Electron Donor For Nitrate Reduction Research Articles

Mixotrophic denitrification driven by plant carbon sources and influent sulphate for enhancing nitrogen removal in constructed wetlands

A lab-scale horizontal baffled subsurface-flow constructed wetland (HBSFCW) packed with plant litter (reed straws (RS)) was built as a sustained-release carbon source, after which it was fed with synthetic wastewater that simulated the low C/N ratios in tailwater from wastewater treatment plants (WWTPs). The purpose of its operation was to explore the relationship between the nitrogen removal performance and variations in microbial community composition, and propose an enhanced nitrogen removal strategy for constructed wetlands (CWs). The results showed that plant carbon resources and influent sulphate can induce changes in redox conditions and organic matter concentrations at CWs, thereby influencing the microbial community composition and denitrification performance. High-throughput sequencing revealed the formation of richer microbial communities in additional RS units. Conventional heterotrophic denitrifying genera (Dechloromonas, Thauera and Ignavibacterium), sulphate-reducing genera (Desulfobacter, Desulfobulbus) and a sulphur-based denitrification genus (Sulfurimonas) were directly or indirectly affected by the addition of RS and became dominant in additional RS units. This study suggests that plant litter in HBSFCWs and sulphate from WWTP tailwater provide an opportunity to utilize sulphate as an electron-acceptor and reduced sulphur as an electron-donor for nitrate reduction, thus driving mixotrophic denitrification.

Microbial nitrate reduction in propane- or butane-based membrane biofilm reactors under oxygen-limiting conditions

Nitrate contamination has been commonly detected in water environments and poses serious hazards to human health. Previously methane was proposed as a promising electron donor to remove nitrate from contaminated water. Compared with pure methane, natural gas, which not only contains methane but also other short chain gaseous alkanes (SCGAs), is less expensive and more widely available, representing a more attractive electron source for removing oxidized contaminants. However, it remains unknown if these SCGAs can be utilized as electron donors for nitrate reduction. Here, two lab-scale membrane biofilm reactors (MBfRs) separately supplied with propane and butane were operated under oxygen-limiting conditions to test its feasibility of microbial nitrate reduction. Long-term performance suggested nitrate could be continuously removed at a rate of ∼40–50 mg N/L/d using propane/butane as electron donors. In the absence of propane/butane, nitrate removal rates significantly decreased both in the long-term operation (∼2–10 and ∼4–9 mg N/L/d for propane- and butane-based MBfRs, respectively) and batch tests, indicating nitrate bio-reduction was driven by propane/butane. The consumption rates of nitrate and propane/butane dramatically decreased under anaerobic conditions, but recovered after resupplying limited oxygen, suggesting oxygen was an essential triggering factor for propane/butane-based nitrate reduction. High-throughput sequencing targeting 16S rRNA, bmoX and narG genes indicated Mycobacterium/Rhodococcus/Thauera were the potential microorganisms oxidizing propane/butane, while various denitrifiers (e.g. Dechloromonas, Denitratisoma, Zoogloea, Acidovorax, Variovorax, Pseudogulbenkiania and Rhodanobacter) might perform nitrate reduction in the biofilms. Our findings provide evidence to link SCGA oxidation with nitrate reduction under oxygen-limiting conditions and may ultimately facilitate the design of cost-effective techniques for ex-situ groundwater remediation using natural gas.

Electrochemical enrichment of marine denitrifying bacteria to enhance nitrate metabolization in seawater

High concentrations of nitrate from industrial discharges to coastal marine environments are a matter of concern owing to their ecological consequences. In the last years, Bioelectrochemical Denitrification Systems (BEDS) have emerged as a promising nitrate removal technology. However, they still have limitations, such as the enrichment strategy for specific microbial communities in the electrodes under natural conditions. In this study, three-electrode electrochemical cells were used to test microbial enrichment from natural seawater by applying three reported potentials associated with the dissimilatory denitrification process (−130, −260, and −570 mV vs. Ag/AgCl). The microbial community analysis showed that by applying −260 mV (vs. Ag/AgCl) to the working electrode, it was possible to significantly enrich denitrifying microorganisms, specifically Marinobacter, in comparison with the control. Furthermore, −260 mV (vs. Ag/AgCl) led to a significantly higher nitrate removal than other conditions, which, combined with cyclic voltammetry analysis, suggested that the polarized electrodes worked as external electron donors for nitrate reduction. Hence, this work demonstrates for the first time that it is possible to enrich marine denitrifying microorganisms by applying an overpotential of −260 mV (vs. Ag/AgCl) without the need for a culture medium, the addition of an exogenous electron donor (i.e., organic matter) or a previously enriched inoculum.

Benzene degradation in contaminated aquifers: Enhancing natural attenuation by injecting nitrate

Natural attenuation processes depend on the availability of suitable electron acceptors. At the megasite Zeitz, concentrations of the main contaminant benzene were observed to increase constantly in the lower aquifer to levels of more than 2.5 mM. This was accompanied by decreasing concentrations of sulphate (SO42−), which has been previously shown to be the main electron acceptor for benzene oxidation at this site, resulting in an electron acceptor-limited, sulphidic benzene plume. Therefore, a field experiment was conducted to stimulate benzene biodegradation by injecting nitrate (NO3−) into the sulphidic benzene plume aiming (i) to recycle sulphate by nitrate-dependent sulphide oxidation, and (ii) to serve as direct electron acceptor for benzene oxidation. Within 60 days, 6.74 tons sodium nitrate (NaNO3) were injected into the lower aquifer, and the resulting biogeochemical effects within the benzene plume were monitored for more than one year by chemical and microbiological analyses of groundwater samples taken from various depths of ten monitoring wells located in three observation lines downstream of nitrate injection. Nitrate was microbiologically consumed, as shown by changes in δ15N-NO3− and δ18O-NO3− values, partial nitrite accumulation, and changing ratios of Na+/NO3−. Main electron donors for nitrate reduction were reduced sulphur compounds, verified by changing δ34S-SO42− and δ18O-SO42− values, partially increasing sulphate concentrations, and strongly increasing abundances of typical sulphur-oxidizing, nitrate-reducing bacterial taxa within the nitrate plume. The general absent hydrogen isotope fractionation of benzene, also in the sulphidic, nitrate-free part of the plume, indicates that benzene was not biodegraded by sulphate-reducing consortia. However, detected small carbon isotope fractionation of benzene points to in situ benzene biodegradation processes in the plume, probably supported by nitrate. In conclusion, nitrate injection resulted in changing redox conditions and recycling of sulphate in the sulphidic, sulphate-depleted benzene plume due to microbial oxidation of reduced sulphur species, leading to presumably favored conditions for in situ benzene biodegradation.

Untangling the nitrate removal pathways for a constructed wetland- sponge iron coupled system and the impacts of sponge iron on a wetland ecosystem

Sponge iron (s-Fe0) is a potential alternative electron donor for nitrate reduction. To gain insight into the mechanism of denitrification in a constructed wetland- sponge iron coupled system (CW-Fe0 system), the removal performance and reduction characteristics of nitrate in constructed wetlands (CWs) with and without s-Fe0 application were compared. Results indicated that s-Fe0 intensified the removal of nitrate with a 6h-HRT. The nitrate removal efficiency was improved by 16–76 % with various influent NO3−-N concentrations (10–30 mg L-1) and at a chemical oxygen demand(COD)/N ratio of 5. The rates of chemical denitrification were positively correlated with the dosage of s-Fe0 and negatively correlated with the influent COD concentration. 16S rDNA sequencing revealed that hydrogen-utilizing autotrophic denitrifier of Hydrogenophaga was highly enriched (accounting for 10 % of the total OTUs) only in CW-Fe0 system. The micro-environment created by s-Fe0 was suitable for heterotrophic denitrifiers of Thauera, Tessaracoccus and Simplicispira. The determination of physiological indicators for plants showed that the application of s-Fe0 causes abiotic stress to wetland plants (Canna indica L.). Nevertheless, s-Fe0 can be used as a substrate for CWs, since it allows a high-efficiency removal of nitrate by mediating chemical denitrification and hydrogen-driven autotrophic denitrification.

Water-soluble bitumen degradation products can fuel nitrate reduction from non-radioactive bituminized waste

An important fraction of the currently stored volume of long-lived intermediate level radioactive waste in Belgium is immobilized as Eurobitum. This type of waste typically contains large amounts of NaNO3 homogeneously dispersed in a hard bituminous matrix. Geological disposal of Eurobitum in a water-saturated sedimentary formation such as Boom Clay will result in the leaching of high concentrations of NaNO3 in the Boom Clay formation. This could cause a geochemical perturbation of the surrounding clay, possibly affecting some of the favorable characteristics of the host formation such as its hydraulic conductivity, sorption potential or the redox conditions. The latter might result in a decrease of its reducing capacity and an increase in the mobility of redox-sensitive radionuclides. Microbial nitrate reduction is a well-known process, which could be stimulated by the chemical and radiolytical water-soluble organic bitumen degradation products. The present study carried out different series of long-term anoxic batch experiments to investigate the ability of the microbial community of Boom Clay borehole water to reduce nitrate, leaching from thermally aged non-radioactive Eurobitum in the presence or absence of acetate, formate and oxalate, being part of the most important organic bitumen degradation products. Obtained results indicate that all three organic bitumen degradation products can be used as electron donor to fuel microbial nitrate reduction, albeit with a different efficiency. The highest nitrate reduction rate was observed in the presence of acetate, while oxalate was the least efficient electron donor for nitrate reduction. Moreover, organic compounds that leached from the Eurobitum during the course of the experiment were used as electron donor for microbial nitrate reduction in all conditions. Furthermore, calcium oxalate crystals were observed, indicating that if oxalate is present, it will probably be less bioavailable compared to other organic compounds.

Uniform and Pitting Corrosion of Carbon Steel by Shewanella oneidensis MR-1 under Nitrate-Reducing Conditions.

Despite observations of steel corrosion in nitrate-reducing environments, processes of nitrate-dependent microbially influenced corrosion (MIC) remain poorly understood and difficult to identify. We evaluated carbon steel corrosion by Shewanella oneidensis MR-1 under nitrate-reducing conditions using a split-chamber/zero-resistance ammetry (ZRA) technique. This approach entails the deployment of two metal (carbon steel 1018 in this case) electrodes into separate chambers of an electrochemical split-chamber unit, where the microbiology or chemistry of the chambers can be manipulated. This approach mimics the conditions of heterogeneous metal coverage that can lead to uniform and pitting corrosion. The current between working electrode 1 (WE1) and WE2 can be used to determine rates, mechanisms, and, we now show, extents of corrosion. When S. oneidensis was incubated in the WE1 chamber with lactate under nitrate-reducing conditions, nitrite transiently accumulated, and electron transfer from WE2 to WE1 occurred as long as nitrite was present. Nitrite in the WE1 chamber (without S. oneidensis) induced electron transfer in the same direction, indicating that nitrite cathodically protected WE1 and accelerated the corrosion of WE2. When S. oneidensis was incubated in the WE1 chamber without an electron donor, nitrate reduction proceeded, and electron transfer from WE2 to WE1 also occurred, indicating that the microorganism could use the carbon steel electrode as an electron donor for nitrate reduction. Our results indicate that under nitrate-reducing conditions, uniform and pitting carbon steel corrosion can occur due to nitrite accumulation and the use of steel-Fe(0) as an electron donor, but conditions of sustained nitrite accumulation can lead to more-aggressive corrosive conditions.IMPORTANCE Microbially influenced corrosion (MIC) causes damage to metals and metal alloys that is estimated to cost over $100 million/year in the United States for prevention, mitigation, and repair. While MIC occurs in a variety of settings and by a variety of organisms, the mechanisms by which microorganisms cause this damage remain unclear. Steel pipe and equipment may be exposed to nitrate, especially in oil and gas production, where this compound is used for corrosion and "souring" control. In this paper, we show uniform and pitting MIC under nitrate-reducing conditions and that a major mechanism by which it occurs is via the heterogeneous cathodic protection of metal surfaces by nitrite as well as by the microbial oxidation of steel-Fe(0).

Sulfate removal and sulfur transformation in constructed wetlands: The roles of filling material and plant biomass

Sulfate in effluent is a challenging issue for wastewater reuse around the world. In this study, sulfur (S) removal and transformation in five batch constructed wetlands (CWs) treating secondary effluent were investigated. The results showed that the presence of the plant cattail (Typha latifolia) had little effect on sulfate removal, while the carbon-rich litter it generated greatly improved sulfate removal, but with limited sulfide accumulation in the pore-water. After sulfate removal, most of the S was deposited with the valence states S (-II) and S (0) on the iron-rich gravel surface, and acid volatile sulfide was the main S sink in the litter-added CWs. High-throughput pyrosequencing revealed that sulfate-reducing bacteria (i.e. Desulfobacter) and sulfide-oxidizing bacteria (i.e. Thiobacillus) were dominant in the litter-added CWs, which led to a sustainable S cycle between sulfate and sulfide. Overall, this study suggests that recycling plant litter and iron-rich filling material in CWs gives an opportunity to utilize the S in the wastewater as both an electron acceptor for sulfate reduction and as an electron donor for nitrate reduction coupled with sulfide oxidation. This leads to the simultaneous removal of sulfate, nitrate, and organics without discharging toxic sulfide into the receiving water body.

Wood-Derived Black Carbon (Biochar) as a Microbial Electron Donor and Acceptor

Research on the environmental impacts of black carbon has focused largely on sorption. Besides being a strong geosorbent, black carbon is redox-active and may facilitate abiotic and microbial transformation. Using a wood-derived black carbon (biochar) and the bacterium Geobacter metallireducens (GS-15), we showed that air-oxidized biochar served as an electron acceptor to enable acetate oxidation, and that chemically or biotically reduced biochar served as an electron donor for nitrate reduction. The bioavailable (to GS-15) electron storage capacities (ESCs) of the biochar, estimated on the basis of acetate oxidation and nitrate reduction, were 0.85 and 0.87 mmol e–/g, respectively, comparable to the ESCs of humic substances and other biochars measured electrochemically. We propose that black carbon should be regarded as a rechargeable reservoir of bioavailable electrons in anaerobic environments. The redox cycling of biochar in natural and engineered systems and its impact on microbial processes and cont...

Nitrate Reduction in an Intensified Electrochemical System: Effects of Different Metallic Particles

The performance of nitrate reduction in an electrochemical system intensified by different metallic particles was evaluated in the present study. In the electrochemical process without metallic particles, the nitrate removal efficiency was found to be 65.7%, while higher nitrate conversion yields of 82.9%, 87.5% and 93.2% were obtained in the developed system in the presence of iron, steel and copper particles, respectively. Moreover, the presence of metallic particles in the electrochemical process significantly reduced 39.3-49.5% of the energy consumptions for nitrate reduction. Mechanisms of the acceleration of nitrate reduction by the three metallic particles were different. Iron and steel particles in the middle of the electric field dissolved and served as electron donors for nitrate reduction, while the copper particles worked as activated metallic catalysts. Taking into account of safety and operating cost, iron was the optimal choice among the three metallic particles studied.

Toluene Depletion in Produced Oil Contributes to Souring Control in a Field Subjected to Nitrate Injection

Souring in the Medicine Hat Glauconitic C field, which has a low bottom-hole temperature (30 °C), results from the presence of 0.8 mM sulfate in the injection water. Inclusion of 2 mM nitrate to decrease souring results in zones of nitrate-reduction, sulfate-reduction, and methanogenesis along the injection water flow path. Microbial community analysis by pyrosequencing indicated dominant community members in each of these zones. Nitrate breakthrough was observed in 2-PW, a major water- and sulfide-producing well, after 4 years of injection. Sulfide concentrations at four other production wells (PWs) also reached zero, causing the average sulfide concentration in 14 PWs to decrease significantly. Interestingly, oil produced by 2-PW was depleted of toluene, the preferred electron donor for nitrate reduction. 2-PW and other PWs with zero sulfide produced 95% water and 5% oil. At 2 mM nitrate and 5 mM toluene, respectively, this represents an excess of electron acceptor over electron donor. Hence, continuous nitrate injection can change the composition of produced oil and nitrate breakthrough is expected first in PWs with a low oil to water ratio, because oil from these wells is treated on average with more nitrate than is oil from PWs with a high oil to water ratio.

Enhanced Nitrogen Removal of Low C/N Wastewater in a Subsequent Denitrifying Reactor Fed by Endogenous Carbon Source Generated from Primary Sludge Fermentation

A novel post-denitrification system fed by carbon source from primary sludge (PS) was used for enhancing biological nitrogen removal (BNR) of low C/N wastewater. This system included one anoxic/oxic (AO) reactor and a special reactor for simultaneous sludge fermentation and denitrification (Sifeden). Ammonia was nitrified to nitrate in AO and then the nitrate was reduced to dinitrogen in Sifeden , into which PS was added intermittently. Results showed that this system had high performance on nitrogen removal. Total nitrogen (TN) removal efficiency was higher than 85% and the effluent TN≤10mg/L in the condition of influent C/N≤2. In Sifeden, volatile fatty acid (VFA) produced from PS fermentation provided electron donor for nitrate reduction, and PS was preliminarily stabilized simultaneously. Oxidation-Reduction Potential (ORP) had a significant correlation with the denitrification performance. TN removal efficiency could be further improved if adopting proper PS addition strategy according to the ORP profiles.

Competitive, Microbially-Mediated Reduction of Nitrate with Sulfide and Aromatic Oil Components in a Low-Temperature, Western Canadian Oil Reservoir

Fields from which oil is produced by injection of sulfate-bearing water often exhibit an increase in sulfide concentration with time (souring). Nitrate added to the injection water lowers the sulfide concentration by the action of sulfide-oxidizing, nitrate-reducing bacteria (SO-NRB). However, the injected nitrate can also be reduced with oil organics by heterotrophic NRB (hNRB). Aqueous volatile fatty acids (VFAs; a mixture of acetate, propionate, and butyrate) are considered important electron donors in this regard. Injection and produced waters from a western Canadian oil field with a low in situ reservoir temperature (30 degrees C) had only 0.1-0.2 mM VFAs. Amendment of these waters with nitrate gave therefore only partial reduction. More nitrate was reduced when 2% (v/v) oil was added, with light oil giving more reduction than heavy oil. GC-MS analysis of in vitro degraded oils and electron balance considerations indicated that toluene served as the primary electron donor for nitrate reduction. The differences in the extent of nitrate reduction were thus related to the toluene content of the light and heavy oil (30 and 5 mM, respectively). Reduction of nitrate with sulfide by SO-NRB always preceded that with oil organics by hNRB, even though microbially catalyzed kinetics with either electron donor were similar. Inhibition of hNRB by sulfide is responsible for this phenomenon. Injected nitrate will thus initially be reduced with sulfide through the action of SO-NRB. However, once sulfide has been eliminated from the near-injection wellbore region, oil organics will be targeted by the action of hNRB. Hence, despite the kinetic advantage of SO-NRB, the nitrate dose required to eliminate sulfide from a reservoir depends on the concentration of hNRB-degradable oil organics, with toluene being the most important in the field under study. Because the toluene concentration is lower in heavy oilthan in light oil, nitrate injection into a heavy-oil-producing field of low temperature is more likely to succeed in containing souring.

Effect of ethanol on nitrate and nitrite reduction and methanogenesis in the ruminal microbiota

ABSTRACTThe effect of ethanol on nitrate and nitrite reduction was examined by conducting in vitro experiments with mixed ruminal microbes. The addition of ethanol to cultures of mixed ruminal microbes stimulated nitrate reduction, and, to a greater extent, nitrite reduction, which resulted in a decrease in nitrite accumulation. However, known nitrate‐reducing ruminal bacteria, such as Selenomonas ruminantium, Veillonella parvula and Wolinella succinogenes, were unable to utilize ethanol directly as an electron donor for nitrate reduction. No nitrate‐reducing bacterium capable of utilizing ethanol was found in the rumen of goats. However, when mixed ethanol‐utilizing, hydrogen gas (H2)‐producing bacteria (Ruminococcus albus and Ruminococcus flavefaciens) were added to the culture of the mixed nitrate‐reducing bacteria described above, nitrate and nitrite reduction was observed. These results suggest that the nitrate‐reducing bacteria utilized the H2 that was produced from ethanol oxidation by the ethanol‐utilizing bacteria as an electron donor. It is conceivable that the stimulation of nitrate and nitrite reduction by ethanol, observed in the culture of mixed ruminal microbes, was a result of electron transfer from ethanol to nitrate, and nitrite through H2, that is, ‘interspecies hydrogen transfer’ from ethanol‐metabolizing bacteria to nitrate‐reducing bacteria. Thus, the addition of ethanol to high‐nitrate diets may be effective for preventing nitrate poisoning. Furthermore, methane production was reduced to less than one‐third by the addition of mixed nitrate‐reducing bacteria to the co‐culture of mixed methanogens with mixed ethanol‐utilizing bacteria incubated in a medium containing ethanol and nitrate. Therefore, the addition of ethanol and nitrate may decrease methanogenesis without suppressing overall fermentation in the rumen.

Use of Multiple Isotope Tracers to Evaluate Denitrification in Ground Water: Study of Nitrate from a Large‐Flux Septic System Plume

AbstractThis study explores the use of multiple isotopic tracers to evaluate the processes involved in nitrate attenuation in ground water. δ15N and δ18O are used to provide information about the role of denitrification on nitrate attenuation, and δ34S, δ18O, and δ13C are used to evaluate the role of reduced sulfur and carbon as electron donors for nitrate reduction. The focus of this study is a zone of significant NO3−1 attenuation occurring in a sand aquifer impacted by septic system contamination. The NO3−1 pattern, the ground water flow system, and changes in other chemical parameters suggest that the NO3−1 depletion is caused by denitrification. This is supported by the nitrate δ15N and δ18O data which both show significant isotopic enrichment as NO3−1 depletion proceeds along the flow path. The increase of sulfate and dissolved inorganic carbon observed in the zone of nitrate attenuation suggests that reduced sulfur in addition to carbon is also involved in denitrification. This is supported by a trend toward depleted sulfate δ34S and δ18O values in the zone of sulfate increase, which reflects the input of sulfate formed by the oxidation of biogenic pyrite present in the aquifer sediments. The trend toward depleted δ13 values in the zone of increasing dissolved inorganic carbon reflects the input of organic carbon into this carbon pool. Chemical mass balance indicates that carbon is the dominant electron donor; however, this study demonstrates the effectiveness of using multiple isotopic tracers for providing insight into the processes affecting nitrate attenuation in ground water.

Structural Studies on Corn Nitrate Reductase: Refined Structure of the CytochromebReductase Fragment at 2.5 Å, its ADP Complex and an Active-site Mutant and Modeling of the CytochromebDomain

The refined crystal structures of the recombinant cytochromebreductase fragment of corn (Zea mays) nitrate reductase, its ADP complex and the active-site mutant Cys242Ser are reported here. The native structure has been refined at 2.5 Å resolution to a crystallographicR-factor of 18.7% with root-mean-square (r.m.s.) deviations from standard bond lengths and angles of 0.013 Å and 2.0°. The diffraction pattern of the crystals is highly anisotropic and correction of this effect lowered the crystallographicR-factor by 5% during the refinement. The structure of the enzyme co-crystallized with ADP has been solved at 2.7 Å resolution and refined to anR-factor of 18.6% with r.m.s. deviations from standard bond lengths and angles of 0.014 Å and 2.1°. It revealed the binding site of the ADP moiety of the NADH cofactor, which is the electron donor for nitrate reduction. Based on this structure, a model of NADH at the active site of the enzyme was built and the implications for electron transfer from NADH to the flavin cofactor are discussed. The crystal structure of an active-site mutant enzyme, Cys242Ser, has been solved by difference Fourier synthesis and refined to anR-factor of 19.0% to 3.0 Å resolution with standard deviations of bond lengths and angles of 0.017 Å and 2.5°. This structure analysis suggests that the observed decrease in catalytic activity of this mutant might be due to misalignment of the nicotinamide ring in its binding site. A model of the heme-containing domain of nitrate reductase has been built based on the X-ray structure of bovine cytochromeb5and has been docked with the cytochromebreductase fragment of nitrate reductase. The model of the complex contains six salt-bridges at the domain-domain interface and a hydrophobic core. In this model, His48, an invariant residue in the cytochromebreductase family, forms an interaction with the propionic acid group of the D-ring of the heme cofactor. This group is in contact with the C-8 methyl group of the flavin ring. Residues that might influence the redox potential of the flavin cofactor are proposed and their possible role in electron transfer is discussed.

Possible interaction between peroxidase and NAD(P)H-dependent nitrate reductase activities of plasma membranes of corn roots

The relationship between the plasma membrane-bound NAD(P)H-nitrate reductase (NR) and a plasma membrane (PM)-bound peroxidase was investigated using highly purified PM vesicles isolated from corn roots. The PM-bound NR activity was strongly enhanced by MnCl 2 and SHAM, which stimulated peroxidase activity. Since both activities, the NAD(P)H-dependent NR and the peroxidase compete for NAD(P)H as electron donor, we propose a model in which a product of peroxidation is able to offer electrons to the nitrate reductase in a more reactive form with respect to NAD(P)H. Out hypothesis was confirmed by experiments in which the effects of inhibitors of peroxidative reactions, catalase, superoxide dismutase, and ascorbate on the PM-bound NR were studied. Results indicate that the putative electron donor for nitrate reduction could be a radicalic species, possibly NAD . . Furthermore, since cytochrome c decreased the activity of the plasma membrane-bound NAD(P)H-dependent NR, cytochrome b 557 might be the site of the enzyme accepting electrons from NAD . . Out results indicate that the PM environment of the NR may be involved in the extent of the membrane-associated nitrate reduction and that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-like NADH-oxidase, can interact.

Membrane-bound nitrite oxidoreductase of Nitrobacter: evidence for a nitrate reductase system

Nitrite oxidoreductase, the essential enzyme complex of nitrite oxidizing membranes, was isolated from cells of the nitrifying bacterium Nitrobacter hamburgensis. The enzyme system was solubilized and purified in the presence of 0.25% sodium deoxycholate. Nitrite oxidoreductase oxidized nitrite to nitrate in the presence of ferricyanide. The pH optimum was 8.0, and the apparent Km value for nitrite amounted to 3.6 mM. With reduced methyl-and benzylviologen nitrite oxidoreductase exhibited nitrate reductase activity with an apparent Km value of 0.9 mM for nitrate. NADH was also a suitable electron donor for nitrate reduction. The pH optimum was 7.0.

Production of nitrous oxide from nitrite in Klebsiella pneumoniae: mutants altered in nitrogen metabolism.

Under anaerobic conditions, Klebsiella pneumoniae reduced nitrite (NO2-), yielding nitrous oxide (N2O) and ammonium ions (NH4+) as products. Nitrous oxide formation accounted for about 5% of the total NO2- reduced, and NH4+ production accounted for the remainder. Glucose and pyruvate were the electron donors for NO2- reduction to N2O by whole cells, whereas glucose, NADH, and NADPH were found to be the electron donors when cell extracts were used. On the one hand, formate failed to serve as an electron donor for NO2- reduction to N2O and NH4+, whereas on the other hand, formate was the best electron donor for nitrate reduction in either whole cells or cell extracts. Mutants that are defective in the reduction of NO2- to NH4+ were isolated, and these strains were found to produce N2O at rates comparable to that of the parent strain. These results suggest that the nitrite reductase producing N2O is distinct from that producing NH4+. Nitrous oxide production from nitric oxide (NO) occurred in all mutants tested, at rates comparable to that of the parent strain. This result suggests that NO reduction to N2O, which also uses NADH as the electron donor, is independent of the protein(s) catalyzing the reduction of NO2- to N2O.

Nitrate reduction in bacteroides multiacidus: Its effect on glucose metabolism and some properties of the reductase.

Bacteroides multiacidus fermented glucose to lactate and succinate as major products and produced small amounts of acetate and pyruvate. During nitrate reduction cell growth increased slightly and the formation of succinate and lactate decreased while acetate increased.The level of nitrate reductase activity in the extracts was several times higher in cells grown on nitrate, while the fumarate reductase level was lowered compared with cells not grown on nitrate. In extracts, nitrate reductase and fumarate reductase occurred mainly in a particulate fraction, whereas nitrite reductase was mostly in a soluble fraction. Addition of tungstate to the culture medium decreased the nitrate reductase activity as well as nitrite formation, suggesting that the enzyme is a molybdoprotein. Ferredoxin from Clostridia and viologen dyes served as electron donors for nitrate reduction. A c-type cytochrome with peaks at 557.5, 527, and 423nm in the extract was partly reduced by NADH and oxidized by fumarate, and to a lesser extent also by nitrate, even in extracts from cells grown in the presence of nitrate. Km′s for substrates and pH optima were also reported for the reductases in crude extracts.