The reduction of nitrate to ammonium by a Clostridium sp. isolated from soil

Cultures of Clostridium KDHS2 reduced 15NO3- to 15NH4+ with a concurrent increase in molar growth yield of 15.7% compared with fermentatively grown bacteria. The bacteria exhibited a Ks (NO3-) of 0.5 mM and reduced NO3- maximally at a rate of 0.1 mumol h(-1) mg dry wt)-1. A partially purified nitrate reductase was obtained which had a Km (NO3-) of 0.15 mM. The reduction of 13NO3- to 13NH4+ by resting bacteria was not inhibited by NH4+, glutamate, glutamine, methionine sulphoximine or azaserine. Glutamine synthetase affected neither the synthesis nor the activity of the NO3(-)-reducing enzymes. The results are consistent with the hypothesis that NO3- reduction to NH4+ in this Clostridium sp. is dissimilative. SO32-, but not SO42-, inhibited the reaction, apparently at the level of NO2- reduction.     

Activation of Nitrite Reductase in a Cell-Free System from the Denitrifier Alcaligenes sp. by Freeze-Thawing

Nitrite reductase (NiR) activity of the cell-free extract orthe soluble fraction prepared from cells of Alcaligenes sp.NC1B 11015 grown anaerobically in the presence of nitrate wasexamined by measuring the rate of nitrite disappearance withdithionitemethyl viologen (MV) as an electron donor. Freezingat — 20?C and subsequent thawing of the fraction resultedin 5-40 times increase of the specific activity of NiR. Fromthe experiments on the effect of freezing conditions on theactivation, the phase change of solvent water due to freezingis considered to play an important role in the activation. Thisactivation occurred with the preparation in the exponentialgrowth phase, but not that in the stationary growth phase. Clearly,the low-molecular-weight (< 12,000) component which was obtainedfrom the soluble fraction through a collodion bag participatedin the activation. The activated enzyme proved to be the dissimilatory NiR, becauseNO production from nitrite, one of the typical characteristicsof the dissimilatory NiR, was also activated when assayed withascorbate-tetramethyl-p-phenylene diamine (TMPD) as an electrondonor. Nevertheless, the reaction products of nitrite reductionwere identified as hydroxylamine and ammonia with dithionite-MV.The possible pathway of nitrite reduction with this electrondonor is discussed. (Received May 26, 1983; Accepted February 2, 1984)     

Light-Mediated Nitrite Accumulation during Denitrification by Pseudomonas sp. Strain JR12

The effect of light on the denitrifying characteristics of a nonphotosynthetic denitrifier, Pseudomonas sp. strain JR12, was examined. Already at low light intensities, nitrite accumulated as a result of light inhibition of nitrite but not of nitrate reduction rates. Exposure of this bacterium to light caused a photooxidation of cytochrome c, an intermediate electron carrier in its respiratory pathway. Photoinhibition of nitrite reduction was reversible, as nitrite reduction rates returned to preillumination levels when light-exposed cells were returned to dark conditions. Antimycin A reversed the inhibitory effect of light on nitrite reduction by preventing a reversed electron flow. Aerobic respiration by this bacterium was not affected by light.     

Nitrite accumulation during anaerobic nitrate reduction by binary suspensions of bacteria isolated from the achlorhydric stomach

Binary suspensions of bacteria isolated from the gastric juice of achlorhydric patients were used to determine conditions which favour nitrite accumulation during nitrate reduction. Suspensions of Veillonella parvula and Haemophilus parainfluenzae accumulated nitrite during nitrate reduction in the absence of nitrite-reducing Neisseria subflava or Streptococcus sanguis. The maximum concentration of nitrite that transiently accumulated decreased predictably as the ratio of nitrite-removing bacteria to nitrite-accumulating bacteria increased. This ratio, but more importantly the bacterial density, determined the duration of nitrite accumulation. These results are correlated with the previously reported tendency of nitrite to accumulate in the gastric juice of hypogammaglobulinaemic and pernicious anaemic patients, and with the extremely high incidence of gastric cancer in the two groups.     

Nitrite reduction in Paracoccus sp. is affected by a novel plasmid pYR1

Two relatively low-copy plasmids of 9 and 16 kb were found to comprise the extrachromosomal DNA of a Paracoccus strain. Reduction of nitrate by plasmid-cured cells resulted in a significant intermediate nitrite accumulation as compared to wild-type cells. By examining nitrate reduction by transformants containing one of the two plasmids, it was found that nitrite accumulation was influenced by the 9.0-kb plasmid, designated as pYR1. Subcloning analysis showed that a 1.8-kb fragment of this plasmid affected nitrite accumulation. Sequence analysis of this fragment revealed the presence of five open reading frames. One of the six deduced proteins showed a strong homology to ABC transporters.     

Nitrite reductase gene from Synechococcus sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite reductases

The gene encoding nitrite reductase (nir) from the cyanobacterium Synechococcus sp. PCC 7942 has been identified and sequenced. This gene comprises 1536 nucleotides and would encode a polypeptide of 56506 Da that shows similarity to nitrite reductase from higher plants and to the sulfite reductase hemoprotein from enteric bacteria. Identities found at positions corresponding to those amino acids which in the above-mentioned proteins hold the Fe₄S₄-siroheme active center suggest that nitrite reductase from Synechococcus bears an active site much alike that present in those reductases. The fact that the Synechococcus and higher-plant nitrite reductases are homologous proteins gives support to the endosymbiont theory for the origin of chloroplasts.     

Nitrite transport is mediated by the nitrite-specific high-affinity NitA transporter and by nitrate transporters NrtA, NrtB in Aspergillus nidulans.

Disruption of the Aspergillus nidulans high-affinity nitrate transporter genes (nrtA and nrtB) prevents growth on nitrate but not nitrite. We identified a distinct nitrite transporter (K(m)=4.2+/-1 microM, V(max)=168+/-21 nmolmg(-1)DW(-1)h(-1)), designated NitA. Disruption of nrtA, nrtB and nitA blocked growth on nitrite, despite low rates of nitrite depletion we ascribe to passive nitrous acid permeation. Growth of the single mutant nitA16 on nitrite was wild-type, suggesting that NrtA and/or NrtB transports nitrite as well as nitrate. Indeed, NrtA and NrtB transport nitrite at higher rates than NitA; K(m) and V(max) values were 16+/-4 microM and 808+/-67 nmolmg(-1)DW(-1)h(-1) (NrtA) and 11+/-1 microM and 979+/-17 nmolmg(-1)DW(-1)h(-1) (NrtB). We suggest that NrtA is a nitrate/nitrite transporter, NrtB absorbs nitrite in preference to nitrate and NitA is exclusively a nitrite transporter.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.     

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c ₁ plus c ₂ and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c ₁ plus c ₂ are oxidized by photosynthesis or respiration.Abbreviations R Rhodobacter - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - HOQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - CCCP carbonyl cyanide m-chlorophenylhydrazone - cytochrome c ₁ cytochrome c ₂ plus cytochrome c ₁     

Mercury bioaccumulation and simultaneous nanoparticle synthesis by Enterobacter sp. cells

A mercury resistant strain of Enterobacter sp. is reported. The strain exhibited a novel property of mercury bioaccumulation with simultaneous synthesis of mercury nanoparticles. The culture conditions viz. pH 8.0 and lower concentration of mercury promotes synthesis of uniform sized 2-5 nm, spherical and monodispersed intracellular mercury nanoparticles. The remediated mercury trapped in the form of nanoparticles is unable to vaporize back into the environment thus, overcoming the major drawback of mercury remediation process. The mercury nanoparticles were recoverable. The nanoparticles have been characterized by high resolution transmission electron microscopy, energy dispersive X-ray analysis, powder X-ray diffraction and atomic force microscopy. The strain can be exploited for metal bioaccumulation from environmental effluent and developing a green process for nanoparticles biosynthesis.     

Dissimilatory Nitrate and Nitrite Reduction under Aerobic Conditions by an Aerobically and Anaerobically Grown Alcaligenes sp. and by Activated Sludge

The oxygen uptake of an Alcaligenes sp., isolated from activated sludge, was inhibited by small amounts of nitric oxide. The occurrence of this inhibition was dependent on the growth conditions and the pretreatment of the cells. Anaerobically grown cells, which had subsequently been aerated in a nitrogen-free medium, accumulated nitric oxide, after the addition of nitrate or nitrite. When the oxygen uptake was inhibited by nitric oxide, dissimilatory reduction of nitrate and nitrite proceeded under aerobic conditions at the same rate as in the absence of oxygen. Activated sludge removed nitric oxide actively under aerobic conditions and as a consequence the oxygen uptake of the sludge was not inhibited in the presence of nitrite. The rate of nitrate reduction under aerobic conditions was about 20% of that in the absence of oxygen.     

Study of Vitamin C ester synthesis by immobilized lipase from Candida sp.

Oleoyl ester of -ascorbic acid was synthesized by using immobilized lipases from Candida sp. A series of solvents, such as ethanol, tetrahydrofuran, pyridine, butanol, tertiary amyl alcohol (t-amyl alcohol), hexanol, octanol and hexane (log P from −0.24 to 3.5) were investigated for the reaction, and t-amyl alcohol was found to be the most suitable from the standpoint of the substrate concentration and the enzyme activity. And the equilibrium of the reaction was affected by the addition of the molecular sieves and the temperature. Reaction carried out at 55 °C and with 50 g/l of 4 Å molecular sieves is good for the enzyme to keep its activity and for making the equilibrium go to the product. The kinetic model was studied and the result showed that the reaction can be described by Ping-Pong mechanism. Parameters value of V_m and K_m′ were obtained. Last, the pure products of the reaction were attained and determined by IR spectra, mass spectrometry and ¹H NMR spectra.     

Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N-acetylgalactosaminidase from Streptomyces sp.

Gal beta-(1-->3)-GalNAc-linked hexapeptide was synthesized by a transglycosylation using Gal beta-(1-->3)-GalNAc beta-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Gal beta-(1-->3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.     

Identification of novel denitrifying bacteria Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13 and application for removal of nitrate from industrial wastewater

Two novel denitrifying bacteria were successfully isolated from industrial wastewater and soil samples. Using morphological, biochemical/biophysical and 16S rRNA gene analyses, these two bacteria were identified as Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13, respectively. Both of these two bacteria showed efficient NO₃ ⁻-N removing abilities under a semi-anaerobic condition without obvious accumulation of NO₂ ⁻-N, N₂O-N and NH₄ ⁺-N. NO₃ ⁻-N removal from paper mill wastewater was also successful by treatments with either a denitrifier or an immobilization method. Therefore, this study provides valuable denitrifying bacteria in biotreatment of industrial wastewater and other environmental pollution caused by NO₃ ⁻/NO₂ ⁻.     

Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.     

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.