Combined Oxygen and Nitrous Oxide Microsensor for Denitrification Studies

The construction of a microsensor which can be used to measure O₂ and N₂O simultaneously is described. The microsensor exhibited a linear response to both O₂ and N₂O, and the response to N₂O was independent of the O₂ concentration and vice versa. The N₂O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter⁻¹. The signals for O₂ and N₂O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O₂ and N₂O were measured in a biofilm which was exposed to acetylene to block the N₂O reductase activity of denitrifying bacteria. O₂ penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N₂O concentration at 1.4 mm depth increased from 32 to 411 μmol liter⁻¹ after the addition of the inhibitor. The shape of the N₂O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm.     

Measurement of Nitrous Oxide Reductase Activity in Aquatic Sediments

Denitrification in aquatic sediments was measured by an N₂O reductase assay. Sediments consumed small added quantities of N₂O over short periods (a few hours). In experiments with sediment slurries, N₂O reductase activity was inhibited by O₂, C₂H₂, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 μM) did not influence activity, and moderate levels (about 150 μM) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N₂O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater (K_m = 2.17 μM), estuarine (K_m = 14.5 μM), and alkaline-saline (K_m = 501 μM) environments. An in situ assay was devised in which a solution of N₂O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N₂O per m² per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N₂O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N₂O per m² per h made with the acetylene block assay.     

Production of Nitric Oxide and Nitrous Oxide During Denitrification by Corynebacterium nephridii

Resting cells of Corynebacterium nephridii reduce nitrate, nitrite, and nitric oxide to nitrous oxide under anaerobic conditions. Nitrous oxide production from nitrite was optimal from pH 7.0 to 7.4. The stoichiometry of nitrous oxide production from nitrite was 99% of the theoretical-two moles of nitrite was used for each mole of nitrous oxide detected. Hydroxylamine increases gas evolution from nitrite but inhibits the reduction of nitric oxide to nitrous oxide. Hydroxylamine is converted to nitrogenous gas(es) by resting cells only in the presence of nitrite. Under certain conditions nitric oxide, as well as nitrous oxide, was detected.     

Sediment Nitrification, Denitrification, and Nitrous Oxide Production in a Deep Arctic Lake

We used a combination of ¹⁵N tracer methods and a C₂H₂ blockage technique to determine the role of sediment nitrification and denitrification in a deep oligotrophic arctic lake. Inorganic nitrogen concentrations ranged between 40 and 600 nmol · cm⁻³, increasing with depth below the sediment-water interface. Nitrate concentrations were at least 10 times lower, and nitrate was only detectable within the top 0 to 6 cm of sediment. Eh and pH profiles showed an oxidized surface zone underlain by more reduced conditions. The lake water never became anoxic. Sediment Eh values ranged from −7 to 484 mV, decreasing with depth, whereas pH ranged from 6.0 to 7.3, usually increasing with depth. The average nitrification rate (49 ng of N · cm⁻³ · day⁻¹) was similar to the average denitrification rate (44 ng of N · cm⁻³ · day⁻¹). In situ N₂O production from nitrification and denitrification ranged from 0 to 25 ng of N · cm⁻³ · day⁻¹. Denitrification appears to depend on the supply of nitrate by nitrification, such that the two processes are coupled functionally in this sediment system. However, the low rates result in only a small nitrogen loss.     

Temporal Change in Nitrous Oxide and Dinitrogen from Denitrification Following Onset of Anaerobiosis

Similar temporal patterns were found in three mineral soils for the composition of the gaseous products of denitrification following the onset of anaerobic conditions. During the early period of anaerobiosis (0 up to 1 to 3 h), N₂ was the dominant product of denitrification. The NO₃⁻ → N₂O activity then increased, but was not accompanied by a corresponding increase in N₂O-reducing activity. This resulted in a relatively extended period of time (1 to 3 up to 16 to 33 h) during which N₂O was a major product. Eventually (after 16 to 33 h), an increase in N₂O-reducing activity occurred without a comparable increase in the N₂O-producing activity. The increase in the rate of N₂O reduction did not occur in the presence of chloramphenicol and required the presence of N₂O or NO₃⁻ during the preceding anaerobic incubation. During the final period (16 to 33, up to 48 h), N₂ was generally the sole product of denitrification, since the rate of N₂O reduction exceeded the rate of N₂O production. A similar sequential pattern was also found for a culture of a denitrifying Flavobacterium sp. shifted to anaerobic growth. A staggered synthesis of the enzymes in the denitrification sequence apparently occurred in response to anoxia, which caused first a net production of N₂O followed by consumption of N₂O.     

Nitrite and Nitrous Oxide Accumulation During Denitrification in the Presence of Pesticide Derivatives

Temporary accumulation of nitrite and nitrous oxide was observed in soil incubated under anaerobic conditions when derivatives of the insecticide chlordimeform [(N-4-chloro-o-tolyl)-N′,N′ -dimethylformamidine] were added. Chlordimeform did not affect the denitrification process, but N-formyl-4-chloro-o-toluidine and 4-chloro-o-toluidine caused an inhibition as determined by the accumulation of nitrite and nitrous oxide. A simultaneous application of the insecticide and its derivatives resulted in a stronger inhibitory effect than the application of each compound separately. Aniline intermediates of other pesticides also inhibited denitrification in soil, and they proved to be more effective than their parent compound.     

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.     

Denitrification, Nitrate Reduction, and Oxygen Consumption in Coastal and Estuarine Sediments

Denitrification and consumption of oxygen and nitrate in sediments from Tama Estuary, Odawa Bay, and Tokyo Bay were measured in an experimental sediment-water system. Filtered seawater containing [¹⁵N]nitrate flowed continuously over undisturbed sediments, and the concentrations of O₂, nitrate, and nitrite in the influent and effluent and of ¹⁵N₂ in the effluent were monitored. Under steady-state conditions, the rate of nitrate consumption was the same order of magnitude as the rate of oxygen consumption in Tama Estuary sediments, whereas the former rate was one order of magnitude lower than the latter rate in Odawa Bay and Tokyo Bay sediments. Denitrification accounted for 27 to 57% of the nitrate consumption.     

Diversity of Nitrous Oxide Reductase (nosZ) Genes in Continental Shelf Sediments

Diversity of the nitrous oxide reductase (nosZ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the nosZ gene were amplified via PCR, using nosZ gene-specific primers. Thirty-seven unique copies of the nosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% ± 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (Cu_Z) of the mature protein. Phylogenetic analysis demonstrated three major clusters of nosZ genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.     

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

The intramolecular distribution of nitrogen isotopes in N₂O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N₂O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N₂O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N₂O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N₂O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ~33‰ and ~0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N₂O.     

Inability of Pseudomonas stutzeri Denitrification Mutants with the Phenotype of Pseudomonas aeruginosa to Grow in Nitrous Oxide

[This corrects the article on p. 1301 in vol. 50.].     

Clinical Trials of Different Concentrations of Oxygen and Nitrous Oxide for Obstetric Analgesia: Report to the Medical Research Council of the Committee on Nitrous Oxide and Oxygen Analgesia in Midwifery

Trials have been organized by a Medical Research Council committee to assess the effectiveness and safety for analgesia in labour of oxygen and nitrous oxide mixtures in different proportions. In a preliminary trial concentrations of 50% and 60% v/v nitrous oxide were compared, but, as the replies of 409 mothers revealed little difference between the two, the results of administering either 50% or 70% nitrous oxide to 778 mothers were then compared. The data relating to normal labour, obtained on 501 of the mothers in this main trial, showed that the relief of pain given was much the same. There was a suggestion, however, that the higher concentration of nitrous oxide might be useful in abnormal labour. The proportion of mothers with normal deliveries who lost consciousness, though very small, was significantly higher with 70% nitrous oxide than with the lower concentration. Ninety-two per cent. of mothers found the gas and oxygen machine helpful, and midwives reported complete or good co-operation by 77% of those using it. It is concluded that the 50% oxygen and 50% nitrous oxide mixture can safely be used by unsupervised midwives.     

Denitrification, Dissimilatory Reduction of Nitrate to Ammonium, and Nitrification in a Bioturbated Estuarine Sediment as Measured with 15N and Microsensor Techniques

Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of ¹⁵N isotope (NO₃^-), specific inhibitor (C₂H₂), and microsensor (N₂O and O₂) techniques in a continuous-flow core system. The estuarine water was NO₃^- rich (125 to 600 μM), and NO₃^- was consistently taken up by the sediment on the four occasions studied. Total NO₃^- uptake (3.6 to 34.0 mmol of N m^-2 day^-1) corresponded closely to N₂ production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO₃^- reduction to NH₄⁺ was insignificant. When C₂H₂ was applied in the flow system, denitrification measured as N₂O production was often less (58 to 100%) than the NO₃^- uptake because of incomplete inhibition of N₂O reduction. The NO₃^- formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from ¹⁵NO₃^- dilution and from changes in NO₃^- uptake before and after C₂H₂ addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m^-2 day^-1 on the four occasions. Attempts to measure total nitrification activity by the difference between NH₄⁺ fluxes before and after C₂H₂ addition failed because of non-steady-state NH₄⁺ fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N₂O and O₂ microelectrodes. The N₂O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N₂O after C₂H₂ was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total O₂ uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.     

Gut-Associated Denitrification and In Vivo Emission of Nitrous Oxide by the Earthworm Families Megascolecidae and Lumbricidae in New Zealand

Previous studies have documented the capacity of European earthworms belonging to the family Lumbricidae to emit the greenhouse gas nitrous oxide (N₂O), an activity attributed primarily to the activation of ingested soil denitrifiers. To extend the information base to earthworms in the Southern Hemisphere, four species of earthworms in New Zealand were examined for gut-associated denitrification. Lumbricus rubellus and Aporrectodea rosea (introduced species of Lumbricidae) emitted N₂O, whereas emission of N₂O by Octolasion cyaneum (an introduced species of Lumbricidae) and emission of N₂O by Octochaetus multiporus (a native species of Megascolecidae) were variable and negligible, respectively. Exposing earthworms to nitrite or nitrate and acetylene significantly increased the amount of N₂O emitted, implicating denitrification as the primary source of N₂O and indicating that earthworms emitted dinitrogen (N₂) in addition to N₂O. The alimentary canal displayed a high capacity to produce N₂O when it was supplemented with nitrite, and alimentary canal contents contained large amounts of carbohydrates and organic acids indicative of fermentation (e.g., succinate, acetate, and formate) that could serve as sources of reductant for denitrification. nosZ encodes a portion of the terminal oxidoreductase used in denitrification. The nosZ sequences detected in the alimentary canals of L. rubellus and O. multiporus were similar to those retrieved from soil and were distantly related to sequences of uncultured soil bacteria and genera common in soils (i.e., Bradyrhizobium, Azospirillum, Rhodopseudomonas, Rhodospirillum, Pseudomonas, Oligotropha, and Sinorhizobium). These findings (i) suggest that the capacity to emit N₂O and N₂ is a general trait of earthworms and not geographically restricted, (ii) indicate that species belonging to different earthworm families (i.e., Megascolecidae and Lumbricidae) may not have equal capacities to emit N₂O, and (iii) also corroborate previous findings that link this capacity to denitrification in the alimentary canal.Earthworms are dominant members of the soil fauna and affect the structure and fertility of soils (5, 20, 22, 23). Various species of European earthworms belonging to the family Lumbricidae (e.g., Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) emit dinitrogen (N₂) and the greenhouse gas nitrous oxide (N₂O), and their burrowing activities and feeding habits in combination with in situ conditions can influence the emission of nitrogenous gases from soils that they inhabit (1, 2, 13, 17, 25, 27, 39).The microbiology of the earthworm alimentary canal has been addressed in numerous studies (3, 4, 6, 9, 14, 16, 32). The alimentary canal of the earthworm is anoxic, in marked contrast to the aerated material that earthworms ingest (14, 39). Anoxia and other in situ conditions of the alimentary canal appear to stimulate soil microbes capable of surviving under anaerobic conditions during passage through the gut (3, 4). Soils are rich in denitrifying bacteria (37), and the capacity of European earthworms to emit nitrogenous gases has been attributed primarily to the in situ activity of ingested denitrifying bacteria that appear to be highly active under the anoxic conditions of the earthworm alimentary canal (12, 15, 17, 25, 39). However, it is not known if the capacity to emit nitrogenous gases is a general trait of earthworms independent of their taxonomic family or geographic location. The main objectives of this study were to examine the capacity of Southern Hemisphere earthworms in New Zealand to emit N₂O and to determine if this capacity was linked to denitrifying bacteria in the alimentary canal.     

Microscale Distribution of Nitrification Activity in Sediment Determined with a Shielded Microsensor for Nitrate

Microprofiles of O₂ and NO₃^- were measured simultaneously in freshwater sediment with microsensors which were completely free from electrical interference because of coaxial designs. Depth profiles of nitrification (NO₃^- production) and denitrification (NO₃^- consumption) were subsequently determined by computer simulation of the measured microprofiles. The nitrifying bacterial community responded very quickly to changes in environmental conditions, and new steady-state microprofiles of O₂ and NO₃^- were usually approached within a few hours after perturbation. Nitrification started quickly after introduction of O₂ in previously anoxic layers, suggesting prolonged survival of the nitrifiers during anaerobiosis. Changes in the availability of O₂ and NH₄⁺ greatly affected the nitrification profile, and there was a high rate of coupled nitrification-denitrification under conditions in which nitrification occurred right above the oxic-anoxic interface. Addition of C₂H₂ rapidly removed the NO₃^- peaks, indicating that NO₃^- production was due mainly to autotrophic nitrification.