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.     

Effects of cultural conditions on denitrification by Pseudomonas stutzeri measured by Membrane Inlet Mass Spectrometry

Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter. Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium. MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen. A nitrate concentration of 15 mmol l⁻¹ and a nitrite concentration of 5 mmol l⁻¹ were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released. Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products. Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps. stutzeri is the amount of nitrite generated at the first enzymatic stage of the process. This appears to cause inhibition of the denitrification pathway above 5 mmol l⁻¹ and at high enough concentrations (15 mmol l⁻¹) restricts growth.     

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.     

Reduction of oxidized inorganic nitrogen compounds by a new strain of Thiobacillus denitrificans

Denitrification by Thiobacillus denitrificans "RT" strain was investigated using manometry and gas chromatography. 1. From nitrate, resting cells produced only nitrogen anaerobically with thiosulfate as the electron donor. The data suggest that nitrate was assimilated and dissimilated by the same nitrate reductase, assayed with benzyl-viologen as the electron donor. 2. From nitrite, whole cells produced nitric oxide, nitrous oxide and nitrogen, using thiosulfate as the electron donor; nitrogen was the final product of the reduction. Crude extract reduced nitrite to nitrogen with p-phenylene-diamine and dimethyl-p-phenylene diamine as the electron donors, and produced nitric oxide, nitrous oxide and nitrogen with tetramethyl-p-phenylene-diamine as the electron donor. Nitrite was reduced to nitric oxide and nitrous oxide by crude extract using ascorbate-phenazine methosulfate as the electron donor. 3. From nitric oxide, whole cells produced nitrous oxide and nitrogen using thiosulfate as the electron donor, nitrogen was the final reduction product. Nitric oxide was reduced to nitrous oxide by crude extract with the ascorbate-phenazine methosulfate system. 4. Whole cells reduced nitrous oxide to nitrogen with thiosulfate as the electron donor. It was not possible to detect any nitrous oxide reductase activity in crude extract. 5. A scheme was of denitrification by Thiobacillus denitrificans "RT" strain.     

Measurement of Denitrification in Two Freshwater Sediments by an In Situ Acetylene Inhibition Method

An acetylene inhibition method was satisfactorily used for the in situ measurement of denitrification in two sediment-water systems incubated for not more than 22 h. In the presence of added nitrate, denitrification acted as a source of nitrous oxide in a drainage pond, but acted as a sink in its absence. The averaged rates of nitrous oxide accumulation with nitrate enrichment in the absence and presence of acetylene were 0.15 and 0.30 mg of N m⁻²h⁻¹, respectively. Acetylene reduction at an average rate of 0.07 mmol of C₂H₄ formed m⁻²h⁻¹ was simultaneously measured in the absence of added nitrate. In a small eutrophic lake where nitrogen was nonlimiting, the in situ rates of sediment denitrification were 0.09 and 0.11 mg of N m⁻²h⁻¹ in the presence and absence of macrophytes, respectively, and no acetylene reduction activity was found.     

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.     

Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle

Here I describe how losses of fixed nitrogen can occur in riparian zones by the activity of denitrifying bacteria associated with methane-oxidizing (methanotrophic) bacteria. Several methanotrophs catalyze nitrogen cycle processes that can occur in riparian buffer zones, including nitrification and nitrogen fixation. Methanotrophs can produce nitric and nitrous oxides during oxidation of ammonium (nitrification), but they cannot carry out denitrification. However, there is good evidence that denitrifying bacteria can be associated with methanotrophs and can use simple carbon compounds released by the methanotrophs as substrates for the denitrification reactions and for growth. Evidence is presented that denitrifiers isolated from methanotrophic gel-stabilized oxygen gradient systems can use methanol, formaldehyde, and formate, all methane oxidation intermediates, to support their denitrification. Such denitrification associated with methanotrophs can release dinitrogen and so contributes to losses of fixed nitrogen, and may also produce the important atmospheric trace gases nitric and nitrous oxides. Data presented also show that some methanotrophs produce nitrogen oxides, including nitrite, nitric oxide, and nitrous oxide, during growth on nitrate. Assimilatory reduction of nitrate appears to be a requirement for the release of these products.     

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.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Denitrification in a nitrogen-limited stream ecosystem

Denitrification was measured in hyporheic, parafluvial, and bank sediments of Sycamore Creek, Arizona, a nitrogen-limited Sonoran Desert stream. We used three variations of the acetylene block technique to estimate denitrification rates, and compared these estimates to rates of nitrate production through nitrification. Subsurface sediments of Sycamore Creek are typically well-oxygenated, relatively low in nitrate, and low in organic carbon, and therefore are seemingly unlikely sites of denitrification. However, we found that denitrification potential (C & N amended, anaerobic incubations) was substantial, and even by our conservative estimates (unamended, oxic incubations and field chamber nitrous oxide accumulation), denitrification consumed 5–40% of nitrate produced by nitrification. We expected that denitrification would increase along hyporheic and parafluvial flowpaths as dissolved oxygen declined and nitrate increased. To the contrary, we found that denitrification was generally highest at the upstream ends of subsurface flowpaths where surface water had just entered the subsurface zone. This suggests that denitrifiers may be dependent on the import of surface-derived organic matter, resulting in highest denitrification rate at locations of surface-subsurface hydrologic exchange. Laboratory experiments showed that denitrification in Sycamore Creek sediments was primarily nitrogen limited and secondarily carbon limited, and was temperature dependent. Overall, the quantity of nitrate removed from the Sycamore Creek ecosystem via denitrification is significant given the nitrogen-limited status of this stream.     

Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.     

Degradation of 2,4-dinitrophenol by two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.     

The application of a laboratory apparatus for the study of nutrient fluxes between sediment and water

Sediments of the river Elbe estuary have been studied to assess their impact on the total nitrogen budget of the estuary. A new laboratory incubation apparatus was used to provide a means of regulating important parameters such as temperature and oxygen concentrations. With this apparatus sediment cores from a typical shallow water area with high organic carbon content were incubated under varying oxygen concentrations in the overlying water. Measurements of ammonium, nitrite, nitrate and nitrous oxide in the water phase were carried out and the fluxes between sediment and water phase calculated. During aerobic conditions in the water phase overall nitrate fluxes between + 4 and –3.5 mmol Nm^–2d^–1 across the sediment/water interface were observed. Under anaerobic conditions the fluxes increased up to –10 mmol Nm^–2 d^–1. Nitrous oxide was formed within the sediment under both aerobic and anaerobic conditions. Fluxes into the water phase were highest when the oxygen concentrations in the water phase were low (between 0.1 and 0.6 mg l^–1).     

Nitrous oxide formation in the Colne estuary, England: the central role of nitrite

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N(2)O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N(2)O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N(2)O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 microM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 micromol N.m(-2).h(-1) with nitrite but only 180 micromol N.m(-2).h(-1) with nitrate. The ratios of rates of nitrous oxide production and denitrification (N(2)O/N(2) x 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N(2) formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N(2) via N(2)O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N(2)O as the end product. Consideration of free-energy changes during N(2)O formation led to the conclusion that N(2)O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N(2)O in high-nutrient-load estuaries.     

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.     

DNA-probing indicates the occurrence of denitrification and nitrogen fixation genes in Hyphomicrobium. Distribution of denitrifying and nitrogen fixing isolates of Hyphomicrobium in a sewage treatment plant

Abstract: Twenty-six Hyphomicrobium isolates from the sewage treatment plant and its receiving water body in Plön (Schleswig-Holstein, Germany) and two culture collection strains were screened for the occurrence of genes coding for denitrification enzymes (dissimilatory nitrate, nitrite and nitrous oxide reductases), for dinitrogen fixation (nitrogenase reductase) and for nitrification (ammonia monooxygenase catalyzing the first stage of this process) by DNA-probing. More than one half of the isolates had genes coding for denitrification enzymes. The DNA-DNA hybridization signals obtained with the gene segments correlated with enzyme activity measurements. The DNA of some isolates distinctly hybridized with the nif H probe indicating the occurrence of nitrogenase in the genus Hyphomicrobium . No signal was detected with the gene probe for nitrification. The results show that probes consisting of gene segments can be employed successfully to monitor the occurrence of genes which can show complex expression and in bacteria growing at low rates. The distribution pattern of the denitrification genes indicates that methylotrophic prosthecate bacteria of the sewage treatment plant and its receiving water body occupy different ecological niches.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.