Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

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.     

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.     

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.     

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.     

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.     

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.