The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.     

Formation of Protoporphyrin from Coproporphyrinogen in Extracts of Various Bacteria

The enzyme system capable of converting coproporphyrinogen to protoporphyrin was demonstrated in the soluble fraction of extracts of Pseudomonas fluorescens grown aerobically, of P. denitrificans grown anaerobically under denitrifying conditions, and of Escherichia coli grown both aerobically and anaerobically. Protoporphyrin accumulation by each of these extracts occurred only if the assay was conducted aerobically. Attempts to replace this oxygen requirement with several alternate electron acceptors were not successful. The conversion of coproporphyrinogen to protoporphyrin could not be demonstrated in extracts of the heme-containing organisms Staphylococcus epidermidis and Bacillus subtilis.     

A cytochrome b-like pigment with a peak at 567 nm in Pseudomonas aeruginosa.

A cytochrome b - like pigment with an absorption peak at 567 nm was detected in Pseudomonas aeruginosa irrespective of whether the organism was grown aerobically or anaerobically under denitrifying conditions. This pigment has not been reported previously for P. aeruginosa but it has been detected in other denitrifying bacteria including closely related Pseudomonas species.     

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.     

Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O

N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed. Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O. Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O. Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O. The pattern with P. aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O. The inability of a number of P. aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity. Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P. aeruginosa and Paracoccus denitrificans. These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration.     

Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O.

N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed. Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O. Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O. Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O. The pattern with P. aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O. The inability of a number of P. aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity. Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P. aeruginosa and Paracoccus denitrificans. These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase

Present knowledge of the different enzymatic steps of the denitrification chains in various bacteria, particularly Paracoccus denitrificans and Pseudomonas aeruginosa has been briefly reviewed. The question whether nitric oxide (NO), nitrous oxide (N2O) and other nitrogen derivatives are obligatory intermediates has been discussed. The second part is an extensive review of the structure and the function of a key enzyme in denitrification, cytochrome c551-nitrite-oxidoreductase from P. aeruginosa. Recent results on the stoichiometry of nitrite reduction have been discussed.     

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.     

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.     

Comparative Effect of Oxygen and Nitrate on Protoporphyrin and Heme Synthesis from Δ-Amino Levulinic Acid in Bacterial Cultures

Pseudomonas aeruginosa and P. denitrificans accumulate more protoheme and considerably more protoporphyrin during anaerobic growth under denitrifying conditions than during aerobic growth. In Escherichia coli, the small accumulation of protoporphyrin and protoheme which occurs during anaerobic growth is slightly stimulated by nitrate and markedly stimulated by oxygen.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

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

The course of denitrification of nitrate, nitrite and both compounds together by static cultures of Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa was studied. These strains represent three different types of denitrification: 1. reduction of nitrate to gaseous nitrogen without accumulation of nitrite (P. denitrificans); 2. partial accumulation of nitrite in growing cultures during reduction of nitrate to gaseous nitrogen (P. aeruginosa) and 3. two-phase denitrification that includes reduction of nitrates at the very beginning of the process, and then, after depletion of the former, the reduction of nitrates to gaseous nitrogen (P. stutzeri). These observations differ from the results reported in the literature and possible reasons are discussed.     

Heterotrophic nitrification among denitrifiers.

Twelve denitrifying bacteria representing six genera were tested for an ability to nitrify pyruvic oxime heterotrophically. Six of these bacteria exhibited appreciable nitrification activity, yielding as much as 5.8 mM nitrite and little or no nitrate when grown in a mineral salts medium containing 7 mM pyruvic oxime and 0.05% yeast extract. Of the six active bacteria, four (Pseudomonas denitrificans, Pseudomonas aeruginosa, and two strains of Pseudomonas fluorescens) could grow on yeast extract but not pyruvic oxime, one (Pseudomonas aureofaciens) could grow slowly on pyruvic oxime, and one (Alcaligenes faecalis) could apparently grow on pyruvic oxime in the presence of yeast extract but not in its absence. Eight of the twelve bacteria in the resting state could oxidize hydroxylamine to nitrite, and P. aureofaciens was remarkably active in this regard. In general, those denitrifiers active in the nitrification of pyruvic oxime or hydroxylamine or both are abundant in soils. A possible advantage of having nitrification and denitrification capabilities in the same organism is discussed.     

Halobacterium denitrificans sp. nov., an extremely halophilic denitrifying bacterium

Halobacterium denitrificans was one of several carbohydrate-utilizing, denitrifying, extremely halophilic bacteria isolated by anaerobic enrichment in the presence of nitrate. Anaerobic growth took place only when nitrate (or nitrite) was present and was accompanied by the production of dinitrogen. In the presence of high concentrations of nitrate (i.e., 0.5%), nitrous oxide and nitrite were also detected. When grown aerobically in a mineral-salts medium containing 0.005% yeast extract, H. denitrificans utilized a variety of carbohydrates as sources of carbon and energy. In every case, carbohydrate utilization was accompanied by acid production. A type culture has been deposited with the American Type Culture Collection, Rockville, Md. (ATCC 35960).     

Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N(2)). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Development and application of a novel and effective screening method for aerobic denitrifying bacteria

Herein we describe a novel and effective screening method for aerobic denitrifying bacteria. For this procedure, we utilized KCN to inhibit the electron transference from Cytaa3 to oxygen in the bacteria respiratory chain. We employed a 3-h aeration operation cycle and intermittent rotations. The resultant bacterial suspensions were plated on a KCN-screening medium and incubated aerobically. Single colonies were selected and incubated in an aerobic culture medium. Culture nitrate and nitrite levels were determined over time, and ultimately four bacterial strains that performed denitrifying under aerobic conditions were identified by this method. Of these, strain Y2-1-1 demonstrated the best aerobic denitrifying ability. In a 5-day test, the NO3--N of the aerobic culture medium was reduced from 282.0+/-8.3 mg L(-1) to 149.2+/-17.1 mg L(-1), with little nitrite or N2O production. The morphological, physiological and biochemical characteristics and the 16S rRNA gene sequence homology comparison data for this strain were consistent with the classification of the genus Pseudomonas. We named this strain Pseudomonas sp. Y2-1-1.     

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.