Denitrification with methanol in the presence of high salt concentrations and at high pH levels

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water, in which nitrate is removed by ion exchange, the resins are regenerated in a closed circuit by a biological denitrification reactor. This denitrification reactor eliminates nitrate from the regenerant. Methanol is used as electron donor for biological denitrification. To obtain sufficient regeneration of the resins within a reasonable time, high NaCl or NaHCO₃ concentrations (10–30 g/l) in the regenerant are necessary. High NaHCO₃ concentrations affected the biological denitrification in three ways: a) a slight decrease in denitrification capacity (30%) was observed; b) the yield coefficient and CH₃OH/NO₃ ^-–N ratio decreased. When high NaHCO₃ concentrations (above 10g NaHCO₃/l) were used, the yield coefficient was 0.10–0.13 g VSS/g NO₃ ^-–N and the CH₃OH/NO₃ ^-–N ratio was 2.00–2.03 g/g; c) high NaHCO₃ concentrations influenced nitrite production. Nitrite is an intermediate product of biological denitrification and with rising NaHCO₃ concentrations nitrite accumulation was suppressed. This was explained by the effect of high NaHCO₃ concentrations on the pH in the microenvironment of the denitrifying organisms. High NaCl concentrations also resulted in a slight decrease in denitrification capacity, but the second and third effects were not observed in the presence of high NaCl concentrations.Although the pH in the regenerant will rise as a result of biological denitrification, the capacity of a denitrification reactor did not decrease significantly when a pH of 8.8–9.2 was reached.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Control of microbial souring by nitrate,nitrite or glutaraldehyde injection in a sandstone column

Microbial souring (production of hydrogen sulfide by sulfate-reducing bacteria, SRB) in crushed Berea sandstone columns with oil field-produced water consortia incubated at 60°C was inhibited by the addition of nitrate (NO₃) or nitrite (NO ₂ ^– ). Added nitrate (as nitrogen) at a concentration of 0.71 mM resulted in the production of 0.57–0.71 mM nitrite by the native microbial population present during souring and suppressed sulfate reduction to below detection limits. Nitrate added at 0.36 mM did not inhibit active souring but was enough to maintain inhibition if the column had been previously treated with 0.71 mM or greater. Continuous addition of 0.71–0.86 mM nitrite also completely inhibited souring in the column. Pulses of nitrite were more effective than the same amount of nitrite added continuously. Nitrite was more effective at inhibiting souring than was glutaraldehyde, and SRB recovery was delayed longer with nitrite than with glutaraldehyde. It was hypothesized that glutaraldehyde killed SRB while nitrite provided a long-term inhibition without cell death. Removal of nitrate after as long as 3 months of continuous addition allowed SRB in a biofilm to return to their previous level of activity. Inhibition was achieved with much lower levels of nitrate and nitrite, and at higher temperatures, than noted by other researchers.     

Mixed culture hydrogenotrophic nitrate reduction in drinking water

Isolation and identification of the bacteria from a hydrogenotrophic reactor for the denitrification of drinking water revealed that several microorganisms are involved. Acinetobacter sp., Aeromonas sp., Pseudomonas sp. and Shewanella putrefaciens were repeatedly isolated from the hydrogenotrophic sludge and postulated to be of primary importance in the process. Nitrate reduction to nitrite appears to be a property of a diverse group of organisms. Nitrite reduction was found to be stimulated by the presence of organic growth factors. Thus, in a mixed culture, hydrogenotrophic denitrification reactor, NO _inf2 ^sup– formed by NO _inf3 ^sup– -reducers can be converted by true denitrifiers thriving on organic growth factors either present in the raw water, or excreted by the microbial community. Mixotrophic growth also contributes to NO _inf2 ^sup– reduction. Finally, chemolithotrophic bacteria participate in the nitrite to nitrogen gas conversion.Offprint requests to: W. Verstraete.     

β-nitropropionic acid and nitrite in relation to nitrate formation by Aspergillus flavus

Summary -nitropropionic acid (BNP) was converted to nitrate in media inoculated with A. flavus spores or with replacement cultures of mycelium pregrown in glucose-peptone medium. Conversion by replacement cultures was rapid: 8–30% in 2 days; influenced by pH: most rapid at pH 3.5; and extensive: as much as 80% BNP nitrogen appeared as nitrate after 14 days. Nitrite was detectable in BNP replacement cultures at low levels or not at all, and nitrate was formed in BNP replacement media with or without glucose. Nitrite was not oxidized in growing cultures inoculated with spores, but replacement cultures oxidized over 50% of added nitrite to nitrate in 8 days. No nitrite or nitrate appeared in replacement systems with pyruvic oxime, oxalacetic acid oxime, acetoxime, ketoglutaric acid oxime, or hydroxylamine.Of the three non-nitrifying mutants of A. flavus obtained, all formed nitrate from BNP in replacement but only one oxidized nitrite to nitrate. No accumulation of free or bound hydroxylamine or of nitrite could be detected in the mutants. BNP was detected by qualitative test in cultures of the wild type but not the mutants. Evidence indicates that the pathway in A. flavus is BNPNO₃ ^- rather than BNPNO₂ ^-NO₃ ^-.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Anaerobic Benzene Biodegradation Linked to Nitrate Reduction

Benzene oxidation to carbon dioxide linked to nitrate reduction was observed in enrichment cultures developed from soil and groundwater microcosms. Benzene biodegradation occurred concurrently with nitrate reduction at a constant ratio of 10 mol of nitrate consumed per mol of benzene degraded. Benzene biodegradation linked to nitrate reduction was associated with cell growth; however, the yield, 8.8 g (dry weight) of cells per mol of benzene, was less than 15% of the predicted yield for benzene biodegradation linked to nitrate reduction. In experiments performed with [¹⁴C]benzene, approximately 92 to 95% of the label was recovered in ¹⁴CO₂, while the remaining 5 to 8% was incorporated into the nonvolatile fraction (presumably biomass), which is consistent with the low measured yield. In benzene-degrading cultures, nitrite accumulated stoichiometrically as nitrate was reduced and then was slowly reduced to nitrogen gas. When nitrate was depleted and only nitrite remained, the rate of benzene degradation decreased to almost zero. Based on electron balances, benzene biodegradation appears to be coupled more tightly to nitrate reduction to nitrite than to further reduction of nitrite to nitrogen gas.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Kinetics of a fluidized-bed reactor for ground-water denitrification

A fluidized-bed reactor, with sand as the carrier and ethanol as the carbon and electron source, was investigated for the biological denitrification of ground water. The paper concentrates on the reactor's kinetics, with special emphasis on nitrite as the intermediate product. Intrinsic zero-order kinetic parameters for both nitrate and nitrite were determined by batch and continuous experiments. Values for the maximum specific nitrate and nitrite removal rates of 11 g and 6 g NO _inf3 ^sup– (g volatile suspended solids)^–1 day^–1, respectively, were obtained. These values were used to interpret nitrate and nitrate concentration profiles in an experimental fluidized-bed reactor operating at different conditions of hydraulic loading and retention time.     

The effect of sodium chlorate and nitrapyrin on the nitrification mediated nitrosation process in soils

Summary The influence of total nitrification to nitrate or partial nitrification to nitrite on the soil organic nitrogen status was examined. NH ₄ ⁺ –¹⁵N was added to the soil in the absence and the presence of NaClO₃, respectively nitrapyrin. The first chemical inhibits only nitrate formation, the second inhibits total nitrification. The accumulation of nitrite nitrogen in the soil at levels up to 5 mg kg^–1 increased the loss of nitrogen. Yet, it did not increase the binding of mineral nitrogen into soil organic matter, relative to the control soil. The data suggest that the biochemistry of the nitrite formation process, rather than the levels of nitrite ions formed, are of primary importance in the role of nitrification mediated nitrosation of soil organic matter.     

Quantification of denitrification by strain T1 during anaerobic degradation of toluene

Summary Strain T1 is a denitrifying bacterium that is capable of toluene degradation under anaerobic conditions. During anaerobic growth on toluene, the specific growth rate of strain T1 was 0.14 h^–1. Nitrite accumulated in the medium stoichiometrically with the depletion of nitrate. When nitrate was nearly depleted from the medium nitrite reduction and dinitrogen formation began. A non-kinetic model was formulated that was based on a hypothesis of non-simultaneous nitrate and nitrite reduction, independent of the concentrations of nitrate and nitrite. The model was verified experimentally over a wide range of conditions that included nitrate and nitrite limitation, toluene limitation, and various ratios of nitrate to nitrite. The model and its experimental verification demonstrated that strain T1 reduces nitrate and nitrite non-simultaneously, even if nitrite is initially present in the medium in addition to nitrate. Offprint requests to: L. Y. Young     

Biotreatment of nitrate-rich industrial effluent by suspended bacterial growth

Of the 29 potentially denitrifying organisms isolated from a denitrifying reactor (DNR) of a fertilizer company, two isolates; I-4 and I-5 were recognized as denitrifiers. Under aerobic conditions, with fusel oil as the carbon source, the organisms decreased nitrate from 1200 mg l^–1 to 100 mg l^–1 in 48 h. Optimal growth conditions for biological removal of nitrate were established in batch culture. The system was scaled up to 4-L and 50-L bioreactors under continuous culture conditions. Up to 95–100% nitrate removal was achieved in the 50-L bioreactor at a COD:NO₃–N ratio of 3.45 with a retention time of 48 h. The isolates showed 1.5 fold higher denitrifying activity than reported previously.     

Influence of bed media characteristics on ammonia and nitrate removal in shallow horizontal subsurface flow constructed wetlands

Two bed media were tested (gravel and Filtralite) in shallow horizontal subsurface flow (HSSF) constructed wetlands in order to evaluate the removal of ammonia and nitrate for different types of wastewater (acetate-based and domestic wastewater) and different COD/N ratios. The use of Filtralite allowed both higher mass removal rates (1.1 g NH₄–N m⁻² d⁻¹ and 3 g NO₃–N m⁻² d⁻¹) and removal efficiencies (>62% for ammonia, 90–100% for nitrate), in less than 2 weeks, when compared to the ones observed with gravel. The COD/N ratio seems to have no significant influence on nitrate removal and the removal of both ammonia and nitrate seems to have involved not only the conventional pathways of nitrification–denitrification. The nitrogen loading rate of both ammonia (0.8–2.4 g NH₄–N m⁻² d⁻¹) and nitrate (0.6–3.2 g NO₃–N m⁻² d⁻¹) seem to have influenced the respective removal rates.     

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.     

Nitrate Uptake by the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> Grown Under Non-Stress and Salt-Stress Conditions

We have compared the characteristics of nitrate uptake by Aphanothece halophytica grown under non-stress and salt-stress conditions. Both cell types showed essentially similar patterns of nitrate uptake toward ammonium, nitrite, and DL-glyceraldehyde. Although the affinities of nitrate to non-stress cells and salt-stress cells were not significantly different, i.e., K_s = 416 and 450 µM, respectively, the V_max value for non-stress cells was about twofold of that for salt-stress cells (9.1 vs 5.3 µmol min^–1 mg^–1 Chl). Nitrate uptake by A. halophytica was found to be dependent on Na⁺. Ammonium inhibited nitrate uptake, and the presence of methionine sulfoximine could not release the inhibition by ammonium. Nitrite appeared to competitively inhibit nitrate uptake with a K_i value of 84 µM. Both chloride and phosphate anions did not affect nitrate uptake. DL-Glyceraldehyde, an inhibitor of CO₂ fixation, caused a reduction in the uptake of nitrate.Received: 22 October 2002 / Accepted: 6 December 2002     

Transformations of inorganic nitrogen by Azospirillum spp.

Azospirillum spp. participate in all steps of the nitrogen cycle except nitrification. They can fix molecular nitrogen and perform assimilatory nitrate reduction and nitrate respiration. Culture conditions have been defined under which nitrate is used both as terminal respiratory electron acceptor and as nitrogen source for growth. Nitrate and, possibly to a very limited extent, nitrite, but not sulfate, iron or fumarate support anaerobic respiration. Under anaerobic conditions, nitrate can also supply energy for nitrogen fixation but without supporting growth. Nitrate-dependent nitrogenase activity lasts only for 3–4 h until the enzymes of assimilatory nitrate reduction are synthesized. Nitrite accumulates during this period and inhibits nitrogenase activity at concentrations of about 1 mM.     

Nitrogenase activity and nitrogen assimilation in Anabaena flos-aquae growing in continuous culture

Summary Anabaena flos-aquae is grown in chemostats under phosphate and urea-limited conditions. Nitrogenase activity in phosphate-limited cells has a maximum activity at a dilution rate of 0.025 h^-1 and is repressed 24-fold by 15 mM KNO₃. Cultures growing on 1.5 mM nitrate obtain 1/2–2/3 of cell nitrogen from N₂. Cells form inducible nitrite assimilating enzymes when grown on nitrate. Algae growing under A or He on limiting urea or phosphate-limited with nitrate have active nitrogenase. The ratio of nitrogenase activity to heterocyst numbers varied 90-fold depending on source of nitrogen, 15 mM KNO₃ gave the smallest ratio. The regulatory mechanisms controlling the activity of nitrogenase in blue-green algae is discussed.     

Nitrogen removal from wastewaters at low C/N ratios with ammonium and acetate as electron donors

A denitrifying upflow anaerobic sludge blanket (UASB) reactor was operated at different nitrate loading rates at a C/N ratio of 1.2, with acetate as an electron donor. This resulted in an increase in the accumulation of nitrite. After this, the UASB reactor was supplemented with 100 mg NH4+-Nl(-1) d(-1), while acetate was gradually limited in the medium. This prevented nitrite accumulation at a C/N ratio of 0.6 due to an enhanced nitrite reduction rate achieved in the reactor. An increasing amount of ammonium was consumed when the C/N ratio was lowered in the medium. This suggested that ammonium was used as an alternative electron donor during denitrification, which is supported by nitrogen balances. Nitrite was shown to be toxic for the nitrogen removal process at 200-400 mg NO2--N(l(-1) when the C/N ratio was decreased to 0.4 leading to formation of ammonium. The present study showed that addition of ammonium as an alternative electron donor for denitrification achieved a nitrogen removal process with negligible accumulation of undesirable intermediates.     

The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions

The assimilation of nitrate under dark-N₂ and dark-O₂ conditions in Zea mays leaf tissue was investigated using colourimetric and ¹⁵N techniques for the determination of organic and inorganic nitrogen. Studies using ¹⁵N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N₂ conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O₂ was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.