Criteria and Methodology for Identifying Respiratory Denitrifiers

Respiratory denitrification is not always adequately established when bacteria are characterized. We have tested a simple method that allows one to evaluate whether the two necessary criteria to claim denitrification have been met, namely, that N(inf2) or N(inf2)O is produced from nitrate or nitrite and that this reduction is coupled to a growth yield increase. Microorganisms were cultured in sealed tubes under a helium headspace and in the presence of 0, 2, 4, 7, and 10 mM nitrate or nitrite. After growth had ceased, N(inf2) and N(inf2)O were quantified by gas chromatography and the final protein concentration was measured. Net protein production was linearly related to nitrate concentration for all denitrifiers tested and ranged from 2 to 6 g of protein per mol of electron equivalent reduced. Nitrogen recovery as N(inf2) plus N(inf2)O from nitrate and nitrite transformed exceeded 80% for all denitrifiers. We also suggest that a rate of N gas production of >10 (mu)mol/min/g of protein can be used as an additional characteristic definitive of denitrification since this process produces gas more rapidly than other processes. These characteristics were established after evaluation of a variety of well-characterized respiratory denitrifiers and other N(inf2)O-producing nitrate reducers. Several poorly characterized denitrifiers were also tested and confirmed as respiratory denitrifiers, including Aquaspirillum itersonii, Aquaspirillum fasciculus, Bacillus azotoformans, and Corynebacterium nephridii. These criteria distinguished respiratory denitrifiers from other groups that reduce nitrate or produce N(inf2)O. Furthermore, they correctly identified respiratory denitrification in weak denitrifiers, a group in which the existence of this process may be overlooked.     

Simultaneous occurrence of denitrification and nitrate ammonification in sediments of the French Mediterranean Coast

Dissimilatory nitrate reductions in coastal marine sediment of Carteau Cove (French Mediterranean Coast) were studied between April 1993 and July 1994. Simultaneous determination of denitrification and dissimilatory nitrate reduction to ammonium was achieved by using a combination of acetylene blockage and 15N techniques. After short incubations (maximum 5 h), a part of 15N labelled nitrate added to the sediment was recovered as ammonium without incorporation in organic matter. The result indicate that a fraction of nitrate was reduced to ammonium by a dissimilatory mechanism instead of denitrifying. Denitrifying and nitrate ammonifying activities ranged from 0 to 19.8 μmol l-1 d-1 and from 2.3 to 83.2 μmol l-1 d-1, respectively. Denitrification rates were highest in early spring whereas nitrate ammonification were highest in fall. The recovery of nitrate reduced as N2O-N plus ammonium was between 40 and 100%, the highest nitrogen losses were recorded in July. Depending on the station and time of year denitrification accounted for between 0 and 43% of the total nitrate reduction whereas dissimilatory nitrate reduction to ammonium (DNRA) accounted for between 18 and 100%. The reduction rate data suggest that the pathway of nitrate reduction to ammonium may be important in coastal sediments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Denitrification kinetics and denitrifier abundances in sediments of lakes receiving atmospheric nitrogen deposition (Colorado, USA)

The transport and deposition of anthropogenic nitrogen (N) to downwind ecosystems is significant and can be a dominant source of new N to many watersheds. Bacterially mediated denitrification in lake sediments may ameliorate the effects of N loading by permanently removing such inputs. We measured denitrification in sediments collected from lakes in the Colorado Rocky Mountains (USA) receiving elevated (5–8?kg?N?ha^?1?y^?1) or low (<2?kg?N?ha^?1?y^?1) inputs of atmospheric N deposition. The nitrate (NO₃ ^?) concentration was significantly greater in high-deposition lakes (11.3?μmol?l^?1) compared to low-deposition lakes (3.3?μmol?l^?1). Background denitrification was positively related to NO₃ ^? concentrations and we estimate that the sampled lakes are capable of removing a significant portion of N inputs via sediment denitrification. We also conducted a dose–response experiment to determine whether chronic N loading has altered sediment denitrification capacity. Under Michaelis–Menten kinetics, the maximum denitrification rate and half-saturation NO₃ ^? concentration did not differ between deposition regions and were 765?μmol?N?m^?2?h^?1 and 293?μmol?l^?1?NO₃ ^?, respectively, for all lakes. We enumerated the abundances of nitrate- and nitrite-reducing bacteria and found no difference between high- and low-deposition lakes. The abundance of these bacteria was related to available light and bulk sediment resources. Our findings support a growing body of evidence that lakes play an important role in N removal and, furthermore, suggest that current levels of N deposition have not altered the abundance of denitrifying bacteria or saturated the capacity for sediment denitrification.     

Electro-Fenton,hydrogenotrophic and Fe2+ ions mediated TOC and nitrate removal from aquaculture system: Different experimental strategies

A number of methods for denitrification were studied including Electro-Fenton method, hydrogenotrophic as well as innovative Fe²⁺ mediated denitrification and their technical feasibility in terms of changes in TOC and nitrate concentrations, effect of different Fenton’s reagent dosage, current and the effect of the pH was investigated. This study was carried out using tailor made electrodialytic reactor. It was found that the highest TOC removal was achieved at pH 2.2 and 2.4 (77.1% and 97.8%, respectively) at the anode and the lowest accumulation of 33% at pH of 6.2 at the cathode. The highest TOC removal in terms of using different H₂O₂ concentrations was achieved at 40 mM reaching as high as 97.3%. Regardless experimental strategy, initially nitrates migrated towards the cathode due to the strong hydraulic gradient under the applied electric current. During the course of experiments, nitrates were transported towards the anode where their concentration decreased. The highest nitrate removal was achieved at 0.12 mA cm^?2 electric current density (94.8%) at the anode and a complete removal at the cathode. Hydrogenotrophic denitrification was the highest reaching 92.5%, however, when Fe²⁺ ions as electron donor was used for the destruction of nitrates, only 66.6% removal was achieved. Denitrification using only Fe²⁺ ions was a factor 1.4 less than using electrically generated hydrogen or a Fenton’s reagent.     

Modeling nitrogen cycling in a coastal fresh water sediment

Increased nitrogen (N) loading to coastal marine and freshwater systems is occurring worldwide as a result of human activities. Diagenetic processes in sediments can change the N availability in these systems, by supporting removal through denitrification and burial of organic N (N_org) or by enhancing N recycling. In this study, we use a reactive transport model (RTM) to examine N transformations in a coastal fresh water sediment and quantify N removal rates. We also assess the response of the sediment N cycle to environmental changes that may result from increased salinity which is planned to occur at the site as a result of an estuarine restoration project. Field results show that much of the N_org deposited on the sediment is currently remineralized to ammonium. A rapid removal of nitrate is observed in the sediment pore water, with the resulting nitrate reduction rate estimated to be 130 μmol N cm⁻² yr⁻¹. A model sensitivity study was conducted altering the distribution of nitrate reduction between dissimilatory nitrate reduction to ammonium (DNRA) and denitrification. These results show a 40% decline in sediment N removal as NO ₃ ⁻ reduction shifts from denitrification to DNRA. This decreased N removal leads to a shift in sediment-water exchange flux of dissolved inorganic nitrogen (DIN) from near zero with denitrification to 133 μmol N cm⁻² yr⁻¹ if DNRA is the dominant pathway. The response to salinization includes a short-term release of adsorbed ammonium. Additional changes expected to result from the estuarine restoration include: lower NO ₃ ⁻ concentrations and greater SO ₄ ²⁻ concentrations in the bottom water, decreased nitrification rates, and increased sediment mixing. The effect of these changes on net DIN flux and N removal vary based on the distribution of DNRA versus denitrification, illustrating the need for a better understanding of factors controlling this competition.     

Growth Yields in Bacterial Denitrification and Nitrate Ammonification

Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H₂ plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.     

Growth yields in bacterial denitrification and nitrate ammonification

Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.     

Linkages between organic matter mineralization and denitrification in eight riparian wetlands

Denitrification (N₂ production) and oxygen consumption rates were measured at ambient field nitrate concentrations during summer in sediments from eight wetlands (mixed hardwood swamps, cedar swamps, heath dominated shrub wetland, herbaceous peatland, and a wetland lacking live vegetation) and two streams. The study sites included wetlands in undisturbed watersheds and in watersheds with considerable agricultural and/or sewage treatment effluent input. Denitrification rates measured in intact cores of water-saturated sediment ranged from 20 to 260 mol N m^-2 h^-1 among the three undisturbed wetlands and were less variable (180 to 260 mol N M^-2 h^-1) among the four disturbed wetlands. Denitrification rates increased when nitrate concentrations in the overlying water were increased experimentally (1 up to 770 M), indicating that nitrate was an important factor controlling denitrification rates. However, rates of nitrate uptake from the overlying water were not a good predictor of denitrification rates because nitrification in the sediments also supplied nitrate for denitrification. Regardless of the dominant vegetation, pH, or degree of disturbance, denitrification rates were best correlated with sediment oxygen consumption rates (r ² = 0.912) indicating a relationship between denitrification and organic matter mineralization and/or sediment nitrification rates. Rates of denitrification in the wetland sediments were similar to those in adjacent stream sediments. Rates of denitrification in these wetlands were within the range of rates previously reported for water-saturated wetland sediments and flooded soils using whole core¹⁵N techniques that quantify coupled nitrification/denitrification, and were higher than rates reported from aerobic (non-saturated) wetland sediments using acetylene block methods.     

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.     

Actinobacterial nitrate reducers and proteobacterial denitrifiers are abundant in N2O-metabolizing palsa peat

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N(2)O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland. In situ N(2)O fluxes were spatially variable, ranging from 0.01 to -0.02 μmol of N(2)O m(-2) h(-1). Fertilization with nitrate stimulated in situ N(2)O emissions and N(2)O production in anoxic microcosms without apparent delay. N(2)O was subsequently consumed in microcosms. Maximal reaction velocities (v(max)) of nitrate-dependent denitrification approximated 3 and 1 nmol of N(2)O per h per gram (dry weight [g(DW)]) in soil from 0 to 20 cm and below 20 cm of depth, respectively. v(max) values of nitrite-dependent denitrification were 2- to 5-fold higher than the v(max) nitrate-dependent denitrification, and v(max) of N(2)O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N(2)O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of narG, nirK and nirS, and nosZ were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as Actino-, Alpha-, and Betaproteobacteria. Copy numbers of nirS always outnumbered those of nirK by 2 orders of magnitude. Copy numbers of nirS tended to be higher, while copy numbers of narG and nosZ tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N(2)O are associated with denitrification, (ii) actinobacterial nitrate reducers and nirS-type and nosZ-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N(2)O fluxes.     

Using the combined bioelectrochemical and sulfur autotrophic denitrification system for groundwater denitrification

A combined bioelectrochemical and sulfur autotrophic denitrification system (CBSAD) was evaluated to treat a groundwater with nitrate contamination (20.9-22.0mgNO(3)(-)-N/L). The reactor was operated continuously for several months with groundwater to maximize treatment efficiency under different hydraulic retention times (HRT) and electric currents. The denitrification rate of sulfur autotrophic part followed a half-order kinetics model. Moreover, the removal efficiency of bioelectrochemical part depended on the electric current. The reactor could be operated efficiently at the HRT ranged from 4.2 to 2.1h (corresponding nitrogen volume-loading rates varied from 0.12 to 0.24 kg N/m(3)d; and optimum current ranged from 30 to 1000 mA), and the NO(3)(-)-N removal rate ranged from 95% to 100% without NO(3)(-)-N accumulation. The pH of effluent was satisfactorily adjusted by bioelectrochemical part, and the sulfate concentration of effluent was lower than 250 mg/L, meeting the drinking water standard of China EPA.     

Vertical distribution of denitrification in an estuarine sediment: integrating sediment flowthrough reactor experiments and microprofiling via reactive transport modeling

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N(2)O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N(2)O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm(-3) h(-1). The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (+/- 10) nmol cm(-2) h(-1), which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.     

Codenitrification and denitrification are dual metabolic pathways through which dinitrogen evolves from nitrate in Streptomyces antibioticus

We screened actinomycete strains for dinitrogen (N(2))-producing activity and discovered that Streptomyces antibioticus B-546 evolves N(2) and some nitrous oxide (N(2)O) from nitrate (NO(3)(-)). Most of the N(2) that evolved from the heavy isotope ([(15)N]NO(3)(-)) was (15)N(14)N, indicating that this nitrogen species consists of two atoms, one arising from NO(3)(-) and the other from different sources. This phenomenon is similar to codenitrification in fungi. The strain also evolved less, but significant, amounts of (15)N(15)N from [(15)N]NO(3)(-) in addition to (15)N(15)NO with concomitant cell growth. Prior to the production of N(2) and N(2)O, NO(3)(-) was rapidly reduced to nitrite (NO(2)(-)) accompanied by distinct cell growth, showing that the actinomycete strain is a facultative anaerobe that depends on denitrification and nitrate respiration for anoxic growth. The cell-free activities of denitrifying enzymes could be reconstituted, supporting the notion that the (15)N(15)N and (15)N(15)NO species are produced by denitrification from NO(3)(-) via NO(2)(-). We therefore demonstrated a unique system in an actinomycete that produces gaseous nitrogen (N(2) and N(2)O) through both denitrification and codenitrification. The predominance of codenitrification over denitrification along with oxygen tolerance is the key feature of nitrate metabolism in this actinomycete.     

Production of N(2) through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments

In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N(2), thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N(2) production in marine sediments. Incubations with (15)N-labeled nitrate or ammonium demonstrated that during this process, N(2) is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N(2) production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N(2) production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N(2) production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N(2), anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.     

Nitrate sources and watershed denitrification inferred from nitrate dual isotopes in the Beijiang River,south China

The great spatial and temporal variability of nitrogen (N) processing introduces large uncertainties for quantifying N cycles in large scales, e.g. a watershed scale, and hence challenges the present techniques in measuring ecosystem N mass balance. The dual isotopes of nitrate (δ¹⁸O and δ¹⁵N) integrate signals for both nitrate sources and N processing, making them promising for studies on large scale N cycling. Here, the dual isotopes, as well as some ion tracers, from a subtropical river in south China were reported to identify the main nitrate sources and to assess the possible occurrence and degree of denitrification in the context of monsoon climate. Our results indicated that nitrification of reduced fertilizer N in soil zones was the main nitrate source, with sewage and manure as another important source in dry winter. Seasonal changes of denitrification was apparent by the ~1:2 enrichment of ¹⁸O and ¹⁵N from April to August, and suggested to occur over the watershed rather than in the river. The lowest denitrification (10%) occurred in April, when the fertilizer application was strongest and the monsoon rainfall abruptly increased, causing enhancement of leaching. The highest denitrification (48%) took place in August due to the high soil temperature and moisture. In December, denitrification was significant (26%) perhaps due to the high enough temperature for microbial activities, whereas the low soil moisture appeared to limit the degree of denitrification. This study suggests that the seasonal variations in denitrification should be taken into account when estimating regional N mass balance.     

Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation

Batch experiments were made to better understand the mechanisms of N2O emissions from activated sludge in denitrifying conditions found in urban WWTPs, i.e. under anoxic and low oxygenation conditions. The results showed that in completely anoxic conditions, denitrification, related to a periplasmic nitrate reductase activity, is the major producer of N2O (100% of the N2O production), whereas the nitrate ammonifying activity is not significant. In a gradient of low oxygenation, the highest N2O emissions (49.7+/-3.8 microg N2O-N/g SS/h on average) occurred at a dissolved-oxygen concentration of around 0.3mg O2/L. Below 0.3mg O2/L, heterotrophic denitrification appeared to be the major process responsible for the N2O emission (100% at zero oxygenation). From 0.4 to 1.1mg O2/L, N2O emissions were due to two processes: (i) heterotrophic denitrification that represented about 40% of the N2O production, and (ii) autotrophic nitrifier denitrification that accounted for about 60%. The N2O emissions from activated sludge represented on average 0.4% of reduced NO3(-) in anoxic conditions. The N2O emissions associated with denitrification of entire nitrogen load would amount to 155 T N2O-N/year, if all the Paris wastewater was treated by a process using activated sludge.     

Estimating denitrification in North Atlantic continental shelf sediments

A model of coupled nitrification/denitrification was developed for continental shelf sediments to estimate the spatial distribution of denitrification throughout shelf regions in the North Atlantic basin. Using data from a wide range of continental shelf regions, we found a linear relationship between denitrification and sediment oxygen uptake. This relationship was applied to specific continental shelf regions by combining it with a second regression relating sediment oxygen uptake to primary production in the overlying water. The combined equation was: denitrification (mmol N m^–2 d^–1)=0.019^* phytoplankton production (mmol C m^–2 d^–1). This relationship suggests that approximately 13% of the N incorporated into phytoplankton in shelf waters is eventually denitrified in the sediments via coupled nitrification/denitrification, assuming a C:N ratio of 6.625:1 for phytoplankton. The model calculated denitrification rates compare favorably with rates reported for several shelf regions in the North Atlantic.The model-predicted average denitrification rate for continental shelf sediments in the North Atlantic Basin is 0.69 mmol N m^{– 2} d^–1. Denitrification rates (per unit area) predicted by the model are highest for the continental shelf region in the western North Atlantic between Cape Hatteras and South Florida and lowest for Hudson Bay, the Baffin Island region, and Greenland. Within latitudinal belts, average denitrification rates were lowest in the high latitudes, intermediate in the tropics and highest in the mid-latitudes. Although denitrification rates per unit area are lowest in the high latitudes, the total N removal by denitrification (53 × 10¹⁰ mol N y^–1) is similar to that in the mid-latitudes (60 × 10¹⁰ mol N y^–1) due to the large area of continental shelf in the high latitudes. The Gulf of St. Lawrence/Grand Banks area and the North Sea are responsible for seventy-five percent of the denitrification in the high latitude region. N removal by denitrification in the western North Atlantic (96 × 10¹⁰ mol N y^–1) is two times greater than in the eastern North Atlantic (47 × 10¹⁰ mol N y^–1). This is primarily due to differences in the area of continental shelf in the two regions, as the average denitrification rate per unit area is similar in the western and eastern North Atlantic.We calculate that a total of 143 × 10¹⁰ mol N y^–1 is removed via coupled nitrification/denitrification on the North Atlantic continental shelf. This estimate is expected to underestimate total sediment denitrification because it does not include direct denitrification of nitrate from the overlying water. The rate of coupled nitrification/denitrification calculated is greater than the nitrogen inputs from atmospheric deposition and river sources combined, and suggests that onwelling of nutrient rich slope water is a major source of N for denitrification in shelf regions. For the two regions where N inputs to a shelf region from onwelling have been measured, onwelling appears to be able to balance the denitrification loss.     

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.     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday