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.     

Reduction of nitrite to nitrous oxide by a cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus.

A cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus reduced nitrite to nitrous oxide in a stoichiometric reaction without nitric oxide as free intermediate. The membrane system had a specific requirement for FMN with NAD(P)H as electron donors. Other electron donors were ascorbate-reduced cytochrome c-551 or phenazine methosulfate. The membrane fraction contained tightly bound cytochrome cd which represented only a small portion of the total cytochrome cd of the cell. As further terminal oxidase cytochrome o was identified. The membrane fraction produced also nitrous oxide from nitric oxide, however, at a substantially lower rate than from nitrite when using ascorbate-reduced phenazine methosulfate as electron donor.     

Effects of sulfide and low redox potential on the inhibition of nitrous oxide reduction by acetylene in Pseudomonas nautica

Membrane introduction mass spectrometry was used to investigate the inhibitory effect of acetylene on the nitrous oxide reductase activity of intact cells of Pseudomonas nautica. We studied the effects of the concentrations of nitrate and sulfide, and the redox potential, which have all been implicated in causing a decrease in the inhibitory effects of acetylene during measurements of denitrification in natural environments. There was no evidence that the concentration of nitrate influenced the effect of acetylene. Lowering the redox potential with the reductant Ti(III)-nitrilotriacetate caused a slight alleviation of acetylene inhibition. Much greater effects at the same redox potential were obtained with concentrations of sulfide in the range 1-10 microM.     

Enhanced reduction of nitrous oxide by Pseudomonas denitrificans with perfluorocarbons

Nitrous oxide reduction and nitrogen production by Pseudomonas denitrificans, as well as culture growth rates all increased 2-3 fold when cultured in the presence of perfluorocarbon emulsions (10% v/v) as compared to control cultures grown in the absence of perfluorocarbons. Initial nitrous oxide concentrations for consecutive experiments were 0.7 and 1.2 mM respectively.     

Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens.

The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied. Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV. The control without reductant had a redox potential near +200 mV. Production of 13NO, [13N]N2O, and [13N]N2 from 13NO3- and 13NO2- was followed. Total gas production was similar for all three treatments. The accumulation of 13NO was most significant in the presence of sulfide. A parallel control with autoclaved cells indicated that the 13NO production was largely biological. The sulfide inhibition was more dramatic at the level of N2O reduction; [13N]N2O became the major product instead of [13N]N2, the dominant product when either no reductant or Ti(III) was present. The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells.     

Nitrate requirement for acetylene inhibition of nitrous oxide reduction in marine sediments

The inhibition of nitrous oxide (N₂O) reduction by acetylene (C₂H₂) in saltmarsh sediment was temporary; we investigated this phenomenon and possible causes. The reduction of N₂O in the presence of C₂H₂ was biological. N₂O consumption in the presence of C₂H₂ began when nitrate concentration became very low. The time course of N₂O consumption after periods of N₂O accumulation was unaffected by initial nitrate concentrations between 16 and 200M, or C₂H₂ concentrations between 10 and 100% of the gas phase. Sulfide had no effect on the kinetics of N₂O reduction in the presence of C₂H₂. In more dilute slurries of saltmarsh sediments and in estuarine sediment, N₂O persisted in the presence of C₂H₂ unless sufficient organic carbon was added to deplete nitrate. In saltmarsh sediments, the rate of N₂O consumption in the presence of C₂H₂ was not changed by preincubation with C₂H₂. Initial positive rates of N₂O production in the presence of C₂H₂ occurred only when the block was apparently effective (i.e., at nitrate concentrations greater than about 5–10M) and appeared to represent a valid estimate of denitrification. Conversely, and in agreement with previous studies, concentrations of NO₃ ^– below these levels resulted in reduced efficiency of C₂H₂ blockage of N₂O reductase.     

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.     

Nitric oxide and nitrous oxide production and cycling during dissimilatory nitrite reduction by Pseudomonas perfectomarina

The denitrifier Pseudomonas perfectomarina reduced nitrite under conditions of kinetic competition between cells and gas sparging for extracellular dissolved nitric and nitrous oxides, NOaq and N2Oaq, in a chemically defined marine medium. Time courses of nitrite reduction and NOg and N2Og alpha removal were integrated to give NOg and N2Og yields. At high sparging rates, the NOg yield was greater than 50% of nitrite-N reduced, and the yield of NOg + N2Og was approximately 75%. Hence interrupted denitrification yields NOaq and N2Oaq as major products. The yields varied with sparging rates in agreement with a quantitative model of denitrification (Betlach, M. P., and Tiedje, J.M. (1981) Appl. Environ. Microbiol. 42, 1074-1084) that applies simplified Michaelis-Menten kinetics to NO2-----NOaq----N2Oaq----N2. The fit gave an estimate of the maximum scavengeable NOaq yield of 73 +/- 8% of nitrite-N. Thus a minor path independent of NOaq is also required. The fit of the model to data at lower sparging rates, where normal denitrification products predominate, implies that the extracellular NOaq pool yield is independent of gas sparging rate. Thus in P. perfectomarina NOaq and N2Oaq are intermediates, or facilely equilibrate with true intermediates, during complete denitrification. The recovery of most nitrite-N as NO and/or N2O under perturbed conditions is not an artifact of irreversible product removal, but an attribute of denitrification in this species, and most probably it is characteristic of denitrification in other species as well.     

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.     

Growth of Pseudomonas aeruginosa on nitrous oxide.

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans.

Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.     

Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans.

Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.     

Modulation by copper of the products of nitrite respiration in Pseudomonas perfectomarinus.

A synthetic growth medium was purified with the chelator 1,5-diphenylthiocarbazone to study the effects of copper on partial reactions and product formation of nitrite respiration in Pseudomonas perfectomarinus. This organism grew anaerobically in a copper-deficient medium with nitrate or nitrite as the terminal electron acceptor. Copper-deficient cells had high activity for reduction of nitrate, nitrite, and nitric oxide, but little activity for nitrous oxide reduction. High rates of nitrous oxide reduction were observed only in cells grown on a copper-sufficient (1 micro M) medium. Copper-deficient cells converted nitrate or nitrite initially to nitrous oxide instead of dinitrogen, the normal end product of nitrite respiration in this organism. In agreement with this was the finding that anaerobic growth of P. perfectomarinus with nitrous oxide as the terminal electron acceptor required copper. This requirement was not satisfied by substitution of molybdenum, zinc, nickel, cobalt, or manganese for copper. Reconstitution of nitrous oxide reduction in copper-deficient cells was rapid on addition of a small amount of copper, even though protein synthesis was inhibited. The results indicate an involvement of copper protein(s) in the last step of nitrite respiration in P. perfectomarinus. In addition we found that nitric oxide, a presumed intermediate of nitrite respiration, inhibited nitrous oxide reduction.     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

Growth of Pseudomonas aeruginosa on nitrous oxide

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria

Acetylene (0.1 atm) caused complete or almost complete inhibition of reduction of N₂O by whole cell suspensions of Pseudomonas perfectomarinus, P. aeruginosa and Micrococcus denitrificans. Acetylene did not inhibit reduction of NO₃^? or NO₂^? by these organisms. In the presence of acetylene there was stoichiometric conversion of NO₃^? or NO₂^? to N₂O with negligible subsequent reduction of the latter. In the absence of acetylene there was no or only transient accumulation of N₂O. The data are consistent with the view that N₂O is an obligatory intermediate in the reduction of NO₂^? to N₂ in all of the three organisms studied.     

Amperometric method for determining nitrous oxide in denitrification and in nitrogenase-catalyzed nitrous oxide reduction

A conventional Clark-type O₂ probe was used to determine N₂O concentrations in suspensions. At a polarizing voltage of–0.95 V versus the reference Ag/AgCl electrode, the probe is almost half as sensitive for N₂O as for O₂, and the detection limit is less than 1 M N₂O. The probe can also be used to determine NO for which the suitable polarizing voltage is–0.7 V. The method was successfully applied for continuously recording dissimilatory formation or utilization of N₂O by intactAzospirillum brasilense Sp 7, NO production by extracts from this bacterium, and N₂O reduction catalyzed by nitrogenase in intactKlebsiella pneumoniae. It is concluded that the probe is useful for measuring N₂O or NO contents in bacterial suspensions when the O₂ level is zero or kept constant during the assays.