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.     

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.     

Relative Rates of Nitric Oxide and Nitrous Oxide Production by Nitrifiers, Denitrifiers, and Nitrate Respirers

Biogenic emissions of nitric and nitrous oxides have important impacts on the photochemistry and chemistry of the atmosphere. Although biogenic production appears to be the overwhelming source of N₂O, the magnitude of the biogenic emission of NO is very uncertain. In soils, possible sources of NO and N₂O include nitrification by autotrophic and heterotrophic nitrifiers, denitrification by nitrifiers and denitrifiers, nitrate respiration by fermenters, and chemodenitrification. The availability of oxygen determines to a large extent the relative activities of these various groups of organisms. To better understand this influence, we investigated the effect of the partial pressure of oxygen (pO₂) on the production of NO and N₂O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO₂ in the range tested (0.5 to 10%), whereas N₂O production was inversely proportional to pO₂. Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N₂O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N₂O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N₂O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N₂O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sparged with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N₂O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.     

Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N₂O) were identified by GC and GC/MS as the main N products formed. NO and N₂O produced from soybean leaflets were both labeled with ¹⁵N when ¹⁵N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N₂O were identified as air (N₂, O₂), CO₂, methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N₂O (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N₂O were not evolved from leaves of the nr₁ soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO_(x) (NO and NO₂). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Merr., and Pueraria thunbergiana Benth. evolved lower levels of NO_(x).     

Production and Loss of Nitric Oxide from Denitrification in Anaerobic Brookston Clay

Nitric oxide, nitrous oxide, and nitrite ion production was measured in a Brookston clay column undergoing anaerobic denitrification. A flow system method was used whereby argon carrier gas continuously stripped soil gases from the column, allowing steady-state rates to be obtained. Over several days the temporal change in rates of these gases and NO₂⁻ followed a pattern of increase and decay which may be expected of a reaction proceeding by several consecutive steps. The method permits observation of the relatively large net production rate of NO, which is normally not observed in static systems based on head space analysis of gaseous denitrification products. In the first several hours after the onset of anoxic conditions, the net rate of NO production, f_NO, increased sharply to a maximum (~1 × 10⁻¹⁰ mol of N/g of soil per min), paralleling the rapid increase in NO₂⁻ level, and then followed a more gradual decline extending over approximately 45 h. A similar but less pronounced pattern was observed for N₂O, with net rates of production being considerably less than for NO. The ratio [NO-N]/[N₂O-N] decreased with time from ~2.5 at 6 h to ~2.0 at 45 h. Estimated rates of N₂ production appeared to be initially high, decreased rapidly within a few hours, and then gradually increased with time after the establishment of anaerobic conditions.     

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.     

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.     

NO和N2O与采后园艺作物的保鲜

合适浓度的NO和N2O可明显延长果蔬和花卉园艺产品的采后货架期,并改善品质,减少水分消耗.NO和N2O的作用机制可能是抑制乙烯的生理效应.该文就这些方面的研究进展作一介绍.     

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.     

Nitrous Oxide Production and Methane Oxidation by Different Ammonia-Oxidizing Bacteria

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N₂O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N₂O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N₂O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N₂O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N₂O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH₄ oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH₄ in soils.     

Production of Nitrous Oxide by Ammonia-Oxidizing Chemoautotrophic Microorganisms in Soil

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N₂O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N₂O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N₂O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.     

Separate Nitrite, Nitric Oxide, and Nitrous Oxide Reducing Fractions from Pseudomonas perfectomarinus

Pseudomonas perfectomarinus was found to grow anaerobically at the expense of nitrate, nitrite, or nitrous oxide but not chlorate or nitric oxide. In several repetitive experiments, anaerobic incubation in culture media containing nitrate revealed that an average of 82% of the cells in aerobically grown populations were converted to the capacity for respiration of nitrate. Although they did not form colonies under these conditions, the bacteria synthesized the denitrifying enzymes within 3 hr in the absence of oxygen or another acceptable inorganic oxidant. This was demonstrated by the ability, after anaerobic incubation, of cells and of extracts to reduce nitrite, nitric oxide, and nitrous oxide to nitrogen. From crude extracts of cells grown on nitrate, nitrite, or nitrous oxide, separate complex fractions were obtained that utilized reduced nicotinamide adenine dinucleotide as the source of electrons for the reduction of (i) nitrite to nitric oxide, (ii) nitric oxide to nitrous oxide, and (iii) nitrous oxide to nitrogen. Gas chromatographic analyses revealed that each of these fractions reduced only one of the nitrogenous oxides.     

Nitrous Oxide Production by Organisms Other than Nitrifiers or Denitrifiers

Heterotrophic bacteria, yeasts, fungi, plants, and animal breath were investigated as possible sources of N₂O. Microbes found to produce N₂O from NO₃⁻ but not consume it were: (i) all of the nitrate-respiring bacteria examined, including strains of Escherichia, Serratia, Klebsiella, Enterobacter, Erwinia, and Bacillus; (ii) one of the assimilatory nitrate-reducing bacteria examined, Azotobacter vinelandii, but not Azotobacter macrocytogenes or Acinetobacter sp.; and (iii) some but not all of the assimilatory nitrate-reducing yeasts and fungi, including strains of Hansenula, Rhodotorula, Aspergillus, Alternaria, and Fusarium. The NO₃⁻-reducing obligate anaerobe Clostridium KDHS2 did not produce N₂O. Production of N₂O occurred only in stationary phase. The nitrate-respiring bacteria produced much more N₂O than the other organisms, with yields of N₂O ranging from 3 to 36% of 3.5 mM NO₃⁻. Production of N₂O was apparently not regulated by ammonium and was not restricted to aerobic or anaerobic conditions. Plants do not appear to produce N₂O, although N₂O was found to arise from some damaged plant tops, probably due to microbial growth. Concentrations of N₂O above the ambient level in the atmosphere were found in human breath and appeared to increase after a meal of high-nitrate food.     

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.     

Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N₂O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 × 10⁻¹⁷ mol of NO cell⁻¹ day⁻¹, mostly after a culture became O₂ limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O₂, and required CH₄. Denitrification (methanol-supported N₂O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd₁ and Cu nitrite reductases, NO reductase, and N₂O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O₂ and nitrate availability occur.     

Expression of Inducible Nitric Oxide Synthase mRNA and Nitric Oxide Production During the Development of Liver Abscess in Hamster Inoculated with Entamoeba histolytica

The present study analyzed iNOS and eNOS mRNA expression and NO production during development of hepatic abscess caused by Entamoeba histolytica trophozoites. One 374-bp sequence, which displayed 88% identity to mammalian iNOS protein, was isolated from LPS-stimulated peritoneal hamster macrophages. A separate 365-bp cDNA sequence showed 99% identity with eNOS protein. iNOS mRNA was detected in hamsters during formation of amoebic liver abscesses, but not in control hamsters. eNOS mRNA expression was not modified. Serum nitrite concentration in hamsters infected with E. histolytica was 33 ± 6 μM, in control hamsters was 20 ± 3 μM. The study shows that iNOS mRNA expression and NO production are induced by E. histolytica trophozoites during amoebic liver abscess formation. However, in spite of iNOS mRNA expression and NO production, E. histolytica trophozoites induced liver abscess formation in hamster.     

Stimulation of Cytokine and Nitric Oxide Production by Acyclic Nucleoside Phosphonates

Abstract

Several antiviral acyclic nucleotide analogues activate expression of genes for cytokines, such as TNF-α, IL-10 in macrophages and IFN-γ in splenocytes. This is an underlying mechanism for substantially enhanced production of nitric oxide generated by IFN-γ. More lipophilic prodrugs, bis-POM-PMEA and bis-POC-PMPA, are cytocidal for macrophages and thus inhibit nitric oxide formation.