Effect of sulfide on nitrate reduction in mixed methanogenic cultures

The effect of sulfide on nitrate reduction and methanogenesis was investigated in two mixed, mesophilic (35 degrees C) methanogenic cultures: sulfide-free and sulfide-acclimated (67 mg S/L total sulfide). A mixture of dextrin/peptone served as the carbon/electron donor source for the two stock cultures, as well as in all assays reported here. The sulfide-free enriched culture was amended with both nitrate (75-350 mg N/L) and sulfide (10-100 mg S/L). Denitrification was the predominant pathway at all sulfide levels tested and methanogenesis did not recover in any of the sulfide- and nitrate-amended cultures, except in the 10 mg S/L culture. Accumulation of denitrification intermediates such as NO and N(2)O took place, which irreversibly inhibited the methanogens and resulted in the complete cessation of methane production. In contrast, conversion of nitrate to nitrite and then to ammonia via dissimilatory nitrate reduction to ammonia (DNRA) prevented the accumulation of denitrification intermediates and led to the recovery of methanogenesis in the nitrate-amended, sulfide-acclimated, mixed methanogenic culture. The effect of the COD/N value on nitrate reduction was assessed with the sulfide-acclimated, methanogenic culture at COD/N values of 10, 20, and 60. As the COD/N value increased, the fraction of nitrate reduced through DNRA also increased. The results of this study have significant implications relative to the combined anaerobic treatment of carbon-, nitrogen-, and/or sulfur-bearing wastes.     

Electron donor effect on nitrate reduction pathway and kinetics in a mixed methanogenic culture

The effect of different electron donors on the pathway and kinetics of nitrate reduction in a sulfide-acclimated mixed, mesophilic (35 degrees C) methanogenic culture was investigated. A mixture of dextrin and peptone, glucose, propionate, acetate, and H(2)/CO(2) were used as substrates at an initial chemical oxygen demand of 1,500 mg/L and the initial nitrate concentration ranged between 0 and 300 mg N/L. The fastest nitrate reduction was observed in the H(2)/CO(2) and acetate-fed cultures. In the case of propionate, nitrate reduction was the slowest followed by partial recovery of methanogenesis and accumulation of volatile fatty acids due to inhibition as a result of accumulation of denitrification intermediates. Similarly, accumulation of nitrite and nitric oxide and partial or complete inhibition of methanogenesis was observed in the H(2)/CO(2)-fed cultures. Methanogenesis completely recovered in the dextrin/peptone-, glucose-, and acetate-fed cultures at all nitrate levels. Denitrification was the dominant pathway of nitrate reduction in the propionate-, acetate-, and H(2)/CO(2)-fed cultures regardless of the COD/N value. However, both denitrification and dissimilatory nitrate reduction to ammonia (DNRA) were observed in the dextrin/peptone- and glucose-fed cultures and the degree of predominance of either of the two pathways was a function of the COD/N value. Therefore, the type of electron donor used affected both the nitrate reduction pathway and kinetics, as well as the recovery of fermentation and/or methanogenesis in the mixed methanogenic culture.     

Inhibition of methanogenesis in salt marsh sediments and whole-cell suspensions of methanogenic bacteria by nitrogen oxides.

Hydrogen-dependent evolution of methane from salt marsh sediments and whole-cell suspensions of Methanobacterium thermoautotrophicum and Methanobacterium fornicicum ceased or decreased after the introduction of nitrate, nitrite, nitric oxide, or nitrous oxide. Sulfite had a similar effect on methanogenesis in the whole-cell suspensions. In salt marsh sediments, nitrous oxide was the strongest inhibitor, followed by nitric oxide, nitrite, and nitrate in decreasing order of inhibition. In whole-cell suspensions, nitric oxide was the strongest inhibitor, followed by nitrous oxide, nitrite, and nitrate. Consideration of the results from experiments using an indicator of oxidation potential, along with the reversed order of effectiveness of the nitrogen oxides in relation to their degree of reduction ,suggests that the inhibitory effect observed was not due to a redox change. Evidence is also presented that suggests that the decrease in the rate of methane production in the presence of oxides of nitrogen was not attributable to competition for methane-producing substrates.     

Nitrate reduction to ammonia: a dissimilatory process in Enterobacter amnigenus

Thirty-four bacterial isolates from an agricultural soil anaerobically preincubated in the presence of glucose were tested for their ability to reduce nitrate to ammonia or to denitrify in two different media: nitrate broth and a minimal medium enriched with glucose. Ten isolates were considered denitrifying bacteria and 7 were dissimilatory ammonia producers. Ammonia production by the isolate identified as Enterobacter amnigenus was quantified and attained 50% of 138?mg?L(-1) of added NO(3)(-) N. The dissimilatory character of this reduction was clearly confirmed by culturing this (15)N-labeled bacterium in the presence of unlabeled nitrite. Nitrous oxide was produced at the same time as nitrite was reduced to ammonia. Increasing nitrate N levels from 48 to 553?mg?L(-1) in culture medium resulted in an increase in the level of nitrite produced and simultaneously a decrease in ammonia and nitrous oxide production. Key words: dissimilatory nitrate reduction, dissimilatory ammonia production, denitrification, Enterobacter amnigenus, (15)N.     

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

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 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.     

Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri

The onset and cessation of the synthesis of denitrification enzymes of Pseudomonas stutzeri were investigated by using continuous culture and defined dissolved oxygen levels covering the full range of transition from air saturation to complete anaerobiosis. Expression of nitrate reductase, nitrite reductase (cytochrome cd1), and N2O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration. N2O reductase was synthesized constitutively at a low level; for enhanced expression, oxygen concentrations were required to decrease below 5 mg of O2 per liter. The threshold values for synthesis of nitrate reductase and cytochrome cd1 in the presence of nitrate were ca. 5 and ca. 2.5 mg of O2 per liter, respectively. With nitrous oxide as the respiratory substrate, nitrite reductase was again the most sensitive to oxygen concentration; however, thresholds for all denitrification enzymes shifted to lower oxygen levels. Whereas the presence of nitrate resulted in maximum expression and nearly uniform induction of all reductases, nitrite and nitrous oxide stimulated preferably the respective enzyme catalyzing reduction. In the absence of a nitrogenous oxide, anaerobiosis did not induce enzyme synthesis to any significant degree. The accumulation of nitrite seen during both the aerobic-anaerobic and anaerobic-aerobic transition phases was caused by the differences in onset or cessation of synthesis of nitrate and nitrite reductases and an inhibitory effect of nitrate on nitrite reduction.     

Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri.

The onset and cessation of the synthesis of denitrification enzymes of Pseudomonas stutzeri were investigated by using continuous culture and defined dissolved oxygen levels covering the full range of transition from air saturation to complete anaerobiosis. Expression of nitrate reductase, nitrite reductase (cytochrome cd1), and N2O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration. N2O reductase was synthesized constitutively at a low level; for enhanced expression, oxygen concentrations were required to decrease below 5 mg of O2 per liter. The threshold values for synthesis of nitrate reductase and cytochrome cd1 in the presence of nitrate were ca. 5 and ca. 2.5 mg of O2 per liter, respectively. With nitrous oxide as the respiratory substrate, nitrite reductase was again the most sensitive to oxygen concentration; however, thresholds for all denitrification enzymes shifted to lower oxygen levels. Whereas the presence of nitrate resulted in maximum expression and nearly uniform induction of all reductases, nitrite and nitrous oxide stimulated preferably the respective enzyme catalyzing reduction. In the absence of a nitrogenous oxide, anaerobiosis did not induce enzyme synthesis to any significant degree. The accumulation of nitrite seen during both the aerobic-anaerobic and anaerobic-aerobic transition phases was caused by the differences in onset or cessation of synthesis of nitrate and nitrite reductases and an inhibitory effect of nitrate on nitrite reduction.     

Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite- and oxide-limited conditions.

The molar growth yields of Pseudomonas denitrificans, for nitrate, nitrite and nitrous oxide, were determined in chemostat culture under electron acceptor-limited conditions. Glutamate was used as the source of energy, carbon and nitrogen. The catabolic pattern was identical, irrespective of the terminal electron acceptors. The molar growth yields, corrected for maintenance energy, were 28-6 g/mol nitrate, 16-9 g/mol nitrite and 8-8 g/mol nitrous oxide. The energy yield, expressed on an electron basis, was proportional to the oxidation number of the nitrogen: nitrate (plus 5), nitrite (plus 3) and nitrous oxide (plus 1). It was concluded that oxidative phosphorylation occurs to a similar extent in each of the electron transport chains associated with the reduction of nitrate to nitrite, nitrite to nitrous oxide and nitrous oxide to nitrogen.     

Sulfide enhances methanogenesis in nitrate-containing methanogenic cultures

The effects of sulfide on nitrate reduction and methanogenesis using butyrate as a carbon source were investigated in a mixed mesophilic, methanogenic culture. In the sulfide-free medium, 25-75 mg l⁻¹ nitrate markedly inhibited the efficiencies of acetogenesis and methanogenesis processes. Adding 25 mg-S l⁻¹ increased methane production in nitrate-amended medium. Low sulfide levels shifted the nitrate reduction pathway from denitrification to dissimilatory nitrate reduction to ammonia (DNRA), thereby reducing the amounts of toxic nitric oxide and nitrous oxide produced that inhibit methanogenesis. The dose of 25 mg l⁻¹ sulfide was oxidized completely, during which heterotrophic DNRA predominated. The oxidized forms of sulfide reformed, limiting induction of the heterotrophic denitrification pathway. The actions of heterotrophic and autotrophic DNRA bacteria, denitrifiers, sulfate-reducing bacteria and methanogens mitigate nitrate toxicity during methanogenesis in an anaerobic process.     

Intermediates of denitrification in the chemoautotroph Thiobacillus denitrificans

Summary Intact cells obtained from Thiobacillus denitrificans grown autotrophically with thiosulfate as the oxidizable substrate and nitrate as the final electron acceptor catalyzed the reduction of nitrate, nitrite and nitric oxide stoichiometrically to nitrogen gas with the concomitant oxidation of thiosulfate. In addition, nitrous oxide was also capable of acting as the terminal oxidant of the respiratory chain with thiosulfate as the reductant. The anaerobic oxidation of thiosulfate by NO₃ ^-, NO, and N₂O was sensitive to the flavoprotein inhibitors, antimycin A or NHQNO, and cyanide or azide thus, implicating the participation of flavins, and cytochromes of b-, c-, and a-types in the denitrification process. The nitrite reductase system, however, was not markedly affected by the electron transport chain inhibitors. The experimental observations suggest that the dissimilatory nitrate reduction in the chemoautotroph T. denitrificans involves nitrite, nitric oxide, and nitrous oxide as theintermediates with nitrogen gas as the final reduction product.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - NHQNO 2-n-nonyl-4-hydroxyquinoline N-oxide     

Aquatic nitrogen transformations at low oxygen concentrations.

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Aquatic nitrogen transformations at low oxygen concentrations

Nitrite and nitrous oxide made up 40% of the hypolimnetic dissolved inorganic nitrogen in mesotrophic Lake Rotoiti, New Zealand, prior to hypolimnetic anoxia. Up to 120 mg of N m-3 as nitrite and 20 mg of N m-3 as nitrous oxide accumulated, whereas dissolved-oxygen concentrations remained between 1.0 and 0.2 g m-3 and were totally consumed when the hypolimnion became completely anoxic. Assays of water column nitrification potentials, together with measurements of the relative rates of nitrate and nitrite reduction, suggested that at low dissolved-oxygen concentrations both nitrite and nitrous oxide were produced mainly by ammonium-oxidizing bacteria, with nitrous oxide being a product of nitrifier denitrification.     

Denitrifying potential of methanogenic sludge

Summary A methanogenic sludge showed denitrifying activity for acetate, glucose and effluents from methanogenic treatments as substrates; denitrifiers were present in a relatively high number. When glucose was used as substrate dissimilatory reduction of nitrate to ammonium occurred. Methane production from acetate was inhibited by denitrification and resumed after nitrite and nitrous oxide depletion.     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens

A pure culture of Pseudomonas fluorescens was used as a model system to study the kinetics of denitrification. An exponentially growing culture was harvested and resuspended in an anoxic acetate solution buffered with K/Na phosphate at pH values of 6.6, 7.0, 7.4, and 7.8. The temperature was kept at 28 degrees C in all assays. Nitrate pulses of approximately 0.2 mg N/L caused nitrite to accumulate due to a faster rate of nitrate reduction over nitrite reduction. The rate of nitrate reduction was observed to depend on its concentration as predicted by the Michaelis-Menten equation. At nonlimiting nitrate concentrations, nitrite reduction was described by the same equation. Otherwise, nitrite reduction also depended on nitrate concentration. Consequently, nitrate and nitrite reductions compete with each other for the oxidation of common electron donors. A kinetic model for nitrate competitive inhibition of nitrite reduction is proposed. The model was used to interpret the nitrate and nitrite profiles observed at the four pH values: the optimum pH value was 7.0 in both cases; the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(3) ) = 0.04 mg N/L); the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(2) ) = 0.06 mg N/L), but it decreased sharply for the pH value of 7.8. Although the ratio between the two maximum reduction rates (V(max NO(2) )/V(max NO(3) )) is constant, nitrite accumulation depends on the medium pH value. Therefore, the regulation mechanism that shifts the electron flow between the two terminal reductases is readily reversible and does not change their relative maximum reduction rates. (c) 1995 John Wiley & Sons, Inc.     

Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli

In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products. The purpose of this study was to determine which of these two agents is more responsible for endogenous nitrosative mutagenesis. An nfi (endonuclease V) mutant was grown anaerobically with nitrate or nitrite, conditions under which it has a high frequency of A:T-to-G:C transition mutations because of a defect in the repair of hypoxanthine (nitrosatively deaminated adenine) in DNA. These mutations could be greatly reduced by two means: (i) introduction of an nirB mutation, which affects the inducible cytoplasmic nitrite reductase, the major source of nitric oxide during nitrate or nitrite metabolism, or (ii) flushing the anaerobic culture with argon (which should purge it of nitric oxide) before it was exposed to air. The results suggest that nitrosative mutagenesis occurs during a shift from nitrate/nitrite-dependent respiration under hypoxic conditions to aerobic respiration, when accumulated nitric oxide reacts with oxygen to form endogenous nitrosating agents such as N2O3. In contrast, mutagenesis of nongrowing cells by nitrous acid was unaffected by an nirB mutation, suggesting that this mutagenesis is mediated by N2O3 that is formed directly by the condensation of nitrous acid.     

N Kinetic Analysis of N(2)O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification

A series of N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the "denitrifying enzyme" nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% N) and 0.4 mM nitrite (99% N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.