Thermophilic Bacillus sp. that shows the denitrification phenotype of Pseudomonas aeruginosa.

A thermophilic Bacillus sp. of marine origin was observed to grow anaerobically on nitrite, nitrous oxide (N2O) in the presence of nitrite, and N2O alone for a few hours after exhaustion of nitrite. This represents the second example of a denitrification phenotype originally observed to occur with Pseudomonas aeruginosa.     

Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions.

Nitrous oxide can be a harmful by-product in nitrogen removal from wastewater. Since wastewater treatment systems operate under different aeration regimens, the influence of different oxygen concentrations and oxygen fluctuations on denitrification was studied. Continuous cultures of Alcaligenes faecalis TUD produced N2O under anaerobic as well as aerobic conditions. Below a dissolved oxygen concentration of 5% air saturation, the relatively highest N2O production was observed. Under these conditions, significant activities of nitrite reductase could be measured. After transition from aerobic to anaerobic conditions, there was insufficient nitrite reductase present to sustain growth and the culture began to wash out. After 20 h, nitrite reductase became detectable and the culture started to recover. Nitrous oxide reductase became measurable only after 27 h, suggesting sequential induction of the denitrification reductases, causing the transient accumulation of N2O. After transition from anaerobic conditions to aerobic conditions, nitrite reduction continued (at a lower rate) for several hours. N2O reduction appeared to stop immediately after the switch, indicating inhibition of nitrous oxide reductase, resulting in high N2O emissions (maximum, 1.4 mmol liter-1 h-1). The nitrite reductase was not inactivated by oxygen, but its synthesis was repressed. A half-life of 16 to 22 h for nitrite reductase under these conditions was calculated. In a dynamic aerobic-anaerobic culture of A. faecalis, a semisteady state in which most of the N2O production took place after the transition from anaerobic to aerobic conditions was obtained. The nitrite consumption rate in this culture was equal to that in an anaerobic culture (0.95 and 0.92 mmol liter-1 h-1, respectively), but the production of N2O was higher in the dynamic culture (28 and 26% of nitrite consumption, respectively).     

Production of NO and N(inf2)O by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration

Peak emissions of NO and N(inf2)O are often observed after wetting of soil. The reactions to sudden changes in the aeration of cultures of nitrifying and denitrifying bacteria with respect to NO and N(inf2)O emissions were compared to obtain more information about the microbiological aspects of peak emissions. In continuous culture, the nitrifier Nitrosomonas europaea and the denitrifiers Alcaligenes eutrophus and Pseudomonas stutzeri were cultured at different levels of aeration (80 to 0% air saturation) and subjected to changes in aeration. The relative production of NO and N(inf2)O by N. europaea, as a percentage of the ammonium conversion, increased from 0.87 and 0.17%, respectively, at 80% air saturation to 2.32 and 0.78%, respectively, at 1% air saturation. At 0% air saturation, ammonium oxidation and N(inf2)O production ceased but NO production was enhanced. Coculturing of N. europaea with the nitrite oxidizer Nitrobacter winogradskyi strongly reduced the relative levels of NO and N(inf2)O production, probably as an effect of the lowered nitrite concentration. After lowering the aeration, N. europaea produced large short-lasting peaks of NO and N(inf2)O emissions in the presence but not in the absence of nitrite. A. eutrophus and P. stutzeri began to denitrify below 1% air saturation, with the former accumulating nitrite and N(inf2)O and the latter reducing nitrate almost completely to N(inf2). Transition of A. eutrophus and P. stutzeri from 80 to 0% air saturation resulted in transient maxima of denitrification intermediates. Such transient maxima were not observed after transition from 1 to 0%. Reduction of nitrate by A. eutrophus continued 48 h after the onset of the aeration, whereas N(inf2)O emission by P. stutzeri increased for only a short period. It was concluded that only in the presence of nitrite are nitrifiers able to dominate the NO and N(inf2)O emissions of soils shortly after a rainfall event.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

Denitrification at extremely high pH values by the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD

Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8(2-)) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd1-like cytochrome and a cytochrome-aa3-like component in the membranes.     

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.     

Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa.

The pathway of anaerobic reduction of nitrite to nitrogen gas (N2) by cell suspensions of the denitrifier, Pseudomonas aeruginosa, was studied using the techniques of gas chromatography and mass spectrometry. While release of nitrous oxide (N2O) is not normally detected during the reduction of nitrite to N2 by this organism, 15N from [15N]nitrite nevertheless can be trapped quantitatively as 15N2O in a pool of added N2O. In such experiments the abundance of 15N in N2O always exceeds that in product N2, consistent with the absence of a major reductive route from nitrite to N2 which by-passes N2O. During the reduction of a mixture of [15N]nitrite and nitric oxide (NO), 15NO produced at most only in trace amounts. The final products are chiefly 15N2 and 14N2 with only a small fraction of the scrambled product, 14N15N. Much of the 14N15N can be accounted for as an artifact caused by traces of molecular oxygen, which promote the conversion of NO to nitrite by autooxidation and thereby degrade slightly the isotopic purity of [15N]nitrite. Nitrous oxide shows all the properties of a free obligatory intermediate during the denitrification of nitrite to N2 by P. aeruginosa, whereas NO does not. The inability to trap 15NO in a pool of NO indicates that NO is not a free obligatory intermediate in the reduction of nitrite. The small mole fractions of 14N15N produced from a mixture of [15N]nitrite and NO require that the main reductive pathways for these nitrogen oxides cannot share any freely diffusible mono-nitrogen intermediate in common. The simplest interpretation is that nitrite and NO are denitrified by separate pathways, at least prior to the formation of the first bi-nitrogen compound.     

Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes

A lab-scale sequencing batch reactor fed with real municipal wastewater was used to study nitrous oxide (N(2)O) emissions from simulated wastewater treatment processes. The experiments were performed under four different controlled conditions as follows: (1) fully aerobic, (2) anoxic-aerobic with high dissolved oxygen (DO) concentration, (3) anoxic-aerobic with low DO concentration, and 4) intermittent aeration. The results indicated that N(2)O production can occur from both incomplete nitrification and incomplete denitrification. N(2)O production from denitrification was observed in both aerobic and anoxic phases. However, N(2)O production from aerobic conditions occurred only when both low DO concentrations and high nitrite concentration existed simultaneously. The magnitude of N(2) O produced via anoxic denitrification was lower than via oxic denitrification and required the presence of nitrite. Changes in DO, ammonium, and nitrite concentrations influenced the magnitude of N(2)O production through denitrification. The results also suggested that N(2)O can be produced from incomplete denitrification and then released to the atmosphere during aeration phase due to air stripping. Therefore, biological nitrogen removal systems should be optimized to promote complete nitrification and denitrification to minimize N(2)O emissions.     

15N tracer studies on the reduction of nitrite by the purified dissimilatory nitrite reductase of Pseudomonas aeruginosa. Evidence for direct production of N2O without free NO as an intermediate

Nitrite reductase (cytochrome c,d1) was purified from Pseudomonas aeruginosa. In the presence of the reducing system, ascorbate-N,N,N',N'-tetramethylphenyl-enediamine, which alone had no ability to reduce nitrite or NO at pH 7.5, the enzyme catalyzed the reduction of nitrite to NO and N2O as major and minor products, respectively, as determined by gas chromatography-mass spectrometry. The rate of reduction of NO to N2O was considerably lower than the rate of reduction of nitrite to N2O and might be zero. The N2O produced in a system containing [15N]nitrite and natural NO was more highly enriched in 15N than was the NO pool and, in this regard, closely resembled the enrichment of the nitrite pool. The amount of 14N in the NO pool changed little, if any, as the result of enzymatic processes. For the enzyme, free NO seems not to be an intermediate between nitrite and N2O, just as was found by this laboratory for certain intact denitrifying bacteria. The results are consistent with reduction of nitrite to enzyme-bound NO, which can partition between release and further reduction.     

Catalysis of intermolecular oxygen atom transfer by nitrite dehydrogenase of Nitrobacter agilis

Nitrobacter agilis, which contains a very active nitrite dehydrogenase, was studied in vivo under anaerobic conditions by the 15N NMR technique. When incubated with equimolar 15NO3- and unlabeled nitrite (or 15NO2- and unlabeled nitrate) the bacterium catalyzed an isotope exchange reaction at rates about 10% those observed in the nitrite oxidase assay. When incubated with 18O-labeled 15NO2- and 18O-labeled 15NO3-, the 18O was observed to exchange at similar rates from both species into water. Finally, when incubated with equimolar [18O]nitrate and 15NO2-, intermolecular 18O transfer was observed to result in formation of double labeled nitrate and nitrite at similar rates. 18O was transferred from nitrate to a 15N species or to water at approximately equal rates under the conditions of the experiments. It is argued that the enzyme responsible for these exchange reactions is nitrite dehydrogenase and not nitrate reductase. This work and the related experiments of DiSpirito and Hooper (DiSpirito, A.A., and Hooper, A.B. (1986) J. Biol. Chem. 261, 10534-10537) represent the first demonstrations of intermolecular oxygen atom transfer among oxotransferases. Mechanistic implications are discussed.     

15N-tracer and NMR studies on the pathway of denitrification. Evidence against trioxodinitrate but for nitroxyl as an intermediate

The effects of four species of denitrifying bacteria on the conversion of [15N]nitrite to trioxodinitrate (HN2O3-) and N2O and of trioxodinitrate to N2O were studied. For all species, the N2O produced in the presence of [15N]nitrite and trioxodinitrate was isotopically randomized throughout the period of incubation and was not composed at the outset predominantly of 14N2O or 14N2O plus 15N2O. The N2O produced was also heavily enriched in 15N at times when the trioxodinitrate pool was only weakly enriched in 15N. By 15N NMR, the N(2) position, but not the N(1) position, of trioxodinitrate was found to become progressively labeled with 15N during incubation with [15N]nitrite. These results argue that (a) the N-N bond of trioxodinitrate is not preserved in its conversion to N2O, (b) trioxodinitrate can be neither a free nor enzyme-bound intermediate in denitrifying bacteria, and (c) the pathways from nitrite and trioxodinitrate involve a common mononitrogen intermediate. The conclusion that this intermediate is probably nitroxyl (HNO), at least with Paracoccus denitrificans and Pseudomonas stutzeri, provides indirect evidence that N-N bond formation in denitrification can occur through the dimerization of nitroxyl.     

Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.     

Low NO concentration dependence of reductive nitrosylation reaction of hemoglobin

The reductive nitrosylation of ferric (met)hemoglobin is of considerable interest and remains incompletely explained. We have previously observed that at low NO concentrations the reaction with tetrameric hemoglobin occurs with an observed rate constant that is at least 5 times faster than that observed at higher concentrations. This was ascribed to a faster reaction of NO with a methemoglobin-nitrite complex. We now report detailed studies of this reaction of low NO with methemoglobin. Nitric oxide paradoxically reacts with ferric hemoglobin with faster observed rate constants at the lower NO concentration in a manner that is not affected by changes in nitrite concentration, suggesting that it is not a competition between NO and nitrite, as we previously hypothesized. By evaluation of the fast reaction in the presence of allosteric effectors and isolated β- and α-chains of hemoglobin, it appears that NO reacts with a subpopulation of β-subunit ferric hemes whose population is influenced by quaternary state, redox potential, and hemoglobin dimerization. To further characterize the role of nitrite, we developed a system that oxidizes nitrite to nitrate to eliminate nitrite contamination. Removal of nitrite does not alter reaction kinetics, but modulates reaction products, with a decrease in the formation of S-nitrosothiols. These results are consistent with the formation of NO(2)/N(2)O(3) in the presence of nitrite. The observed fast reductive nitrosylation observed at low NO concentrations may function to preserve NO bioactivity via primary oxidation of NO to form nitrite or in the presence of nitrite to form N(2)O(3) and S-nitrosothiols.     

Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase (cytochrome c,d1)

The dissimilatory nitrite reductase (cytochrome c,d1) from Pseudomonas aeruginosa was observed at pH 7.5 to catalyze nitrosyl transfer (nitrosation) between [15N]nitrite and several N-nucleophiles or H2 18O, with rate enhancement of the order of 10(8) relative to analogous chemical reactions. The reducing system (ascorbate, N,N,N',N'-tetramethylphenylenediamine) could reduce nitrite (but not NO) enzymatically and had essentially no direct chemical reactivity toward nitrite or NO. The N-nitrosations showed saturation kinetics with respect to the nucleophile and, while exhibiting Vmax values which varied by about 40-fold, nevertheless showed little or no dependence of Vmax on nucleophile pKa. The N-nitrosations and NO-2/H2O-18O exchange required the reducing system, whereas NO/H2O-18O exchange was inhibited by the reducing system. NO was not detected to serve as a nitrosyl donor to N-nucleophiles. These and other kinetic observations suggest that the enzymatic nitrosyl donor is an enzyme-bound species derived from reduced enzyme and one molecule of nitrite, possibly a heme-nitrosyl compound (E-FeII X NO+) for which there is precedence. Nitrosyl transfer to N-nucleophiles may occur within a ternary complex of enzyme, nitrite, and nucleophile. Catalysis of nitrosyl transfer by nitrite reductase represents a new class of enzymatic reactions and may present another example of electrophilic catalysis by a metal center. The nitrosyl donor trapped by these reactions is believed to represent an intermediate in the reduction of nitrite by cytochrome c,d1.     

Nitrite and nitrous oxide production by Methylosinus trichosporium

Conditions for the production of nitrite and nitrous oxide by an obligate methanotroph, Methylosinus trichosporium (OB 3b), were studied. The rate of nitrite production (V NO2-) was correlated with the concentration of ammonia up to 20 mM in the presence of sufficient amounts of oxygen and inversely correlated with the amounts of methane in the system. The rate of nitrous oxide (N2O) production (V N2O) was correlated positively with V NO2- and the amount of nitrite produced and inversely with the oxygen concentration in the system. Nitrite started to disappear when either oxygen or methane or both were depleted, but only a part of the loss could be accounted for by an increase in N2O. Maximum rates of nitrite and N2O production by Ms. trichosporium were 6.9 X 10(-16) and 2.2 X 10(-17) mol . cell-1 X day-1, respectively. These values are about 0.2 and 1.6% of the values for Nitrosomonas europaea. Therefore, production of nitrite and N2O by methanotrophs in aquatic environments may not be as significant as that of Nitrosomonas.     

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.     

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.     

Isotope labeling studies on the mechanism of N-N bond formation in denitrification

The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).     

Denitrification by fungi

Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N2O), and/or dinitrogen (N2). Several strains could reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nitrite. Several fungi could also form N2. When [15]nitrite was used as substrate for the N2-forming denitrification, 15N2O, 15NO, and 14N15N were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen.     

N2O-producing microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria

The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N(2)O) and (ii) to characterize the N(2)O-producing bacteria of the earthworm gut. The production of N(2)O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N(2)O were transient products when supplemental nitrate was reduced to N(2) by gut content homogenates. In contrast, nitrite and N(2)O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N(2)O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N(2)O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N(2)O or N(2). The initial N(2)O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N(2)O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N(2)O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.