Dissimilatory ammonia production vs. denitrification in vitro and in inoculated agricultural soil samples

A dissimilatory ammonia-producing isolate identified as Enterobacter amnigenus and a denitrifier identified as Agrobacterium radiobacter isolated from the same soil were studied. The products of nitrate reduction in a minimal medium, enriched with glucose and containing nitrate N as the sole nitrogen source, were quantified when each of these isolates was cultured anaerobically, alone or mixed together in the presence or absence of C(2)H(2). When they were cultured together, ammonia was the principal product of nitrate reduction. The distribution between denitrification and dissimilatory ammonia production (DAP) for nonsterilised soil samples inoculated with E. amnigenus or A. radiobacter, or a mixture of these two isolates, was also investigated. Production of NH(4)(+) was increased under these conditions (strict anaerobiosis and much available fermentable carbon), but the inoculation of soil samples with 1.2?×?10(7) cells of E. amnigenus·g dried soil(-1) was not sufficient to shift nitrate reduction from nitrous oxide (denitrification) to ammonia production, suggesting that inoculation with a greater number of DAP bacteria than introduced would probably be required to enable ammonia production to exceed nitrous oxide release. Key words: dissimilatory ammonia production, denitrification, Enterobacter amnigenus, Agrobacterium radiobacter.     

Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway

Abstract: A total of 28 nitrate-reducing bacteria were isolated from marine sediment (Mediterranean coast of France) in which dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. ¹⁵N ammonium production from ¹⁵N nitrate by an Enterobacter sp. and a Vibrio sp., the predominant bacteria involved in nitrate ammonification in marine sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (1 mM) produced ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in the presence of nitrate, ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium. These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate formation, instead of ethanol formation which was favoured in the absence of 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.     

Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium

Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite and produce N(2). They reside in many natural ecosystems and contribute significantly to the cycling of marine nitrogen. Anammox bacteria generally live under ammonium limitation, and it was assumed that in nature anammox bacteria depend on other biochemical processes for ammonium. In this study we investigated the possibility of dissimilatory nitrate reduction to ammonium by anammox bacteria. Physically purified Kuenenia stuttgartiensis cells reduced (15)NO(3) (-) to (15)NH(4) (+) via (15)NO(2) (-) as the intermediate. This was followed by the anaerobic oxidation of the produced ammonium and nitrite. The overall end-product of this metabolism of anammox bacteria was (15)N(15)N dinitrogen gas. The nitrate reduction to nitrite proceeds at a rate of 0.3 +/- 0.02 fmol cell(-1) day(-1) (10% of the 'normal' anammox rate). A calcium-dependent cytochrome c protein with a high (305 mumol min(-1) mg protein(-1)) rate of nitrite reduction to ammonium was partially purified. We present evidence that dissimilatory nitrate reduction to ammonium occurs in Benguela upwelling system at the same site where anammox bacteria were previously detected. This indicates that anammox bacteria could be mediating dissimilatory nitrate reduction to ammonium in natural ecosystems.     

Nitrate Reduction to Nitrite,Nitric Oxide and Ammonia by Gut Bacteria under Physiological Conditions

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.     

Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens

The influence of redox potential on dissimilatory nitrate reduction to ammonium was investigated on a marine bacterium, Pseudomonas putrefaciens. Nitrate was consumed (3.1 mmol liter-1), and ammonium was produced in cultures with glucose and without sodium thioglycolate. When sodium thioglycolate was added, nitrate was consumed at a lower rate (1.1 mmol liter-1), and no significant amounts of nitrite or ammonium were produced. No growth was detected in glucose media either with or without sodium thioglycolate. When grown on tryptic soy broth, the production of nitrous oxide paralleled growth. In the same medium, but with sodium thioglycolate, nitrous oxide was first produced during growth and then consumed. Acetylene caused the nitrous oxide to accumulate. These results and the mass balance calculations for different nitrogen components indicate that P. putrefaciens has the capacity to dissimilate nitrate to ammonium as well as to dinitrogen gas and nitrous oxide (denitrification). The dissimilatory pathway to ammonium dominates except when sodium thioglycolate is added to the medium.     

Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens.

The influence of redox potential on dissimilatory nitrate reduction to ammonium was investigated on a marine bacterium, Pseudomonas putrefaciens. Nitrate was consumed (3.1 mmol liter-1), and ammonium was produced in cultures with glucose and without sodium thioglycolate. When sodium thioglycolate was added, nitrate was consumed at a lower rate (1.1 mmol liter-1), and no significant amounts of nitrite or ammonium were produced. No growth was detected in glucose media either with or without sodium thioglycolate. When grown on tryptic soy broth, the production of nitrous oxide paralleled growth. In the same medium, but with sodium thioglycolate, nitrous oxide was first produced during growth and then consumed. Acetylene caused the nitrous oxide to accumulate. These results and the mass balance calculations for different nitrogen components indicate that P. putrefaciens has the capacity to dissimilate nitrate to ammonium as well as to dinitrogen gas and nitrous oxide (denitrification). The dissimilatory pathway to ammonium dominates except when sodium thioglycolate is added to the medium.     

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.     

Inhibitory effects of nitrogen oxides on a mixed methanogenic culture

The effect of nitrate, nitrite, nitric oxide (NO), and nitrous oxide on a mixed, mesophilic (35 degrees C) methanogenic culture was investigated. Short-term inhibition assays were conducted at a concentration range of 10-350 mg N/L nitrate, 17-500 mg N/L nitrite, 0.02-0.8 mg N/L aqueous NO, and 19-191 mg N/L aqueous nitrous oxide. Simultaneous methane production and N-oxide reduction was observed in 10 and 30 mg N/L nitrate and 0.02 mg N/L aqueous NO-amended cultures. However, addition of N-oxide resulted in immediate cessation of methanogenesis in all other cultures. Methanogenesis completely recovered subsequent to the complete reduction of N-oxides to nitrogen gas in all N-oxide-amended cultures, with the exception of the 500 mg N/L nitrite- and 0.8 mg N/L aqueous NO-amended cultures. Partial recovery of methanogenesis was observed in the 500 mg N/L nitrite-amended culture in contrast to complete inhibition of methanogenesis in the 0.8 mg N/L aqueous NO-amended culture. Accumulation of volatile fatty acids was observed in both cultures at the end of the incubation period. Among all N-oxides, NO exerted the most and nitrate exerted the least inhibitory effect on the fermentative/methanogenic consortia. The effect of multiple additions of nitrate (300 mg N/L) on the same methanogenic culture was also investigated. Long-term exposure of the methanogenic culture to nitrate resulted in an increase of N-oxide reduction rates and decrease of methane production rates, which was attributed to changes in the microbial community structure due to nitrate addition.     

Anaerobic energy metabolism of the sulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitrate reduction to ammonia

In a batch culture experiment the microaerophilic Campylobacter-like bacterium “Spirillum” 5175 derived its energy for growth from the reduction of nitrate to nitrite and nitrite to ammonia. Hereby, formate served as electron donor, acetate as carbon source, and l-cysteine as sulfur source. Nitrite was quantitatively accumulated in the medium during the reduction of nitrate; reduction of nitrite began only after nitrate was exhausted from the medium. The molar growth yield per mol formate consumed, Y_m, was 2.4g/mol for the reduction of nitrate to nitrite and 2.0 g/mol for the conversion of nitrite to ammonia. The gain of ATP per mol of oxidized formate was 20% higher for the reduction of nitrate to nitrite, compared to the reduction of nitrite to ammonia. With succinate as carbon source and nitrite as electron acceptor, Y_m was 3.2g/mol formate, i.e. 60% higher than with acetate as carbon source. No significant amount of nitrous oxide or dinitrogen was produced during growth with nitrate or nitrite both in the presence or absence of acetylene. No growth on nitrous oxide was found. The hexaheme c nitrite reductase of “Spirillum” 5175 was an inducible enzyme. It was present in cells cultivated with nitrate or nitrite as electron acceptor. It was absent in cells grown with fumarate, but appeared in high concentration in “Spirillum” 5175 grown on elemental sulfur. Furthermore, the dissimilatory enzymes nitrate reductase and hexaheme c nitrite reductase were localized in the periplasmic part of the cytoplasmic membrane.     

Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction.

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.     

Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

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.     

Dissimilatory nitrate reduction by Propionibacterium acnes.

Propionibacterium acnes P13 was isolated from human feces. The bacterium produced a particulate nitrate reductase and a soluble nitrite reductase when grown with nitrate or nitrite. Reduced viologen dyes were the preferred electron donors for both enzymes. Nitrous oxide reductase was never detected. Specific growth rates were increased by nitrate during growth in batch culture. Culture pH strongly influenced the products of dissimilatory nitrate reduction. Nitrate was principally converted to nitrite at alkaline pH, whereas nitrous oxide was the major product of nitrate reduction when the bacteria were grown at pH 6.0. Growth yields were increased by nitrate in electron acceptor-limited chemostats, where nitrate was reduced to nitrite, showing that dissimilatory nitrate reduction was an energetically favorable process in P. acnes. Nitrate had little effect on the amounts of fermentation products formed, but molar ratios of acetate to propionate were higher in the nitrate chemostats. Low concentrations of nitrite (ca. 0.2 mM) inhibited growth of P. acnes in batch culture. The nitrite was slowly reduced to nitrous oxide, enabling growth to occur, suggesting that denitrification functions as a detoxification mechanism.     

Dissimilatory nitrate reduction by Propionibacterium acnes

Propionibacterium acnes P13 was isolated from human feces. The bacterium produced a particulate nitrate reductase and a soluble nitrite reductase when grown with nitrate or nitrite. Reduced viologen dyes were the preferred electron donors for both enzymes. Nitrous oxide reductase was never detected. Specific growth rates were increased by nitrate during growth in batch culture. Culture pH strongly influenced the products of dissimilatory nitrate reduction. Nitrate was principally converted to nitrite at alkaline pH, whereas nitrous oxide was the major product of nitrate reduction when the bacteria were grown at pH 6.0. Growth yields were increased by nitrate in electron acceptor-limited chemostats, where nitrate was reduced to nitrite, showing that dissimilatory nitrate reduction was an energetically favorable process in P. acnes. Nitrate had little effect on the amounts of fermentation products formed, but molar ratios of acetate to propionate were higher in the nitrate chemostats. Low concentrations of nitrite (ca. 0.2 mM) inhibited growth of P. acnes in batch culture. The nitrite was slowly reduced to nitrous oxide, enabling growth to occur, suggesting that denitrification functions as a detoxification mechanism.     

N2O reduction by Vibrio succinogenes.

Vibrio succinogenes grew anaerobically at the expense of formate oxidation, with nitrous oxide (N2O) serving a terminal oxidant. N2O was quantitatively reduced to dinitrogen (N2). In the presence of 5 x 10(-2) atm (ca. 5 kPa) of acetylene (C2H2), which inhibits the reduction of N2O, growth of V. succinogenes was completely inhibited. Nitrate was reduced to nitrite or to ammonia, depending on the extent of availability of formate, but N2 was not produced by reduction of nitrate. During the reduction of nitrate to ammonia, all eight electrons transported to a molecule of nitrate appeared to be coupled for energy-yielding reactions.     

真菌异化硝酸盐还原机理的研究进展

真菌异化硝酸盐还原途径的发现打破了反硝化仅存在于原核细胞这一传统观念。真菌异化硝酸盐还原途径是在环境中氧供给受限的情况下发生的, 包括反硝化和氨的发酵。硝酸盐能诱导产生反硝化作用的酶, 其中, 硝酸盐还原酶与亚硝酸还原酶位于线粒体中, 它们所催化的酶促反应能偶联呼吸链ATP合成酶合成ATP, 同时产生NO。与参与反硝化作用前两个酶不同, 真菌NO还原酶能以NADH为直接电子供体将NO还原为N2O, 在NAD+的再生和自由基NO的脱毒中起着重要作用。氨发酵则将硝酸盐还原成NH4+, 同时偶联乙酸的生成和底物水平磷酸化。此文从参与该过程的关键酶、关键酶的表达调节、真菌与细菌异化硝酸盐还原的比较等角度综述了真菌异化硝酸盐还原的最新研究进展。     

Influence of oxygen tension on nitrate reduction by a Klebsiella sp. growing in chemostat culture.

At dissolved oxygen tensions of 15 mmHg (2 kPa) and below, nitrate-limited continuous cultures of Klebsiella K312 synthesized nitrate reductase (NR) and nitrite reductase (NiR) and excreted ammonia. Under anaerobic conditions over 60% of the nitrate-nitrogen utilized was excreted as ammonia. In contrast, carbon-limited cultures excreted nitrite at dissolved oxygen tensions of 15 mmHg or below and synthesized NR but not NiR. Ammonia repressed neither NR nor NiR synthesis. These observations indicate that below a critical oxygen tension of 15 mmHg Klebsiella K312 utilizes oxygen and nitrate as electron acceptors. This oxygen tension correlates well with the critical oxygen tension observed for a change from oxidative to fermentative metabolism in cultures of Klebsiella aerogenes. The product of dissimilatory nitrate reduction is ammonia in nitrate-limited cultures but principally nitrite in carbon-limited (nitrate excess) cultures.