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.     

The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea

Nitrite reductase has been separated from cell-free extracts of Nitrosomonas and partially purified from hydroxylamine oxidase by polyacrylamide-gel electrophoresis. In its oxidized state the enzyme, which did not contain haem, had an extinction maximum at 590nm, which was abolished on reduction. Sodium diethyldithiocarbamate was a potent inhibitor of nitrite reductase. Enzyme activity was stimulated 2.5-fold when remixed with hydroxylamine oxidase, but was unaffected by mammalian cytochrome c. The enzyme also exhibited a low hydroxylamine-dependent nitrite reductase activity. The results suggest that this enzyme is similar to the copper-containing ;denitrifying enzyme' of Pseudomonas denitrificans. A dithionite-reduced, 465nm-absorbing haemoprotein was associated with homogeneous preparations of hydroxylamine oxidase. The band at 465nm maximum was not reduced during the oxidation of hydroxylamine although the extinction was abolished on addition of hydroxylamine, NO(2) (-) or CO. These last-named compounds when added to the oxidized enzyme precluded the appearance of the 465nm-absorption band on addition of dithionite. Several properties of 465nm-absorbing haemoprotein are described.     

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.     

Growth of Nitrosomonas europaea on hydroxylamine

Abstract Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite, but until now it has not been possible to grow Nitrosomonas europaea on hydroxylamine. This study demonstrates that cells of N. europaea are capable of growing mixotrophically on ammonia and hydroxylamine. The molar growth yield on hydroxylamine (4.74 g mol⁻¹ at a growth rate of 0.03 h⁻¹) was higher than expected. Aerobically growing cells of N. europaea oxidized ammonia to nitrite with little loss of inorganic nitrogen, while significant inorganic nitrogen losses occurred when cells were growing mixotrophically on ammonia and hydroxylamine. In the absence of oxygen, hydroxylamine was oxidized with nitrite as electron acceptor, while nitrous oxide was produced. Anaerobic growth of N. europaea on ammonium, hydroxylamine and nitrite could not be observed at growth rates of 0.03 h⁻¹ and 0.01 h⁻¹.     

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.     

Photoinactivation of Ammonia Oxidation in Nitrosomonas

Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml.     

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twentyone of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.In memoriam of Professor R. Y. Stanier     

Bacterial cytochrome c nitrite reductase: new structural and functional aspects

Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia as a key step within the biological nitrogen cycle. Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution. This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme. In the crystal, the protein was a homodimer with ten hemes in very close packing. The strong interaction between the nitrite reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration. Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity. Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity. In addition to nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates. With N2O the activity was much lower; most likely dinitrogen was the product in this case. Cytochrome c nitrite reductase exhibited a remarkably high sulfite reductase activity, with hydrogen sulfide as the product. A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with nitrite. This potential reaction intermediate of the reduction of nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

Characteristics of nitrogenous substrate conversion by anammox enrichment

The characteristics of nitrogenous substrates conversion by anammox enrichment were investigated using batch experiments. The anammox enrichment was proved able to convert hydroxylamine to hydrazine, as well as convert hydrazine to ammonia anaerobically, with the average conversion rates of 0.207 and 0.031 mmol gVSS⁻¹ h⁻¹. It could convert hydroxylamine and nitrite simultaneously, with ammonia as an intermediate product. The maximum conversion rates of hydroxylamine and nitrite were 0.535 and 0.145 mmol gVSS⁻¹ h⁻¹, respectively. Their conversion rates were enhanced by 26.7% and 120.7%, respectively, by raising the ratio of hydroxylamine to nitrite from 1:1 to 2:1. The characteristics of nitrogenous substrate conversion by anammox enrichment could be explained using the speculative anammox pathway based on van de Graaf model.     

Nitrite Formation from Hydroxylamine and Oximes by Pseudomonas aeruginosa

Nitrite was formed from hydroxylamine and several oximes by intact cells and extracts of Pseudomonas aeruginosa. The activity was induced by the presence of oximes in the culture medium. Nitroalkanes were not intermediates in the conversion of acetaldoxime, acetone oxime, or butanone oxime to nitrite, since nitromethane inhibited the formation of nitrite from the nitro compounds but not from the corresponding oximes. The oxime apparently functions as a constant source of hydroxylamine during growth of the bacterium. Hydroxylamine at low concentration was converted stoichiometrically to nitrite by extracts of the bacterium; high concentrations were inhibitory. Nicotinamide adenine dinucleotide phosphate, oxygen, and other unidentified cofactors were necessary for the reaction. Actively nitrifying extracts possessed no hydroxylamine-cytochrome c reductase activity. Hyponitrite, nitrous oxide, and nitric oxide were not metabolized.     

Competition between Nitrospira spp. and Nitrobacter spp. in nitrite-oxidizing bioreactors

In this work the question was addressed if in nitrite-oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculated with nitrifying-activated sludge containing Nitrospira and operated under identical conditions. After addition of Nitrobacter to both chemostats, the nitrite concentration in the influent of one of the chemostats was increased such that nitrite peaks in the bulk liquid of this reactor were detected. The other chemostat served as control reactor, which always had a constant nitrite influent concentration. The relative cellular area (RCA) of Nitrospira and Nitrobacter was determined by quantitative fluorescence in situ hybridization (FISH). The nitrite perturbation stimulated the growth of Nitrobacter while in the undisturbed control chemostat Nitrospira dominated. Overall, the results of this experimental study support the hypothesis that Nitrobacter is a superior competitor when resources are abundant, while Nitrospira thrive under conditions of resource scarcity. Interestingly, the dominance of Nitrobacter over Nitrospira, caused by the elevated nitrite concentrations, could not be reverted by lowering the available nitrite concentration to the original level. One possible explanation for this result is that when Nitrobacter is present at a certain cell density it is able to inhibit the growth of Nitrospira. An alternative explanation would be that the length of the experimental period was not long enough to observe an increase of the Nitrospira population.     

Response of anaerobic ammonium-oxidizing bacteria to hydroxylamine

Anaerobic ammonium oxidation is a recent addition to the microbial nitrogen cycle, and its metabolic pathway, including the production and conversion of its intermediate hydrazine, is not well understood. Therefore, the effect of hydroxylamine addition on the hydrazine metabolism of anaerobic ammonium-oxidizing (anammox) bacteria was studied both experimentally and by mathematical modeling. It was observed that hydroxylamine was disproportionated biologically in the absence of nitrite into dinitrogen gas and ammonium. Little hydrazine accumulated during this process; however, rapid hydrazine production was observed when nearly all hydroxylamine was consumed. A mechanistic model is proposed in which hydrazine is suggested to be continuously produced from ammonium and hydroxylamine (possibly via nitric oxide) and subsequently oxidized to N(2). The electron acceptor for hydrazine oxidation is hydroxylamine, which is reduced to ammonium. A decrease in the hydroxylamine reduction rate, therefore, leads to a decrease in the hydrazine oxidation rate, resulting in the observed hydrazine accumulation. The proposed mechanism was verified by a mathematical model which could explain and predict most of the experimental data.     

Cytochrome oxidase from Pseudomonas aeruginosa. III. Reduction of hydroxylamine

Pseudomonas aeruginosa cytochrome oxidase, which reduces nitrite and oxygen, is also capable of reducing hydroxylamine to ammonia.The K_m for hydroxylamine reduction is 6 · 10^?4M compared to 5 · 10^?5M for nitrite reduction. NADH, NADPH, reduced P. aeruginosa cytochrome c₅₅₁, and reduced P. aeruginosa copper protein were ineffective as electron donors for hydroxylamine reduction whereas reduced pyocyanine and methylene blue acted as electron mediators.Hydroxylamine reduction did not require the presence of Mn²⁺ of FAD and was not inhibited by prolonged dialysis versus sodium diethyldithiocarbamate. Cyanide, nitrite, and CO were very effective inhibitors.Removal of heme d and its reconstitution, as well as inhibition by CO, suggest that the reduction of hydroxylamine, like the reduction of nitrite or oxygen, proceeds via the heme d.     

Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions.

The effciency of denitrification, or anaerobic respiration, in Pseudomonas denitrificans was investigated, using growth yield as an index. Glutamate was mainly used as the sole source of energy and carbon. In batch culture, the growth yield per mole of electrons transported through the respiratory system under denitrifying conditions was about half that under aerobic conditions. Similar figures were also obtained in chemostat cultures under glutamate-limited conditions. The decrease in growth yield under denitrifying conditions could be due to the restriction of phosphorylation associated with nitrate reduction to nitrogen gas.     

Mechanism of Nitrification by Arthrobacter sp

Resting cells of Arthrobacter sp. excrete as much as 60 mug of hydroxylamine-nitrogen per ml when supplied with ammonium. An organic carbon source in abundant supply is necessary for the oxidation. Resting cells oxidize hydroxylamine to nitrite and 1-nitrosoethanol, the former accumulating only when an exogenous carbon source is available. Cell-free extracts contain an enzyme catalyzing the formation of hydroxylamine from acetohydroxamic acid, a hydroxylamine-nitrite oxido-reductase, and an enzyme producing nitrite and nitrate from various primary nitro compounds. Nitrite is not produced from hydroxylamine by the extracts, but 1-nitrosoethanol is formed from hydroxylamine in the presence of acetate. 1-Nitrosoethanol is also produced from acetohydroxamic acid by these preparations. Nitrite was formed from hydroxylamine, however, by extracellular enzymes excreted by the bacterium.     

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.     

Intracellular appearance of nitrite and nitrate in nitrogen-starved cells of Ankistrodesmus braunii

Summary The occurrence of heterotrophic nitrification in nitrogen-starved cells of Ankistrodesmus braunii was confirmed. The levels of nitrate and nitrite were measured over a period of four weeks. The validity of quantitative determinations in the presence of highly active nitrate and nitrite reductases is discussed. Whereas free hydroxylamine as an intermediate could not be detected, increased hydroxylamine oxidase activity was found in nitrogen-starved cultures. Nitrite reductase and hydroxylamine oxidase can be assigned to particles by sucrose density gradient centrifugation. The possible involvement of microbodies, which were found to be present in Ankistrodesmus, in metabolic processes during nitrogen starvation is discussed.Abbreviations NR nitrate reductase - NiR nitrite reductase - NNEDA N-(1-naphthyl)ethylenediaminedihydrochloride - DCPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - TCA trichloroacetic acid - DAB 3,3-diaminobenzidine - AT 3-amino-1H-1,2,4-triazole - AMP 2-amino-2-methyl-1,3-propanediol     

5,7-Dodecadien-1-ol: Candidate for the Sex Pheromone of the Pine Moth

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86,000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690 nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic of a high spin Fe³⁺ -siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7 g-atoms of iron per 86,000 g of protein.     

Physiological and Molecular Biological Characterization of Ammonia Oxidation of the Heterotrophic Nitrifier Pseudomonas putida

The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N₂O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized γ-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C₂H₂, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. Received: 9 April 1998 / Accepted: 15 May 1998