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.     

Nitrification of Aspartate by Aspergillus flavus

Heterotrophic conversion of l-aspartic acid to nitrification products by Aspergillus flavus was studied in a replacement incubation system. Numerous amino acids supported nitrification; aspartate and glutamate were about equivalent as the best sources of nitrate. Addition of sodium bicarbonate to the incubation system substantially enhanced nitrate formation for all nitrifiable amino acids except aspartic acid, but the basis for the bicarbonate effect is obscure. The yield of nitrate from l-aspartate was not approached by forms of aspartic acid resulting from substitution on the beta carbon, the amino nitrogen, or the gamma carboxyl group or by aspartate presented as the d-configuration. There was no relationship between nitrate formation and the occurrence of such possible intermediates as nitrite, bound hydroxylamine, ammonia, aspergillic acid, and beta-nitropropionic acid. Uniformly labeled (14)C-l-aspartate that was nitrified in replacement incubation led to no accumulation of label in possible nitrification products in the culture filtrate. Label was found in components of the mycelium after acid hydrolysis, with heaviest accumulation in what appeared to be glucosamine and an unidentified compound, possibly acetylglucosamine. Detectable label was redistributed into serine, glycine, and threonine.     

Heterotrophic Nitrification in an Acid Forest Soil and by an Acid-Tolerant Fungus

Nitrate was formed from ammonium at pH 3.2 to 6.1 in suspensions of a naturally acid forest soil; the maximum rates of formation occurred at ca. pH 4 to 5. Nitrate was also formed from soil nitrogen in suspensions incubated at 50°C. Autotrophic nitrifying bacteria could not be isolated from this soil. Enrichment cultures produced nitrate in a medium with β-alanine if much soil was added to the medium, and nitrite but not nitrate was formed in the presence of small amounts of soil. Nitrification by these enrichments was abolished by eucaryotic but not procaryotic inhibitors. A strain of Absidia cylindrospora isolated from this soil was found to produce nitrate and nitrite in a medium with β-alanine at pH values ranging from 4.0 to 4.8. Nitrate production by A. cylindrospora required the presence of sterile soil. Free and bound hydroxylamine, hydroxamic acids, and primary aliphatic nitro compounds did not accumulate during the conversion of β-alanine to nitrite by the fungus. The organism also formed nitrite from ammonium in a medium containing acetate. We suggest that nitrification in this soil is a heterotrophic process catalyzed by acid-tolerant fungi and not by autotrophs or heterotrophs in nonacid microsites.     

Transformation of hemoglobin a into methemoglobin by sesamol

Sesamol (3,4-methylenedioxyphenol), a monophenolic antioxidant in sesame iol, produced methemoglobin from hemoglobin A (oxyhemoglobin and deoxyhemoglobin) and from red cells. The activity of the compound was more extensive than the polyphenolic compounds. The profiles of the methemoglobin formation by the compound were compared with those by nitrite and hydroxylamine. The formation of methemoglobin from oxyhemoglobin by the compound was rather slowly progressed, but the amount of methemoglobin formed was proportional to the concentration of oxyhemoglobin even when the concentration of the compound was low. The sesamol-induced methemoglobin formation was influenced by inositol hexaphosphate, an allosteric effector of hemoglobin. Thus, the phosphate enhanced the transformation of oxyhemoglobin and inhibited the transformation of deoxyhemoglobin.     

Nitrite and nitrate synthesis from pyruvic-oxime by anAlcaligenes sp.

AnAlcaligenes sp. isolated from soil was characterized as to its ability to oxidize and grow on pyruvic-oxime. Abundant nitrification of pyruvic-oxime was demonstrated with maximal nitrite and nitrate production of 1867 mg NO₂ ^–-N per liter and 42 mg NO₃ ^–-N per liter. TheAlcaligenes sp. oxidized hydroxylamine and this metabolism was stimulated when either acetate or pyruvate was present. This organism was also capable of limited pyruvic-oxime oxidation when cultured in an acidic medium. The metabolism of pyruvic-oxime and nitrification by theAlcaligenes sp. in the environment are discussed.     

Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V_max) of 0.066 and 0.003 μmol of N × min⁻¹ × mg of protein⁻¹, respectively, at 22°C. K_m values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.     

硝化基质和产物对发光细菌的急性毒性

摘要：【目的】对硝化基质和产物对硝化过程的影响进行初步研究。【方法】采用发光细菌法,在pH=7.0的条件下,测定了氨、羟胺、亚硝酸和硝酸对发光细菌的急性毒性（15min-半抑制浓度（the half inhibitory concentration,IC50））。【结果】单一物质的毒性试验结果表明,硝化基质和产物对发光细菌的毒性随浓度的升高而增大,且具有较好的线性关系;氨、羟胺、亚硝酸和硝酸的IC50分别为2180.2 mg/L、6.2740 mg/L、1207.2 mg/L和3140.3 mg/L;其毒性大小顺序为：羟胺 >亚硝酸 >氨 >硝酸。按等效浓度混合法测定硝化基质和产物的联合毒性,结果表明：氨与羟胺、氨与亚硝酸、羟胺与亚硝酸对发光细菌的联合毒性呈相加作用;氨与硝酸、羟胺与硝酸、亚硝酸与硝酸对发光细菌的联合毒性呈独立作用;氨、羟胺、亚硝酸、硝酸四元混合物的联合毒性也呈相加作用。【结论】根据硝化基质和产物对发光细菌和硝化细菌抑制浓度的相关性,可用发光细菌发光强度的变化指示硝化基质和产物的抑制作用。     

Nitrite: a key compound in N loss processes under acid conditions?

Summary Nitrite is very important in N transformation processes because it is an intermediate product in the aerobic nitrification as well as in the anaerobic denitrification process. Under soil conditions whereby aerobic and anaerobic zones are close to each other, the mobile nitrite can be a link between both N transformation processes. Because of its low stability in acid conditions, nitrite can be a key compound in N loss processes.The results are presented in three sets of incubation experiments using soil+added nitrite before and after oxidation of organic matter; soil+added nitrite and various iron oxide minerals; nitrite solutions without soil but with added ferrous iron.It was found that under acid conditions, soil organic matter as well as the soil mineral phase have a stimulating effect on the nitrite decomposition. Conditions favouring the solubility of Fe(III)-compounds and promoting the formation of Fe²⁺ increase the nitrite decomposition, even under slightly acid conditions. Of the gaseous decomposition products, only trace amounts of NO₂ occur while NO is the major component. Conditions whereby NO and NO₂ cannot escape from the medium promote production of some nitrite.     

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.     

Heterotrophic nitrification and aerobic denitrification inAlcaligenes faecalis strain TUD

Heterotrophic nitrification and aerobic and anaerobic denitrification byAlcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (K_I=109 M acetate). Denitrifying or assimilatory nitrate reductases were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized byA. faecalis strain TUD, with a maximum oxygen uptake rate of 140–170nmol O₂·min^-1·mg prot^-1. Comparison of the behaviour ofA. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.     

Studies on the oxidation of ammonia by Nitrosomonas

1. Free-energy calculations for pH7 showed that the oxidation of ammonia to hydroxylamine is endergonic and that the oxidations of hydroxylamine to nitrite and hydrazine to nitrogen are exergonic. It is suggested that the oxidation of ammonia requires the expenditure of energy. 2. The anaerobic dehydrogenation of hydrazine to nitrogen by extracts of the autotrophic nitrifying micro-organism, Nitrosomonas, in the presence of methylene blue as electron acceptor, was less rapid than the anaerobic dehydrogenation of hydroxylamine to nitric oxide. The inhibition by hydrazine of the dehydrogenation of hydroxylamine was attributed to substrate competition. 3. Whole cells in air did not produce nitrite from hydrazine. They produced nitrite from low concentrations of hydroxylamine more rapidly than from equimolar concentrations of ammonia; this result is consistent if hydroxylamine is an intermediate of the oxidation of ammonia. 4. The production of nitrite from hydroxylamine by whole cells was slightly inhibited by hydrazine, but the production of nitrite from ammonia was greatly inhibited and small amounts of hydroxylamine were formed. These results suggested that the dehydrogenation of hydroxylamine supplied energy required for the oxidation of ammonia and that hydroxylamine appeared because the energy production was replaced by that of the dehydrogenation of hydrazine. 5. The oxidation of hydroxylamine by whole cells was not inhibited by thiourea, but micromolar concentrations of the metal-binding agent markedly inhibited the oxidation of ammonia to hydroxylamine, suggesting that the oxidation of ammonia involved copper. A possible mechanism for the activation of ammonia is suggested.     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

Formation of Nitrate from 3-Nitropropionate by Aspergillus flavus

Extracts of the hyphae of a nitrifying strain of Aspergillus flavus formed nitrite and nitrate from 3-nitropropionate. Nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotide enhanced the production of nitrate but not nitrite, whereas cysteine and diethyldithiocarbamate increased nitrite but diminished nitrate synthesis. Quinacrine reduced the extent of conversion of the nitro compound to nitrite and nitrate, but only the inhibition of nitrite formation was completely reversed by flavine coenzymes. Molecular oxygen was essential for this part of the nitrification sequence. 3-Chloropropionate stimulated the oxidation of nitrite by hyphae or enzyme preparations. Although the fungus contained a noncytochrome-linked nitrite-oxidizing enzyme, partially purified preparations free of this enzyme formed both nitrite and nitrate from 3-nitropropionate. Possible mechanisms of this latter stage of heterotrophic nitrification are discussed.     

Metabolism of hydroxylamine by spinach leaf discs and its relationship to nitrate reduction

Nitrate reduction in vivo by spinach leaf discs was shown to be inhibited by hydroxylamine when this was included in the nitrate reductase assay solutions or introduced to the tissue during a preincubation period. The sensitivity of nitrate reduction to hydroxylamine was not sufficient to suggest a natural process, considering the small endogenous concentrations of hydroxylamine in the leaves. Inhibition of nitrate reduction in vivo could be approximately related to rates of in vitro inhibition of nitrate reductase by this compound. There was no need to suppose conversion of hydroxylamine to cyanide to inhibit nitrate reduction. Some of the in vivo and in vitro characteristics of hydroxylamine inhibition of nitrate reductase are described. Hydroxylamine was metabolised by discs at rates comparable to nitrate reduction. Rates of metabolism of hydroxylamine, and its accumulation in the tissues from an external solution were both enhanced by light but little affected by anaerobiosis.Abbreviations NR nitrate reductase     

Fatty Acid Hydroxamate Formation and Ester Hydrolysis by Cell-free Extracts of Micrococcus sp.

When Micrococcus sp. which was isolated from soil assimilated azelaic acid as a sole carbon source, cell-free extract of the organism catalyzed enzymic fatty acid hydroxamate formation. The enzyme was effective only for mono-carboxylic acid, but not for di-carboxylic acids such as azelaic acid. The activity was high with higher fatty acid such as oleic acid. Some of the properties of higher fatty acid hydroxamate formation were investigated.

Olelylhydroxamate was formed with the high concentration of hydroxylamine. The reaction was inhibited by PCMB, but recovered by the addition of SH-compounds (such as cysteine).

On the other hand, when methylacetate was used as a sole carbon source and cell-free extract of Micrococcus sp. hydrolyzed several fatty acid esters. The fatty acid hydroxamate degradation by esterolysis are also discussed.     

The acylphosphate present in chromaffin granule membrane preparations is not associated with the proton-pump

Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low temperature. A phosphorylated compound was rapidly formed which was stable in 10% trichloroacetic acid at 0 degree C. The lability of this compound in the presence of hydroxylamine or hot trichloroacetic acid indicated an acylphosphate, i.e., an ATPase phosphointermediate. Vanadate but not N-ethylmaleimide inhibited the formation of this derivative. Since the ATP-dependent generation of a transmembrane potential in chromaffin granule vesicles by the H+-pump was inhibited by N-ethylmaleimide but not by vanadate, the acylphosphate should not be associated with the H+-pump, i.e. ATPase I. We suggest that it is associated with ATPase II, an ATPase of unknown function present in chromaffin granule membrane preparations. This hypothesis is supported by the fact that ATPase II is vanadate sensitive and has a molecular mass of 140 kDa, properties similar to those of the phosphorylated intermediate.     

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     

Destruction and formation of toxins by one bacterial species affect biodegradation by a second species

Two strains of Pseudomonas able to grow on phenol or p-nitrophenol (PNP) were isolated from sewage. Pseudomonas sp. PN101 mineralized and formed nitrite from PNP but did not mineralize phenol, and Pseudomonas sp. PH111 mineralized phenol but not PNP. Phenol increased the lag period before Pseudomonas sp. PN101 grew on and mineralized PNP, but this toxicity was reduced by inoculation of the medium with Pseudomonas sp. PH111. PNP inhibited growth of Pseudomonas sp. PH111 and slightly increased the length of the acclimation period for the mineralization of phenol by the bacterium. Inoculation of Pseudomonas sp. PN101 into solutions containing PNP and phenol increased the lag period prior to growth of Pseudomonas sp. PH111 on phenol and markedly lengthened the lag period for its mineralization of phenol. Coinciding with this delay in the onset of phenol degradation was the accumulation of an organic compound formed from PNP by Pseudomonas sp. PN101. This compound was not mineralized by the phenol-degrading bacterium. The data suggest that bacteria may interact during the decomposition of chemical mixtures by destroying or by forming toxins that affect the biodegradation of individual components of those mixtures.     

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.     

Metabolism of 4-Chloronitrobenzene by the Yeast Rhodosporidium sp

The yeast Rhodosporidium sp. metabolized 4-chloronitrobenzene by a reductive pathway to give 4-chloroacetanilide and 4-chloro-2-hydroxyacetanilide as the major final metabolites. The intermediate production of 4-chloronitrosobenzene, 4-chlorophenylhydroxylamine, and 4-chloroaniline was demonstrated by high-pressure liquid chromatography. Additional studies with selected metabolites established that the metabolite 4-chloro-2-hydroxyacetanilide was produced by an initial Bamberger rearrangement of the hydroxylamine metabolite, followed by acetylation. Direct C hydroxylation of the aromatic ring was not observed in this species. No hydroxamic acid production was detected, even though significant concentrations of the nitroso and hydroxylamine precursors to this functional group were observed.