Aerobic denitrifying bacteria that produce low levels of nitrous oxide

Most denitrifiers produce nitrous oxide (N(2)O) instead of dinitrogen (N(2)) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N(2)O under aerobic conditions. We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures. Both strains reduced nitrate (NO(3)(-)) to N(2) at rates of 0.9 and 0.03 micro mol min(-1) unit of optical density at 540 nm(-1) at dissolved oxygen (O(2)) (DO) concentrations of 39 and 38 micro mol liter(-1), respectively. At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N(2) but evolved more than 10-fold more N(2)O than strains TR2 and K50 evolved. The isolates denitrified NO(3)(-) with concomitant consumption of O(2). These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the beta subclass of the class Proteobacteria and were identified as P. stutzeri TR2 and Pseudomonas sp. strain K50. These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N(2)O emission, the single-stage process for nitrogen removal, and microbial N(2)O emission into the ecosystem.     

Hybrid respiration in the denitrifying mitochondria of Fusarium oxysporum

Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)). Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors. The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase. We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria.     

Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-atoms of N m(-2) h(-1)). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Kinetics of denitrification using sulphur compounds: effects of S/N ratio, endogenous and exogenous compounds

The influence of different sulphur to nitrogen (S/N) ratios on the specific autotrophic denitrification activity was studied in batch experiments using thiosulphate and nitrate as substrates. Transitory accumulations of nitrite were observed for assays with S/N ratios of 3.70 and 6.67 g/g, probably due to the higher specific reduction rate of nitrate compared to that of nitrite. Nitrite was the main end product when S/N ratios of 1.16 and 2.44 g/g were tested. The effects of endogenous (NO(3)(-),NO(2)(-),S(2)O(3)(2-)and SO(4)(2-)) and exogenous compounds (acetate and NaCl) on the specific denitrifying activity of the sludge were tested. Nitrite and sulphate did exert clear inhibitory effects over the process while thiosulphate, acetate and NaCl did not have strong effects at the concentrations tested. Similar experiments also showed that sulphur was not a suitable electron donor for these microorganisms, but sulphide was used successfully.     

Oxygen requirement for denitrification by the fungus Fusarium oxysporum

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.     

Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.     

A Microsensor for Nitrate Based on Immobilized Denitrifying Bacteria

A biosensor for NO(inf3)(sup-) was constructed by attaching a 30- to 70-(mu)m-wide capillary with immobilized denitrifying bacteria in front of an N(inf2)O microsensor. These bacteria reduced O(inf2) so that only bacteria in the very tip of the sensor were exposed to O(inf2) whereas bacteria at a greater depth could carry out the anaerobic process of denitrification. In the presence of acetylene, which inhibits nitrous oxide reductase, bacteria reduced NO(inf3)(sup-) (or NO(inf2)(sup-)) from the surrounding medium to N(inf2)O and the concentration sensed by the N(inf2)O microsensor was directly proportional to the concentration of NO(inf3)(sup-) in the medium. By applying a 250-(mu)m-long capillary in front of the N(inf2)O microsensor, the 90% response time of the biosensor was 50 s. Biosensors may also be made with nitrous oxide-deficient strains so that acetylene inhibition can be omitted.     

Nitrogen Transformations in Wetland Soil Cores Measured by (sup15)N Isotope Pairing and Dilution at Four Infiltration Rates

The effect of water infiltration rate (IR) on nitrogen cycling in a saturated wetland soil was investigated by applying a (sup15)N isotope dilution and pairing method. Water containing [(sup15)N]nitrate was infiltrated through 10-cm-long cores of sieved and homogenized soil at rates of 72, 168, 267, and 638 mm day(sup-1). Then the frequencies of (sup30)N(inf2), (sup29)N(inf2), (sup15)NO(inf3)(sup-), and (sup15)NH(inf4)(sup+) in the outflow water were measured. This method allowed simultaneous determination of nitrification, coupled and uncoupled denitrification, and nitrate assimilation rates. From 3% (at the highest IR) to 95% (at the lowest IR) of nitrate was removed from the water, mainly by denitrification. The nitrate removal was compensated for by the net release of ammonium and dissolved organic nitrogen. Lower oxygen concentrations in the soil at lower IRs led to a sharper decrease in the nitrification rate than in the ammonification rate, and, consequently, more ammonium leaked from the soil. The decreasing organic-carbon-to-nitrogen ratio (from 12.8 to 5.1) and the increasing light A(inf250)/A(inf365) ratio (from 4.5 to 5.2) indicated an increasing bioavailability of the outflowing dissolved organic matter with increasing IR. The efflux of nitrous oxide was also very sensitive to IR and increased severalfold when a zone of low oxygen concentration was close to the outlet of the soil cores. N(inf2)O then constituted 8% of the total gaseous N lost from the soil.     

Denitrification of wastewater containing high nitrate and calcium concentrations

The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentrations led to complete denitrification in 6h (denitrification rate: 22.8mg NO(3)(-)-N/gVSSh), using methanol as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150mg/L. Higher calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7h at a maximum rate of 30.4mg NO(3)(-)-N/gVSSh and very low residual COD in the effluent.     

Aerobic denitrification in soils and sediments: From fallacies to factx

Denitrification is the key step of the nitrogen cycle in which gaseous end products are released from the nitrate of terrestrial and aquatic environments. Although this process has always been regarded as an anaerobic one, recent research indicates that aerobic denitrification can be demonstrated with laboratory cultures and suggests that it may be widespread environmentally. Thus, denitrifying bacteria are both taxonomically and physiologically diverse, and may be predominantly aerobic. Simultaneous use of O(2) and NO(3)(-) as alternative terminal oxidants is not precluded in many bacteria, although the use of NO(3)(-) when O(2) is available is of no known advantage.     

Effects of nitrite and ammonium on methane-dependent denitrification

For effective application of methane-dependent denitrification (MDD) in the treatment of wastewater containing NO(2)(-) or NH(4)(+), the effect of these inorganic nitrogen compounds on MDD activity needs to be clarified. The MDD activity of sludge acclimatized with CH(4) and O(2) was determined with mineral media of different nitrogen-compound compositions in the presence of 0.21 atm CH(4) and 0.20 atm O(2). Incubations with media containing only NO(2)(-) or two of the three inorganic nitrogen compounds (NO(3)(-)+NO(2)(-), NO(2)(-)+NH(4)(+) or NH(4)(+)+NO(3)(-)) resulted in MDD activity equal to or higher than that with media containing only NO(3)(-). However, there was no MDD activity in media containing NO(2)(-) at 10 degrees C, probably because of serious inhibition of NO(2)(-) on methane oxidation. MDD occurred in media containing only NH(4)(+), although the total nitrogen removal efficiency was very low. These results show that NO(2)(-) and NH(4)(+), in the presence of NO(x)(-), do not inhibit but rather promote MDD. Consequently, NH(4)(+) does not need to be completely oxidized to NO(3)(-) in the nitrification reactor before MDD. However, under psychrophilic conditions, NO(2)(-) seriously inhibited MDD. Therefore, the nitrification reactor must not discharge effluent containing NO(2)(-) under psychrophilic conditions.     

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.     

Selection of organic substrates as potential reactive materials for use in a denitrification permeable reactive barrier (PRB)

The aim of the present study was to select a suitable natural organic substrate as a potential carbon source for use in a denitrification permeable reactive barrier (PRB). A number of seven organic substrates were first tested in batch tests. The materials attained varying degrees of success at promoting denitrification. Some of the organic substrates performed very well, achieving complete nitrate removal (>98%), while others were considered unsuitable for a variety of reasons, including: insufficient nitrate or nitrogen removal, excessive release of leachable nitrogen from the substrate or excessive reduction of nitrate to ammonium rather than removing it as gaseous N2. The top performing substrate in terms of denitrification extent (>98%) and rate (0.067 mgNO3(-)-N dm(-3)d(-1)g(sub)(-1)) was then selected for two bench-scale column experiments in an attempt to simulate the PRB. The inlet concentration was 50 mg dm(-3) NO3(-)-N and the columns operated at two different flow rates: 0.3 cm3 min(-1) (Column 1) and 1.1cm3 min(-1) (Column 2). The two columns showed different general patterns, making it clear that the flow rate was a key factor at the nitrate removal. Nitrate was completely removed (>96%) by the passage through Column 1, while only partially removed in Column 2 (66%). The results indicated that the selected organic substrate (Softwood) was applicable for further use as a filling material for a PRB.     

Anaerobic ammonium oxidation measured in sediments along the Thames estuary, United Kingdom

Until recently, denitrification was thought to be the only significant pathway for N(2) formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of (15)N-labeled NH(4)(+), NO(3)(-), and NO(2)(-) (and (14)N analogues), production of (29)N(2) and (30)N(2) was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of (29)N(2) in the presence of (15)NH(4)(+) and either (14)NO(3)(-) or (14)NO(2)(-) confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO(2)(-). Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO(2)(-) or indirect reduction, following the initial reduction of NO(3)(-). Whether NO(2)(-) was directly present at 800 micro M or it accumulated at 3 to 20 micro M (from the reduction of NO(3)(-)), the rate of (29)N(2) formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO(2)(-). We observed a shift in the significance of anaerobic ammonium oxidation to N(2) formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.     

Nitrosospira spp. can produce nitrous oxide via a nitrifier denitrification pathway

Nitrous oxide (N(2)O) emission from soils is a major contributor to the atmospheric loading of this potent greenhouse gas. It is thought that autotrophic ammonia oxidizing bacteria (AOB) are a significant source of soil-derived N(2)O and a denitrification pathway (i.e. reduction of NO(2) (-) to NO and N(2)O), so-called nitrifier denitrification, has been demonstrated as a N(2)O production mechanism in Nitrosomonas europaea. It is thought that Nitrosospira spp. are the dominant AOB in soil, but little information is available on their ability to produce N(2)O or on the existence of a nitrifier denitrification pathway in this lineage. This study aims to characterize N(2)O production and nitrifier denitrification in seven strains of AOB representative of clusters 0, 2 and 3 in the cultured Nitrosospira lineage. Nitrosomonas europaea ATCC 19718 and ATCC 25978 were analysed for comparison. The aerobically incubated test strains produced significant (P < 0.001) amounts of N(2)O and total N(2)O production rates ranged from 2.0 amol cell(-1) h(-1), in Nitrosospira tenuis strain NV12, to 58.0 amol cell(-1) h(-1), in N. europaea ATCC 19718. Nitrosomonas europaea ATCC 19718 was atypical in that it produced four times more N(2)O than the next highest producing strain. All AOB tested were able to carry out nitrifier denitrification under aerobic conditions, as determined by production of (15)N-N(2)O from applied (15)N-NO(2) (-). Up to 13.5% of the N(2)O produced was derived from the exogenously applied (15)N-NO(2) (-). The results suggest that nitrifier denitrification could be a universal trait in the betaproteobacterial AOB and its potential ecological significance is discussed.     

Hexadecane mineralization and denitrification in two diesel fuel-contaminated soils

The effect of nitrate, ammonium and urea on the mineralization of [(14)C]hexadecane (C(16)H(34)) and on denitrification was evaluated in two soils contaminated with diesel fuel. In soil A, addition of N fertilizers did not stimulate or inhibit background hexadecane mineralization (4.3 mg C(16)H(34) kg(-1) day(-1)). In soil B, only NaNO(3) stimulated hexadecane mineralization (0.91 mg C(16)H(34) kg(-1) day(-1)) compared to soil not supplemented with any nitrogen nutrient (0.17 mg C(16)H(34) kg(-1) day(-1)). Hexadecane mineralization was not stimulated in this soil by NH(4)NO(3) (0.13 mg C(16)H(34) kg(-1) day(-1)), but the addition of NH(4)Cl or urea suppressed hexadecane mineralization (0.015 mg C(16)H(34) kg(-1) day(-1)). Addition of 2 kPa C(2)H(2) did not inhibit the mineralization process in either soil. Denitrification occurred in both soils studied when supplemented with NaNO(3) and NH(4)NO(3), but was not detected with other N sources. Denitrification started after a longer lag in soil A (10 days) than in soil B (4 days). In soil A microcosms supplemented with NaNO(3) or NH(4)NO(3), rates of denitrification were 20.6 and 13.6 mg NO(3)(-) kg(-1) day(-1), respectively, and in soil B, they were 18.5 and 12.5 mg NO(3)(-) kg(-1) day(-1), respectively. We conclude that denitrification may lead to a substantial loss of nitrate, making it unavailable to the mineralizing bacterial population. Nitrous oxide was an important end-product accounting for 30-100% of total denitrification. These results indicate the need for preliminary treatability studies before implementing full-scale treatment processes incorporating commercial fertilizers.     

Fungal denitrification and nitric oxide reductase cytochrome P450nor

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.     

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.