Nitrate reduction and assimilation in Chlorella

1. Nitrate reduction and assimilation have been studied in Chlorella pyrenoidosa under growth conditions by observing effects on the CO(2)/O(2) gas exchange quotient. 2. During assimilation of glucose in the dark, nitrate reduction is noted as an increase in the R.Q. to about 1.6 caused by an increased rate of carbon dioxide production. 3. During photosynthesis at low light intensity nitrate reduction is evidenced by a reduction in the CO(2)O(2) quotient to about 0.7 caused by a decreased rate of carbon dioxide uptake. 4. Chlorella will assimilate nitrogen from either nitrate or ammonia. When both sources are supplied, only ammonia is utilized and no nitrate reduction occurs. It is inferred that under the usual conditions of growth nitrate is reduced only at a rate required for subsequent cellular syntheses. The effect of nitrate reduction on the CO(2)O(2) quotient therefore provides a measure of the relative rate of nitrogen assimilation. 5. Over-all photosynthetic metabolism may be described from elementary analysis of the cells since excretory products are negligible. The gas exchange predicted in this way is in good agreement with the observed CO(2)/O(2) quotients.     

Inhibition of nitrate and nitrite reduction by 2,4-dinitrophenol in ankistrodesmus

Summary As Kessler (1955, 1959) has shown, nitrite reduction by the green alga, Ankistrodesmus braunii is completely inhibited by 10^-3 m 2,4-dinitrophenol. However, although nitrite accumulates in the medium when cultures are supplied with nitrate and dinitrophenol, the reduction of nitrate is not completely insensitive to the inhibitor.Direct measurements show that 2,4-dinitrophenol inhibits nitrate disappearance from the medium by 65–80%. The degree of inhibition increases when the initial nitrate concentration is decreased.It is suggested that inhibition of nitrate assimilation by dinitrophenol is due to inhibition of an active uptake of nitrate by the cells, and that at high nitrate concentrations, a dinitrophenol-insensitive uptake process increases in importance.     

Inhibition of nitrate consumption by nitrite in entrapped Chlamydomonas reinhardtii cells.

Whereas in freely suspended cell cultures growing photoautotrophically under non-limiting carbon conditions nitrite and nitrate were simultaneously consumed after ammonium consumption was complete, in alginate-entrapped cell cultures a sequential consumption of nitrite (first) and nitrate was observed after ammonium had almost been fully removed. In this paper results are reported that show inhibition of nitrate consumption by nitrite in immobilized cells. However no inhibition of nitrate active transport was observed. The sequential consumption of ammonium, nitrite and nitrate by Ca-alginate immobilized cells is explained on the basis of local ammonium accumulation due to its photoproduction by photorespiration, that could be caused by the increase of the O2/CO2 ratio around the entrapped cells. Measurements of light-dependent oxygen production (LDOP) and activity levels of nitrogen assimilation enzymes, including nitrite reductase (NiR) and glutamine synthetase (GS) in immobilized cells, determined under photorespiration stimulating conditions, are shown that support this explanation.     

Production of NO and N(inf2)O by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration

Peak emissions of NO and N(inf2)O are often observed after wetting of soil. The reactions to sudden changes in the aeration of cultures of nitrifying and denitrifying bacteria with respect to NO and N(inf2)O emissions were compared to obtain more information about the microbiological aspects of peak emissions. In continuous culture, the nitrifier Nitrosomonas europaea and the denitrifiers Alcaligenes eutrophus and Pseudomonas stutzeri were cultured at different levels of aeration (80 to 0% air saturation) and subjected to changes in aeration. The relative production of NO and N(inf2)O by N. europaea, as a percentage of the ammonium conversion, increased from 0.87 and 0.17%, respectively, at 80% air saturation to 2.32 and 0.78%, respectively, at 1% air saturation. At 0% air saturation, ammonium oxidation and N(inf2)O production ceased but NO production was enhanced. Coculturing of N. europaea with the nitrite oxidizer Nitrobacter winogradskyi strongly reduced the relative levels of NO and N(inf2)O production, probably as an effect of the lowered nitrite concentration. After lowering the aeration, N. europaea produced large short-lasting peaks of NO and N(inf2)O emissions in the presence but not in the absence of nitrite. A. eutrophus and P. stutzeri began to denitrify below 1% air saturation, with the former accumulating nitrite and N(inf2)O and the latter reducing nitrate almost completely to N(inf2). Transition of A. eutrophus and P. stutzeri from 80 to 0% air saturation resulted in transient maxima of denitrification intermediates. Such transient maxima were not observed after transition from 1 to 0%. Reduction of nitrate by A. eutrophus continued 48 h after the onset of the aeration, whereas N(inf2)O emission by P. stutzeri increased for only a short period. It was concluded that only in the presence of nitrite are nitrifiers able to dominate the NO and N(inf2)O emissions of soils shortly after a rainfall event.     

Effect of CO2 and phosphate deprivation on the control of nitrate, nitrite and ammonium metabolism in Chlorella

Chlorella vulgaris Beijerinck, strain 211/12, uses nitrate, nitrite and ammonium at pH 8.2 but not at pH 6.4 when kept under conditions of CO₂-deprivation, as observed in cell suspensions aerated with CO₂-free air during a 20–30. h period Most of the nitrate absorbed at pH 8.2, however, was not assimilated but was released into the external medium as nitrite and ammonium. Cells of Chlorella previously grown in phosphate-limited continuous cultures were unable to absorb nitrate, nitrite or ammonium under conditions of phosphate starvation at either pH 6.4 or 8.2 in cell suspensions flushed with air containing 5% CO₂, However, in cell suspensions flushed with CO₂-free air, the capacity of the alga to absorb and reduce nitrate and to excrete nitrite and ammonium at pH 8.2 was restored.
It is hypothesized that in Chlorella the metabolism of nitrate, nitrite and ammonium is influenced by the availability of other nutrients and controlled by the cell's carbon status at the level of ion entry into the cell. With respect to nitrate this carbon-dependent control is distinct and works independently of that triggered by the cell's nitrogen status.     

Blue light photoreactivation of nitrate reductase from green algae and higher plants

The inactive form of NADH-nitrate reductase from spinach and $\text{Chlorella fusca}$ is fully reactivated in short periods of time when the enzyme-complex is illuminated with white or blue light but not with red light. Flavin nucleotides greatly accelerate the photoreactivation process. The results suggest that blue light might act as a modulating agent in the assimilation of nitrate in green algae and higher plants.     

Photosynthetic Nitrite Reduction as Influenced by the Internal Inorganic Carbon Pool in Air-Grown Cells of Synechococcus UTEX 625

Photosynthetic reduction of NO2- was studied in air-grown cells of a cyanobacterium, Synechococcus UTEX 625. Addition of NO2- resulted in significant amounts of chlorophyll a fluorescence quenching both in the absence and presence of CO2, fixation inhibitors, glycolaldehyde or iodoacetamide. The degree of NO2- quenching was insensitive to the O2 concentration in the medium. Addition of 100 [mu]M inorganic carbon in the presence of glycolaldehyde and O2, leading to formation of the carbon pool within the cells, resulted in pronounced fluorescence quenching. Removal of O2 from the medium restored the fluorescence yield completely, and the subsequent addition of NO2- quenched 36% of the variable fluorescence. From the response to added 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the quenching by NO2- appeared to be photochemical quenching, and nonphotochemical quenching did not seem to be present. The reduction of NO2- observed on its addition to inorganic carbon-depleted cells remained uninfluenced by O2 or glycolaldehyde. The internal inorganic carbon pool in the cells stimulated NO2- reduction, both in the presence and absence of O2, by 4.8-fold. An increase in NO2- reduction by 0.5-fold was also observed in the presence of O2 during simultaneous assimilation of carbon and nitrogen in inorganic carbon-depleted cells. Contrary to this, under anaerobiosis, NO2- reduction was suppressed when carbon and nitrogen assimilation occurred together.     

Product formation and kinetic simulations in the pH range 1-14 account for a free-radical mechanism of peroxynitrite decomposition

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).     

Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans

The photosynthetic nature of the initial stages of nitrate assimilation, namely, uptake and reduction of nitrate, has been investigated in cells of the cyanobacterium Anacystis nidulans treated with l-methionine dl-sulfoximine to prevent further assimilation of the ammonium resulting from nitrate reduction. The light-driven utilization of nitrate or nitrite by these cells results in ammonium release and is associated with concomitant oxygen evolution. Stoichiometry values of about 2 mol oxygen evolved per mol nitrate reduced to ammonium and 1.5 mol oxygen per mol nitrite have been determined in the presence of CO₂, as well as in its absence, with nitrate or nitrite as the only Hill reagent. This indicates that in A. nidulans water photolysis directly provides, without the need for carbon metabolites, the reducing power required for the in vivo reduction of nitrate and nitrite to ammonium, processes which are besides strongly inhibited when the operation of the photosynthetic noncyclic electron flow is blocked. Evidence indicating the participation of concentrative transport system(s) in the uptake of nitrate and nitrite by A. nidulans is also presented. The operation of these energy-requiring systems seems to account for the sensitivity to ATP-synthesis inhibitors exhibited by nitrate and nitrite utilization in l-methionine dl-sulfoximine-treated cells. The utilization of nitrate by A. nidulans cells, concomitant with oxygen evolution, can therefore be considered as a genuinely CO₂-independent photosynthetic process that makes direct use of photosynthetically generated assimilatory power.     

Factors promoting emissions of nitrous oxide and nitric oxide from denitrifying sequencing batch reactors operated with methanol and ethanol as electron donors

The emissions of nitrous oxide (N₂O) and nitric oxide (NO) from biological nitrogen removal (BNR) operations via nitrification and denitrification is gaining increased prominence. While many factors relevant to the operation of denitrifying reactors can influence N₂O and NO emissions from them, the role of different organic carbon sources on these emissions has not been systematically addressed or interpreted. The overall goal of this study was to evaluate the impact of three factors, organic carbon limitation, nitrite concentrations, and dissolved oxygen concentrations on gaseous N₂O and NO emissions from two sequencing batch reactors (SBRs), operated, respectively, with methanol and ethanol as electron donors. During undisturbed ultimate‐state operation, emissions of both N₂O and NO from either reactor were minimal and in the range of <0.2% of influent nitrate‐N load. Subsequently, the two reactors were challenged with transient organic carbon limitation and nitrite pulses, both of which had little impact on N₂O or NO emissions for either electron donor. In contrast, transient exposure to oxygen led to increased production of N₂O (up to 7.1% of influent nitrate‐N load) from ethanol grown cultures, owing to their higher kinetics and potentially lower susceptibility to oxygen inhibition. A similar increase in N₂O production was not observed from methanol grown cultures. These results suggest that for dissolved oxygen, but not for carbon limitation or nitrite exposure, N₂O emission from heterotrophic denitrification reactors can vary as a function of the electron donor used. Biotechnol. Bioeng. 2010; 106: 390–398. © 2010 Wiley Periodicals, Inc.     

Effect of nitrite and nitrate on chlorophyll fluorescence in green algae

Summary The influence of nitrite and nitrate on chlorophyll fluorescence, a very sensitive indicator for the redox state of the primary acceptor of photosystem II of photosynthesis, was studied in green algae (several species of Chlorella, and Ankistrodesmus braunii). In phosphate solution under an atmosphere of nitrogen (i.e., in the absence of O₂ and CO₂, and without nitrite or nitrate), fluorescence shows a pronounced induction and then rises to a high steady-state level. In the presence of nitrite, however, fluorescence decreases after a rather short induction peak to a much lower steady-state. Nitrate, on the other hand, does not have any influence on either induction or steady-state of fluorescence. These results clearly demonstrate that nitrite reduction in the light is very closely coupled to the photosynthetic electron transport system, whereas nitrate is not reduced photosynthetically in vivo.     

Effect of Chloramphenicol on Denitrification in Flexibacter canadensis and "Pseudomonas denitrificans"

It was recently reported that chloramphenicol inhibits existing denitrification enzyme activity in sediments and carbon-starved cultures of "Pseudomonas denitrificans." Therefore, we studied the effect of chloramphenicol on denitrification by Flexibacter canadensis and "P. denitrificans." Production of N(inf2)O from nitrate by F. canadensis cells decreased as the concentration of chloramphenicol was increased, and 10.0 mM chloramphenicol completely inhibited N(inf2)O production. "P. denitrificans" was less sensitive to chloramphenicol, and production of N(inf2)O from nitrate was inhibited by only about 50% even in the presence of 10.0 mM chloramphenicol. These results suggested that inhibition of denitrification enzyme activity depended on the concentration of chloramphenicol. Increasing the concentration of chloramphenicol decreased the rate of production of nitrite from nitrate by F. canadensis cells, and the concentration of chloramphenicol which resulted in 50% inhibition of production of nitrite from nitrate was 2.5 mM. In contrast, the rates of production of nitrite from nitrate by intact cells and cell extracts of "P. denitrificans" were inhibited by only 58 and 54%, respectively, at a chloramphenicol concentration of 10.0 mM. Chloramphenicol caused accumulation of NO from nitrite but not from nitrate and inhibited NO consumption in F. canadensis; however, it had neither effect in "P. denitrificans." Chloramphenicol did not affect N(inf2)O consumption by these organisms. We concluded that chloramphenicol inhibits denitrification at the level of nitrate reduction and, in F. canadensis, also at the level of NO reduction.     

Über die energetische Kopplung der Nitritassimilation von Grünalgen an Atmung und Photosynthese

Summary Inhibitors and uncouplers of phosphorylation, i.e., arsenate, 2.4-dinitrophenol (DNP), pentachlorophenol (PCP), and carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibit the assimilation of nitrite by the green alga Ankistrodesmus braunii in the dark and in the light. In a medium containing nitrate, these inhibitors interrupt nitrate reduction at the level of nitrite. In phosphatedeficient algae, the assimilation of nitrite can be decreased by a concomitant, energy-dependent uptake of chloride and phosphate ions. These results support the assumption that high-energy phosphate is required for the assimilation of nitrite.CO₂ and glucose (after pre-illumination) increase nitrite assimilation in the light. Photosynthetic nitrite reduction is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), an inhibitor of oxygen evolution, and by disalicylidene-propanediamine-(1,3) (DSPD), an inhibitor of the photosynthetic reduction of ferredoxin.

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Abkürzungen CCCP Carbonylcyanid-m-chlorphenylhydrazon - DCMU 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff - DNP 2,4-Dinitrophenol - DSPD Disalicylidenpropandiamin-(1,3) - PCP Pentachlorphenol - JAA Jodacetamid     

Production of nitric oxide by human salivary peroxidase and by bovine lactoperoxidase

Peroxidases catalyze the oxidation of nitrite to nitrate in the presence of hydrogen peroxide. Two pathways may occur: one entailing the intermediate formation of NO(2) and the other implying the generation of peroxynitrite. The products of nitrite (NO(2) (-) ) oxidation by salivary peroxidase (SPO) and commercial bovine lactoperoxidase (LPO) are studied by utilizing an electrochemical assay that allows the direct, continuous monitoring of NO and/or NO(2) and by HPLC to assess nitrates at the end of the reaction. Dialyzed saliva and LPO, in the presence of H(2) O(2) , convert nitrite into nitrate and form some NO, with a molar ratio of 10(3) . In our experimental conditions, no NO(2) was detectable among the products of nitrite oxidation. SCN(-) inhibits NO formation and so does I(-) , although at higher concentrations. No effects are observed with Cl(-) or Br(-) . We conclude that SPO and LPO transform NO(2) (-) into nitrate-forming small amounts of NO in the presence of H(2) O(2) as an intermediate or a by-product, synthesized through the peroxynitrite pathway.     

The interaction of nitrogen assimilation with photosynthesis in nitrogen deficient cells of Chlorella

Nitrogen-limited chemostat cultures of Chlorella fusca var. vacuolata, when given nitrogen in the inorganic forms of nitrate, nitrite and ammonium divert photo-generated electrons, from CO₂ fixation to nitrogen assimilation. Addition of nitrate or nitrite, but not ammonium, stimulates rate of oxygen evolution. All but the most severely nitrogen-deficient culture have increased dark respiration rates after addition of inorganic nitrogen. The nitrite reduction step of nitrogen assimilation is the most light-dependent reaction.Abbreviation DCMU 3-(3, 4-dichloro)-1-1-dimethyl urea - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures.

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Influence of Water Hyacinth Cover on the Physico-chemical Characteristics of Water and Phytoplankton Composition in a Reservoir near Jaipur (India)

Water hyacinth (Eichhornia crassipes (Mart.) Solms.) invaded a eutrophic reservoir receiving domestic sewage near Jaipur (India) during 1975 and gradually developed a complete thick cover over the whole water body during Sept.–Oct. 1978. The physico-chemical characteristics of the water and the phytoplankton composition were studied during Sept. 1977–Sept. 1979 by fortnightly sampling. The changes observed during the second year of study are ascribed to the water hyacinth cover. The important changes were: lowering of water temperature, pH, dissolved oxygen content and nitrate nitrogen, and increase in total alkalinity, free carbon dioxide, chemical oxygen demand, ammonia nitrogen, sulphides, calcium, magnesium and phosphate phosphorus. The changes in the phytoplankton were both qualitative and quantitative. The green algae, particularly the species of Ankistrodesmus, Chlorella, Crucigenia and Selenastrum, increased considerably and replaced the blue-green algae, of which Oscillatoria and Microcystis disappeared totally. The densities of several other taxa changed significantly.