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.     

Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle

Here I describe how losses of fixed nitrogen can occur in riparian zones by the activity of denitrifying bacteria associated with methane-oxidizing (methanotrophic) bacteria. Several methanotrophs catalyze nitrogen cycle processes that can occur in riparian buffer zones, including nitrification and nitrogen fixation. Methanotrophs can produce nitric and nitrous oxides during oxidation of ammonium (nitrification), but they cannot carry out denitrification. However, there is good evidence that denitrifying bacteria can be associated with methanotrophs and can use simple carbon compounds released by the methanotrophs as substrates for the denitrification reactions and for growth. Evidence is presented that denitrifiers isolated from methanotrophic gel-stabilized oxygen gradient systems can use methanol, formaldehyde, and formate, all methane oxidation intermediates, to support their denitrification. Such denitrification associated with methanotrophs can release dinitrogen and so contributes to losses of fixed nitrogen, and may also produce the important atmospheric trace gases nitric and nitrous oxides. Data presented also show that some methanotrophs produce nitrogen oxides, including nitrite, nitric oxide, and nitrous oxide, during growth on nitrate. Assimilatory reduction of nitrate appears to be a requirement for the release of these products.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

The expression of redox proteins of denitrification inThiosphaera pantotropha grown with oxygen,nitrate, and nitrous oxide as electron acceptors

The redox proteins and enzymes involved in denitrification inThiosphaera pantotropha exhibited a differential expression in response to oxygen. Pseudoazurin was completely repressed during batch or continuous culture under oxic conditions. Cytochromecd ₁ nitrite reductase was also heavily repressed after aerobic growth. Nitrite, nitric oxide, and nitrous oxide reductase activities were detected in intact cells under some conditions of aerobic growth, indicating that aerobic denitrification might occur in some circumstances. However, the rates of denitrification were much lower after aerobic growth than after anaerobic growth. Growth with nitrous oxide as sole electron acceptor mimicked aerobic growth in some respects, implying that expression of parts of the denitrification apparatus might be controlled by the redox state of a component of the electron transport chain rather than by oxygen itself. Nevertheless, the regulation of expression of nitrous oxide reductase was linked to the oxygen concentration.     

Metabolic regulation including anaerobic metabolism inParacoccus denitrificans

Under anaerobic circumstances in the presence of nitrateParacoccus denitrificans is able to denitrify. The properties of the reductases involved in nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase are described. For that purpose not only the properties of the enzymes ofP. denitrificans are considered but also those fromEscherichia coli, Pseudomonas aeruginosa, andPseudomonas stutzeri. Nitrate reductase consists of three subunits: the subunit contains the molybdenum cofactor, the subunit contains the iron sulfur clusters, and the subunit is a special cytochromeb. Nitrate is reduced at the cytoplasmic side of the membrane and evidence for the presence of a nitrate-nitrite antiporter is presented. Electron flow is from ubiquinol via the specific cytochromeb to the nitrate reductase. Nitrite reductase (which is identical to cytochromecd ₁) and nitrous oxide reductase are periplasmic proteins. Nitric oxide reductase is a membrane-bound enzyme. Thebc ₁ complex is involved in electron flow to these reductases and the whole reaction takes place at the periplasmic side of the membrane. It is now firmly established that NO is an obligatory intermediate between nitrite and nitrous oxide. Nitrous oxide reductase is a multi-copper protein. A large number of genes is involved in the acquisition of molybdenum and copper, the formation of the molybdenum cofactor, and the insertion of the metals. It is estimated that at least 40 genes are involved in the process of denitrification. The control of the expression of these genes inP. denitrificans is totally unknown. As an example of such complex regulatory systems the function of thefnr, narX, andnarL gene products in the expression of nitrate reductase inE. coli is described. The control of the effects of oxygen on the reduction of nitrate, nitrite, and nitrous oxide are discussed. Oxygen inhibits reduction of nitrate by prevention of nitrate uptake in the cell. In the case of nitrite and nitrous oxide a competition between reductases and oxidases for a limited supply of electrons from primary dehydrogenases seems to play an important role. Under some circumstances NO formed from nitrite may inhibit oxidases, resulting in a redistribution of electron flow from oxygen to nitrite.P. denitrificans contains three main oxidases: cytochromeaa ₃, cytochromeo, and cytochromeco. Cytochromeo is proton translocating and receives its electrons from ubiquinol. Some properties of cytochromeco, which receives its electrons from cytochromec, are reported. The control of the formation of these various oxidases is unknown, as well as the control of electron flow in the branched respiratory chain. Schemes for aerobic and anaerobic electron transport are given. Proton translocation and charge separation during electron transport from various electron donors and by various electron transfer pathways to oxygen and nitrogenous oxide are given. The extent of energy conservation during denitrification is about 70% of that during aerobic respiration. In sulfate-limited cultures (in which proton translocation in the NADH-ubiquinone segment of the respiratory chain is lost) the extent of energy conservation is about 60% of that under substrate-limited conditions. These conclusions are in accordance with measurements of molar growth yields.     

Acetylene inhibition technique underestimates in situ denitrification rates in intact cores of freshwater sediment

Abstract Denitrification in intact sediment cores was measured by the acetylene inhibition technique and compared with the nitrate flux between water and sediment. Less than half of the nitrate-N consumed by the sediment could be recovered as nitrous oxide-N. The low recovery rate of nitrous oxide from intact sediment cores indicated losses of nitrous oxide by diffusion down to nitrate-free sediment layers, where reduction of nitrous oxide may take place. In sediment slurries 100% of nitrate-N could be recovered as nitrous oxide-N as long as the nitrate concentration in the liquid phase was above 10 μM. Nitrous oxide added to nitrate-free sediment slurries was reduced regardless of whether acetylene was present or not. Therefore denitrification may be significantly underestimated by this method.     

The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment

Abstract Experiments were carried out with slurries of saltmarsh sediment to which varying concentrations of nitrate were added. The acetylene blocking technique was used to measure denitrification by accumulation of nitrous oxide, while reduction of nitrate to nitrite and ammonium was also measured. There was good recovery of reduced nitrate and at the smallest concentration of nitrate used (250 μM) there was approximately equal reduction to either ammonium or nitrous oxide (denitrification). Nitrite was only a minor end-product of nitrate reduction. As the nitrate concentration was increased the proportion of the nitrate which was denitrified to nitrous oxide increased, to 83% at the greatest nitrate concentration used (2 mM), while reduction to ammonium correspondingly decreased. This change was attributed either to a greater competitiveness by the denitrifiers for nitrate as the ratio of electron donor to electron acceptor decreased; or to the increased production of nitrite rather than ammonium by fermentative bacteria under high nitrate, the nitrite then being reduced to nitrous oxide by denitrifying bacteria.     

Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene.

15N tracer methods and gas chromatography coupled to an electron capture detector were used to investigate dissimilatory reduction of nitrate and nitrite by the rumen microbiota of a fistulated cow. Ammonium was the only 15N-labeled end product of quantitative significance. Only traces of nitrous oxide were detected as a product of nitrate reduction; but in experiments with nitrite, up to 0.3% of the added nitrogen accumulated as nitrous oxide, but it was not further reduced. Furthermore, when 13NO3- was incubated with rumen microbiota virtually no [13N]N2 was produced. Acetylene partially inhibited the reduction of nitrite to ammonium as well as the formation of nitrous oxide. It is suggested that in the rumen ecosystem nitrous oxide is a byproduct of dissimilatory nitrite reduction to ammonium rather than a product of denitrification and that the latter process is absent from the rumen habitat.     

Denitrification in lucerne nodules and bacteroids supplied with nitrate

Nodulated lucerne plants ( Medicago sativa L. cv. Aragón) were supplied with 20 m M nitrate. Anaerobically isolated bacteroids of Rhizobium meliloti from these plants were able to denitrify after 48 h treatment. R. meliloti bacteroids behave as total denitrifiers, reducing nitrate to dinitrogen: when acetylene was omitted from the assay medium very little nitrous oxide was recovered. The onset of denitrification activity was coincident with the induction of nitrite reductase activity (EC 1.7.99.3) whereas nitrate reductase activity (EC 1.7.99.4) was constitutive. Whole nodules from plants receiving several doses of nitrate were assayed, in a nitrate-free medium, to monitor denitrification activity dependent on nitrate within the nodules. Denitrification activity was detected after 2 days of 20 m M nitrate supply or after 3 days in the presence of 10 or 5 m M nitrate. These results are discussed in relation to current controversy about nitrate entry into the infection region of nodules. It is concluded that this process occurs more rapidly than suggested in recent research.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Structural aspects of denitrifying enzymes

The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.     

Respiratory nitrate ammonification by Shewanella oneidensis MR-1

Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Specific sodium dependence of a nitrate reductase in a marine bacterium

Abstract The two key enzymes of denitrification, the nitrate and nitrate reductases, were studied in a marine organism, Pseudomonas nautica , strain 617. Both enzymes were grown under anaerobic conditions with either nitrate or nitrous oxide as electron acceptor. The effect of sodium on these enzymes was studied. Only sodium activated the membranous and purified nitrate reductase, and none of the salts affected nitrite reductase.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Dissimilatory nitrate reduction by Propionibacterium acnes

Propionibacterium acnes P13 was isolated from human feces. The bacterium produced a particulate nitrate reductase and a soluble nitrite reductase when grown with nitrate or nitrite. Reduced viologen dyes were the preferred electron donors for both enzymes. Nitrous oxide reductase was never detected. Specific growth rates were increased by nitrate during growth in batch culture. Culture pH strongly influenced the products of dissimilatory nitrate reduction. Nitrate was principally converted to nitrite at alkaline pH, whereas nitrous oxide was the major product of nitrate reduction when the bacteria were grown at pH 6.0. Growth yields were increased by nitrate in electron acceptor-limited chemostats, where nitrate was reduced to nitrite, showing that dissimilatory nitrate reduction was an energetically favorable process in P. acnes. Nitrate had little effect on the amounts of fermentation products formed, but molar ratios of acetate to propionate were higher in the nitrate chemostats. Low concentrations of nitrite (ca. 0.2 mM) inhibited growth of P. acnes in batch culture. The nitrite was slowly reduced to nitrous oxide, enabling growth to occur, suggesting that denitrification functions as a detoxification mechanism.     

Dissimilatory nitrate reduction by Propionibacterium acnes.

Propionibacterium acnes P13 was isolated from human feces. The bacterium produced a particulate nitrate reductase and a soluble nitrite reductase when grown with nitrate or nitrite. Reduced viologen dyes were the preferred electron donors for both enzymes. Nitrous oxide reductase was never detected. Specific growth rates were increased by nitrate during growth in batch culture. Culture pH strongly influenced the products of dissimilatory nitrate reduction. Nitrate was principally converted to nitrite at alkaline pH, whereas nitrous oxide was the major product of nitrate reduction when the bacteria were grown at pH 6.0. Growth yields were increased by nitrate in electron acceptor-limited chemostats, where nitrate was reduced to nitrite, showing that dissimilatory nitrate reduction was an energetically favorable process in P. acnes. Nitrate had little effect on the amounts of fermentation products formed, but molar ratios of acetate to propionate were higher in the nitrate chemostats. Low concentrations of nitrite (ca. 0.2 mM) inhibited growth of P. acnes in batch culture. The nitrite was slowly reduced to nitrous oxide, enabling growth to occur, suggesting that denitrification functions as a detoxification mechanism.     

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.