The effect of light on nitrate and nitrite assimilation by chlorella and ankistrodesmus

Light stimulates the assimilation of nitrate and nitrite by two green algae, Chlorella pyrenoidosa and Ankistrodesmus braunii. Assimilation can be observed when the algae are illuminated in the absence of carbon dioxide under both aerobic and anaerobic conditions. The rates of assimilation by Chlorella do not depend on the presence of carbon dioxide, but Ankistrodesmus assimilates nitrate and nitrite more rapidly when cultures are illuminated in the presence of carbon dioxide than in its absence. The ratios of O(2) : NO(3') and O(2) : NO(2') vary from one experiment to the other and, with the exception of Chlorella cultures reducing nitrite they are higher than the 'expected' values of 2.0 and 1.5 respectively. Oxygen evolution accompanying nitrate and nitrite by algae illuminated in the absence of carbon dioxide is completely inhibited by DCMU at concentrations of 4 × 10(-6) M. However, nitrite assimilation by both Ankistrodesmus and Chlorella and nitrate assimilation by Ankistrodesmus are less sensitive to the inhibitor.     

Temperature-influenced species competition in mass cultures of marine phytoplankton

Five marine phytoplankton species (Phaeodactylum tricornutum, Thalassiosira pseudonana, Skeletonema costatum, Monochrysis lutheri, and Dunaliella tertiolecta) were grown in enriched laboratory continuous cultures and natural populations were mass cultured outdoors for 16 months. Competition among the species was shown to be highly dependent on temperature, although the actual production of plant organic matter at the low growth rates used was relatively independent of this variable. Control of marine species in mass cultures does not appear economically feasible, but this drawback may be overcome by selecting herbivorous shellfish that are capable of assimilating those temperature-dependent phytoplankton species dominating in a particular locale.     

Influence of oxygen tension on nitrate reduction by a Klebsiella sp. growing in chemostat culture.

At dissolved oxygen tensions of 15 mmHg (2 kPa) and below, nitrate-limited continuous cultures of Klebsiella K312 synthesized nitrate reductase (NR) and nitrite reductase (NiR) and excreted ammonia. Under anaerobic conditions over 60% of the nitrate-nitrogen utilized was excreted as ammonia. In contrast, carbon-limited cultures excreted nitrite at dissolved oxygen tensions of 15 mmHg or below and synthesized NR but not NiR. Ammonia repressed neither NR nor NiR synthesis. These observations indicate that below a critical oxygen tension of 15 mmHg Klebsiella K312 utilizes oxygen and nitrate as electron acceptors. This oxygen tension correlates well with the critical oxygen tension observed for a change from oxidative to fermentative metabolism in cultures of Klebsiella aerogenes. The product of dissimilatory nitrate reduction is ammonia in nitrate-limited cultures but principally nitrite in carbon-limited (nitrate excess) cultures.     

Diel periodicity in uptake of nitrate and nitrite by reservoir phytoplankton

Diel periodicity in the uptake of nitrate, and nitrite as measured by the ¹⁵N technique, occurs in reservoir phytoplankton. The time course of changes in the rate of nitrate uptake generally paralleled changes in irradiance. Uptake of nitrate and nitrite occurred in the dark, but at low rates. Periodicity in nitrate uptake needs to be considered in models of primary production where nitrogen is the limiting nutrient.     

Dilution of 15N in Dunaliella tertiolecta by uptake of an unidentified compound following nitrate exhaustion

Nitrate (about 20 μM) was added as ¹⁵NO₃ to a nitrate-limited continuous culture of Dunaliella tertiolecta at steady-state. Nitrate uptake was then estimated from the decrease in nitrate in the medium, the incorporation of ¹⁵N into cells, and the increase in cellular nitrogen. Although the overall nitrogen budget over 5 h was balanced, there were large differences in estimates (up to a factor of five) of nitrate assimilation by the three methods on shorter time scale. After nitrate was exhausted from the medium, cellular nitrogen continued to increase while the ¹⁵N content of the particulate matter decreased over the next 1.5 h. This indicated that an unidentified, unlabelled nitrogen form, which was neither nitrite, ammonium nor dissolved free amino acids, was being taken up by the cells, at rates comparable to those of nitrate. This phenomenon leads to an underestimation of new biomass production when assessed through ¹⁵N incorporation into cells.     

GLUTAMINE SYNTHETASE ASE ACTYVITY IN IN CHAETOCEROS AFFINIS (BACILLARIOPHYCEAE): COMPARISON WITH OTHER ESTIMATES OF NITROGEN UTILIZATION DURING NUTRIENT PERTURBATION1

Glutamine synthftase (GS) activity was investigated in a nitratt limited continuous culture of the marine diatom Chaeloccros afTinis (Lauder) Hustedt before and after the perturbation of the culture medium with 10 μM of ¹⁵ N labelled nitrate. Parallel studies were carried out on nitrate reductase(NR). nitrate uptake and assimilation, and Ievels of cellular nitrogen containing compounds with the objective to determine the validity of the GS assay as a measure of nitrate utilization. Activities in N-deficient cells, grown at steady state, correlated well with uptake and assimilation rates. In N-sufftcient celts, however, during the nitrate pertirbation period, they accounted only for about 10% of the two latter rates, when ambient nitrate concentrations were high (0. 7-10 μ). It is proposed that under these growth conditions an alternative pathway via glutamate dehydrogenase (GDH) was operative. At low ambient nitrate concentrations (0.1-0.7 μM), GS activities, uptake and assimilation rates again balanced rather well. Thus, the data support the view that GDH activity is associated with high levels and GS with low levels of external or internal nitrogen.     

A multiparameter phytoplankton culture system driven by microcomputer

A computer-controlled phytoplankton culture system is described which is regulated as a chemostat. Measurements of temperature, pH, size distribution, culture density,in vivo fluorescence, nitrate and nitrite, are automatic and programmable, thus obviating manual errors. The system has been developed successfully to survey long-term cultures of the dinoflagellateProrocentrum minimum. author for correspondence     

Inhibitory effects of nitrogen oxides on a mixed methanogenic culture

The effect of nitrate, nitrite, nitric oxide (NO), and nitrous oxide on a mixed, mesophilic (35 degrees C) methanogenic culture was investigated. Short-term inhibition assays were conducted at a concentration range of 10-350 mg N/L nitrate, 17-500 mg N/L nitrite, 0.02-0.8 mg N/L aqueous NO, and 19-191 mg N/L aqueous nitrous oxide. Simultaneous methane production and N-oxide reduction was observed in 10 and 30 mg N/L nitrate and 0.02 mg N/L aqueous NO-amended cultures. However, addition of N-oxide resulted in immediate cessation of methanogenesis in all other cultures. Methanogenesis completely recovered subsequent to the complete reduction of N-oxides to nitrogen gas in all N-oxide-amended cultures, with the exception of the 500 mg N/L nitrite- and 0.8 mg N/L aqueous NO-amended cultures. Partial recovery of methanogenesis was observed in the 500 mg N/L nitrite-amended culture in contrast to complete inhibition of methanogenesis in the 0.8 mg N/L aqueous NO-amended culture. Accumulation of volatile fatty acids was observed in both cultures at the end of the incubation period. Among all N-oxides, NO exerted the most and nitrate exerted the least inhibitory effect on the fermentative/methanogenic consortia. The effect of multiple additions of nitrate (300 mg N/L) on the same methanogenic culture was also investigated. Long-term exposure of the methanogenic culture to nitrate resulted in an increase of N-oxide reduction rates and decrease of methane production rates, which was attributed to changes in the microbial community structure due to nitrate addition.     

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.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Nitrate reductase and glutamate dehydrogenase activities in Skeletonema costatum as measures of nitrogen assimilation rates

Nitrate reductase (NADH-NR) and glutamate dehydrogenase (NADPH-GDH)activities were measured in Skeletonema costatum (Grev.) Clevein ammonium and nitrate limited continuous cultures before andafter additions of nitrate and/or ammonium. Comparisons of enzymicactivity with nitrogen uptake and assimilation rates, externaland internal nitrate concentrations, and external ammonium concentrationswere made in order to assess the roles of NR and GDH in nitrogenassimilation and to determine their suitability as measuresof nitrogen assimilation rates. NR activity appeared to be inducedby internal rather than external nitrate concentrations. Ammoniumin the medium reduced NR activity under some environmental conditions,but not others. However, ammonium acted indirectly, perhapsby causing the accumulation of an internal pool of an intermediateof ammonium assimilation. NR activity was found to approximatenitrate assimilation rates during growth limited by the nitratesupply and undeT some conditions in the presence of high nitrateand ammonium concentrations in the medium. Under other environmentalconditions, NR activity did not agree with nitrate assimilationrates; a second nitrate reducing mechanism may operate whenthese conditions prevail. GDH activities were consistently low,representing less than 5% of the ammonium uptake and assimilationrates. Consequently, it is proposed that ODH is not the primaryammonium assimilating enzyme under most environmental conditionsand cannot be used as a measure of ammonium assimilation. ¹ Contribution number 1095 from the Department of Oceanography,University of Washington     

Assimilation of [N]Nitrate and [N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [¹⁵N]nitrate and [¹⁵N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [¹⁵N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [¹⁵N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.

In dark anaerobic conditions, the assimilation of [¹⁵N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [¹⁵N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [¹⁵N]nitrite assimilation.

     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Interactions Between Nitrate Assimilation and 2,4-Dinitrophenol Cometabolism in Rhodobacter capsulatus E1F1

The phototrophic, nitrate-photoassimilating bacterium Rhodobacter capsulatus E1F1 cometabolizes 2,4-dinitrophenol (DNP) by photoreducing it to 2-amino-4-nitrophenol under anaerobic conditions. DNP uptake and nitrate metabolism share some biochemical features, and in this article we show that both processes are influenced by each other. Thus, as was demonstrated for nitrate assimilation, DNP uptake requires a thermolabile periplasmic component. Nitrate assimilation is inhibited by DNP, which probably affects the nitrite reduction step because neither nitrate reductase activity nor the transport of nitrate or nitrite is inhibited. On the other hand, DNP uptake is competitively inhibited by nitrate, probably at the transport level, because the nitroreductase activity is not inhibited in vitro by nitrate, nitrite, or ammonium. In addition, the decrease in the intracellular DNP concentration in the presence of nitrate probably inactivates the nitroreductase. These results allow prediction of a negative environmental effect if nitrate and DNP are released together to natural habitats, because it may lead to a lower rate of DNP metabolism and to nitrite accumulation.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO₂ and high CO₂ conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO₂ conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and \textP_\textm^\textchla {\text{P}}_{\text{m}}^{{\text{chl}}a} , \textR_\textd^\textchla {\text{R}}_{\text{d}}^{{\text{chl}}a} , α^chla than under low CO₂ conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.     

Selenite reduction by a denitrifying culture: batch- and packed-bed reactor studies

Selenite reduction by a bacterial consortium enriched from an oil refinery waste sludge was studied under denitrifying conditions using acetate as the electron donor. Fed-batch studies with nitrate as the primary electron acceptor showed that accumulation of nitrite led to a decrease in the extent of selenite reduction. Also, when nitrite was added as the primary electron acceptor, rapid selenite reduction was observed only after nitrite was significantly depleted from the medium. These results indicate that selenite reduction was inhibited at high nitrite concentrations. In addition to batch experiments, continuous-flow selenite reduction experiments were performed in packed-bed columns using immobilized enrichment cultures. These experiments were carried out in three phases: in phase I, a continuous nitrate feed with different inlet selenite concentration was applied; in phase II, nitrate was fed in a pulsed fashion; and in phase III, nitrate was fed in a continuous mode but at much lower concentrations than the other two phases. During the phase I experiments, little selenite was removed from the influent. However, when the column was operated in the pulse feed strategy (phase II) or in the continuous mode with low nitrate levels (phase III), significant quantities of selenium were removed from solution and retained in the immobilization matrix in the column. Thus, immobilized denitrifying cultures can be effective in removing selenium from waste streams, but nitrate-limited operating conditions might be required.