other CuNIRs but appears to be oxygen tolerant. Crystal structures of oxidized and reduced NeNIR reveal a substrate channel to the active site that is much more restricted than channels in typical CuNIRs. In addition, there is a second fully hydrated channel leading to the active site that likely acts a water exit pathway. The structure is minimally affected by changes in pH. Taken together, these findings provide insight into the molecular basis for NeNIR oxygen tolerance.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.     

Simultaneous Nitrification and Denitrification in Aerobic Chemostat Cultures of Thiosphaera pantotropha

Thiosphaera pantotropha is capable of simultaneous heterotrophic nitrification and aerobic denitrification. Consequently, its nitrification potential could not be judged from nitrite accumulation, but was estimated from complete nitrogen balances. The maximum rate of nitrification obtained during these experiments was 93.9 nmol min⁻¹ mg of protein⁻¹. The nitrification rate could be reduced by the provision of nitrate, nitrite, or thiosulfate to the culture medium. Both nitrification and denitrification increased as the dissolved oxygen concentration fell, until a critical level was reached at approximately 25% of air saturation. At this point, the rate of (aerobic) denitrification was equivalent to the anaerobic rate. At this dissolved oxygen concentration, the combined nitrification and denitrification was such that cultures receiving ammonium as their sole source of nitrogen appeared to become oxygen limited and the nitrification rate fell. It appeared that, under carbon-and energy-limited conditions, a high nitrification rate was correlated with a reduced biomass yield. To facilitate experimental design, a working hypothesis for the mechanism behind nitrification and denitrification by T. pantotropha was formulated. This involved the basic assumption that this species has a “bottleneck” in its cytochrome chain to oxygen and that denitrification and nitrification are used to overcome this. The nitrification potential of other heterotrophic nitrifiers has been reconsidered. Several species considered to be “poor” nitrifiers also simultaneously nitrify and denitrify, thus giving a falsely low nitrification potential.     

The pathway of nitrogen and reductive enzymes of denitrification

Some recent studies on the pathway of nitrogen and the reductases of denitrification are reviewed. The available evidence suggests that while the intermediates of denitrification can remain enzyme-bound (presumably to nitrite reductase) prior to formation of N₂O, NO and nitroxyl (HNO) can be released in part by certain bacteria. Release of NO is recognized by a nitrite/NO?¹⁵N exchange reaction and isotopic scrambling in product N₂O; release of nitroxyl by Pseudomonas stutzeri is recognized by isotopic scrambling of nitrite and NO in product N₂O in absence of exchange and affords evidence that the first N?N bond forms in denitrification at the N¹⁺ redox level. The recent purification and partial characterization of nitrous oxide reductase are described. The ability of the dissimilatory nitrite reductase to activate nitrite for nitrosyl transfer affords a new chemical probe into the mechanism of action of this central enzyme. It would appear that reduction of nitrite is subject to electrophilic catalysis. ¹⁸O studies show that dissociation of nitrite from nitrite reductase can be slow relative to competing reduction or nitrosyl transfer.     

Nitrous oxide production, its source and distribution in urine patches on grassland on peat soil

Urine patches are considered to be important sites for nitrous oxide (N₂O) production through nitrification and denitrification due to their high concentration of nitrogen (N). The aim of the present study was to determine the microbial source and size of production of N₂O in different zones of a urine patch on grassland on peat soil. Artificial urine was applied in elongated patches of 4.5 m. Four lateral zones were distinguished and sampled for four weeks using an intact soil core incubation method. Incubation of soil cores took place without any additions to the headspace to determine total N₂O production, with acetylene addition to determine total denitrification (N₂O+N₂), and with methyl fluoride to determine the N₂O produced through denitrification.Nitrous oxide production was largest in the centre and decreased towards the edge of the patch. Maximum N₂O production was about 50 mg N m^–2 d^–1 and maximum denitrification activity was 70 mg N m^–2 d^–1. Nitrification was the main N₂O producing process. Nitrous oxide production through denitrification was only of significance when denitrification activity was high. Total N loss through nitrification and denitrification over 31 days was 4.1 g N per patch which was 2.2% of the total applied urine-N.     

Aquatic nitrogen transformations at low oxygen concentrations.

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Aquatic nitrogen transformations at low oxygen concentrations

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Nitrous Oxide Formation in the Colne Estuary, England: the Central Role of Nitrite

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N₂O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N₂O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N₂O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 μM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 μmol N · m⁻² · h⁻¹ with nitrite but only 180 μmol N · m⁻² · h⁻¹ with nitrate. The ratios of rates of nitrous oxide production and denitrification (N₂O/N₂ × 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N₂ formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N₂ via N₂O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N₂O as the end product. Consideration of free-energy changes during N₂O formation led to the conclusion that N₂O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N₂O in high-nutrient-load estuaries.     

Simultaneous nitrification,denitrification, and phosphorus removal in a lab-scale sequencing batch reactor

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic-anoxic-enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the energy and COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab-scale sequencing batch reactor (SBR) was operated in alternating anaerobic-aerobic mode with a low dissolved oxygen (DO) concentration (0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHAs), accompanied by phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to <0.5 mg/L by the end of the cycle. Ammonia was also oxidized during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off-gas analysis showed that the final denitrification product was mainly nitrous oxide (N(2)O), not N(2). Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen-accumulating organisms (DGAOs), rather than denitrifying polyphosphate-accumulating organisms (DPAOs), were responsible for the denitrification activity.     

Simultaneous nitrification and denitrification with different mixed nitrogen loads by a hypothermia aerobic bacterium

Microorganism with simultaneous nitrification and denitrification ability plays a significant role in nitrogen removal process, especially in the eutrophic waters with excessive nitrogen loads. The nitrogen removal capacity of microorganism may suffer from low temperature or nitrite nitrogen source. In this study, a hypothermia aerobic nitrite-denitrifying bacterium, Pseudomonas tolaasii strain Y-11, was selected to determine the simultaneous nitrification and denitrification ability with mixed nitrogen source at 15 °C. The sole nitrogen removal efficiencies of strain Y-11 in simulated wastewater were obtained. After 24 h of incubation at 15 °C, the ammonium nitrogen fell below the detection limit from an initial value of 10.99 mg/L. Approximately 88.0 ± 0.33% of nitrate nitrogen was removed with the initial concentration of 11.78 mg/L and the nitrite nitrogen was not detected with the initial concentration of 10.75 mg/L after 48 h of incubation at 15 °C. Additionally, the simultaneous nitrification and denitrification nitrogen removal ability of P. tolaasii strain Y-11 was evaluated using low concentration of mixed NH₄⁺-N and NO₃^?–N/NO₂^?–N (about 5 mg/L-N each) and high concentration of mixed NH₄⁺–N and NO₃^?–N/NO₂^?–N (about 100 mg/L-N each). There was no nitrite nitrogen accumulation at the time of evaluation. The results demonstrated that P. tolaasii strain Y-11 had higher simultaneous nitrification and denitrification capacity with low concentration of mixed inorganic nitrogen sources and may be applied in low temperature wastewater treatment.     

Reduction of the Powerful Greenhouse Gas N2O in the South-Eastern Indian Ocean

Nitrous oxide (N₂O) is a powerful greenhouse gas and a key catalyst of stratospheric ozone depletion. Yet, little data exist about the sink and source terms of the production and reduction of N₂O outside the well-known oxygen minimum zones (OMZ). Here we show the presence of functional marker genes for the reduction of N₂O in the last step of the denitrification process (nitrous oxide reductase genes; nosZ) in oxygenated surface waters (180–250 O₂ μmol.kg^-1) in the south-eastern Indian Ocean. Overall copy numbers indicated that nosZ genes represented a significant proportion of the microbial community, which is unexpected in these oxygenated waters. Our data show strong temperature sensitivity for nosZ genes and reaction rates along a vast latitudinal gradient (32°S-12°S). These data suggest a large N₂O sink in the warmer Tropical waters of the south-eastern Indian Ocean. Clone sequencing from PCR products revealed that most denitrification genes belonged to Rhodobacteraceae. Our work highlights the need to investigate the feedback and tight linkages between nitrification and denitrification (both sources of N₂O, but the latter also a source of bioavailable N losses) in the understudied yet strategic Indian Ocean and other oligotrophic systems.     

Nitrous oxide formation in the Colne estuary, England: the central role of nitrite

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N(2)O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N(2)O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N(2)O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 microM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 micromol N.m(-2).h(-1) with nitrite but only 180 micromol N.m(-2).h(-1) with nitrate. The ratios of rates of nitrous oxide production and denitrification (N(2)O/N(2) x 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N(2) formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N(2) via N(2)O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N(2)O as the end product. Consideration of free-energy changes during N(2)O formation led to the conclusion that N(2)O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N(2)O in high-nutrient-load estuaries.     

Effect of ammonium concentration on the emission of N(2)O under oxygen-limited autotrophic wastewater nitrification

A significant amount of nitrous oxide (N(2)O), which is one of the serious greenhouse gases, is emitted from nitrification and denitrification of wastewater. Batch wastewater nitrifications with enriched nitrifiers were carried out under oxygen-limited condition with synthetic (without organic carbon) and real wastewater (with organic carbon) in order to find out the effect of ammonium concentration on N(2)O emission. Cumulated N(2)O-N emission reached 3.0, 5.7, 6.2, and 13.5 mg from 0.4 l of the synthetic wastewater with 50, 100, 200, and 500 mg/l NH(4)(+)-N, respectively, and 1.0 mg from the real wastewater with 125 mg/l NH(4)(+)-N. The results indicate that N(2)O emission increased with ammonium concentration and the load. The ammonium removal rate and nitrite concentration also increased N(2)O emission. Comparative analysis of N(2)O emission from synthetic and real wastewaters revealed that wastewater nitrification under oxygen-limited condition emitted more N(2)O than that of heterotrophic denitrification. Summarizing the results, it can be concluded that denitrification by autotrophic nitrifiers contributes significantly to the N(2)O emission from wastewater nitrification.     

Selective Inhibition of Ammonium Oxidation and Nitrification-Linked N2O Formation by Methyl Fluoride and Dimethyl Ether

Methyl fluoride (CH₃F) and dimethyl ether (DME) inhibited nitrification in washed-cell suspensions of Nitrosomonas europaea and in a variety of oxygenated soils and sediments. Headspace additions of CH₃F (10% [vol/vol]) and DME (25% [vol/vol]) fully inhibited NO₂^- and N₂O production from NH₄⁺ in incubations of N. europaea, while lower concentrations of these gases resulted in partial inhibition. Oxidation of hydroxylamine (NH₂OH) by N. europaea and oxidation of NO₂^- by a Nitrobacter sp. were unaffected by CH₃F or DME. In nitrifying soils, CH₃F and DME inhibited N₂O production. In field experiments with surface flux chambers and intact cores, CH₃F reduced the release of N₂O from soils to the atmosphere by 20- to 30-fold. Inhibition by CH₃F also resulted in decreased NO₃^- + NO₂^- levels and increased NH₄⁺ levels in soils. CH₃F did not affect patterns of dissimilatory nitrate reduction to ammonia in cell suspensions of a nitrate-respiring bacterium, nor did it affect N₂O metabolism in denitrifying soils. CH₃F and DME will be useful in discriminating N₂O production via nitrification and denitrification when both processes occur and in decoupling these processes by blocking NO₂^- and NO₃^- production.