Denitrification Intermediates Research Articles

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired.

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a β-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Genotypic and phenotypic characterization of hydrogenotrophic denitrifiers.

Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO3 - , but it often entails accumulation of the cytotoxic intermediate NO2 - and the greenhouse gas N2 O. To explore if these high NO2 - and N2 O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole-genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO2 - , NO, N2 O, N2 and O2 as they depleted O2 and transitioned to denitrification with NO3 - as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO2 - accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO3 - in the narG-containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napA-containing D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N2 O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N2 O reductase expression.

Linking meta-omics to the kinetics of denitrification intermediates reveals pH-dependent causes of N2O emissions and nitrite accumulation in soil

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N2O reduction despite early transcription of nosZ (mainly clade I), encoding N2O reductase, by diverse denitrifiers. This shows that post-transcriptionally hampered maturation of the NosZ apo-protein at low pH is a generic phenomenon. Identification of transcript reads of several accessory genes in the nos cluster indicated that enzymes for NosZ maturation were present across a range of organisms, eliminating their absence as an explanation for the failure to produce a functional enzyme. nir transcript abundances (for NO2− reductase) in SoilA suggest that low NO2− concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to biological activity. The accumulation of NO2− in neutral soil was ascribed to high nar expression (nitrate reductase). The -omics results revealed dominance of nirK over nirS in both soils while qPCR showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK pool. qnor encoding NO reductase was strongly expressed in SoilA, implying an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2− accumulating soil, was estimated to be ten times higher from SoilA than from SoilN. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

Integrated omics analyses reveal differential gene expression and potential for cooperation between denitrifying polyphosphate and glycogen accumulating organisms

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2 O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The six transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA and IC, a competing glycogen accumulating organism (GAO) affiliated with Competibacteraceae (GAO1), a Gammaproteobacteria PR6 and an Anaerolineae CH7. Strongly elevated expression of nitrite reductase genes compared to nitrous oxide reductase genes was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2 O accumulation. Surprisingly, while ~90% of the nirS gene transcripts were expressed by the three co-occurring PAO populations, ~93% of the norB gene transcripts were expressed by GAO1 and ~75% of the norZ gene transcripts were mapped to PR6 and several other non-PAO flanking populations. This suggests the potential for cooperation between flanking populations and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide.

The effect of a biochar temperature series on denitrification: which biochar properties matter?

Biochar has been shown to reduce nitrous oxide (N2O) emissions from soils, but the effect is highly variable across studies and the mechanisms are under debate. To improve our mechanistic understanding of biochar effects on N2O emission, we monitored kinetics of NO, N2O and N2 accumulation in anoxic slurries of a peat and a mineral soil, spiked with nitrate and amended with feedstock dried at 105 °C and biochar produced at 372, 416, 562 and 796 °C at five different doses. Both soils accumulated consistently less N2O and NO in the presence of high-temperature chars (BC562 and BC796), which stimulated reduction of denitrification intermediates to N2, particularly in the acid peat. This effect appeared to be strongly linked to the degree of biochar carbonisation as predicted by the H:C ratio of the char. In addition, biochar surface area and pH were identified as important factors, whereas ash content and CEC played a minor role. At low pyrolysis temperature, the biochar effect was soil dependent, suppressing N2O accumulation in the mineral soil, but enhancing it in the peat soil. This contrast was likely due to the labile carbon content of low temperature chars, which contributed to immobilise N in the mineral soil, but stimulated denitrification and N2O emission in the peat soil. We conclude that biochar with a high degree of carbonisation, high pH and high surface area is best suited to supress N2O emission from denitrification, while low temperature chars risk supporting incomplete denitrification.

Substrate Diffusion within Biofilms Significantly Influencing the Electron Competition during Denitrification.

A common and long-existing operational issue of wastewater denitrification is the unexpected accumulation of nitrite (NO2-) that could suppress the activity of various microorganisms involved in biological wastewater treatment process and nitrous oxide (N2O) that could emit as a potent greenhouse gas. Recently, it has been confirmed that the accumulation of these denitrification intermediates in biological wastewater treatment process is greatly influenced by the electron competition between the four denitrification steps. However, little is known about this in biofilm systems. In this work, we applied a mathematical model that links carbon oxidation and nitrogen reduction processes through a pool of electron carriers, to assess electron competition in denitrifying biofilms. Simulations were performed comprehensively at seven combinations of electron acceptor addition scheme (i.e., simultaneous addition of one, two or three among nitrate (NO3-), NO2-, and N2O) to compare the effect of electron competition on NO3-, NO2- and N2O reduction. Overall, the effects of substrate loading, biofilm thickness and effective diffusion coefficients on electron competition are not always intuitive. Model simulations show that electron competition was intensified due to the substrate load limitation (from 120 to 20 mg COD/L) and increasing biofilm thicknesses (from 0.1 to 1.6 mm) in most cases, where electrons were prioritized to nitrite reductase because of the insufficient electron donor availability in the biofilm. In contrast, increasing effective diffusion coefficients did not pose a significant effect on electron competition and only increased electrons distributed to nitrite reductase when both NO2- and N2O are added.

Denitrification of metallurgic wastewater: mechanisms of inhibition by Fe, Cr and Ni

AbstractBACKGROUNDMetallurgic wastewaters are acidic effluents containing large amounts of nitrate and heavy metals. Citric acid is one of the acids used by this industry and forms stable complexes with metal ions. The aim of this study was to elucidate the chemical aspects driving inhibitory or stimulatory effects of heavy metals on denitrifying processes, based on speciation analysis and monitoring key denitrification intermediates (nitrite and N2O).RESULTSDenitrifying sludge incubations were conducted with iron, chromium and nickel, in single and multi‐metal assays, using citrate and ethanol as electron donors. Ferric‐citrate complex, [Fe‐cit](aq), was readily consumed, while complexes of divalent metals, [Fe‐cit]‐ and [Ni‐cit2]4‐, remained very stable affecting denitrification. Nitrate reduction was affected by Ni2+, while nitrite and N2O accumulation was observed with NiCO3 and Ni(OH)2 oversaturation. [Cr‐cit2]4‐ resulted in overall denitrification inhibition, while species of [Cr‐cit]2‐ caused denitrification stimulation.CONCLUSIONFe and Cr inhibited the overall denitrification process, while Ni caused accumulation of intermediates. Synergistic inhibition imposed by multi‐metal systems revealed lower inhibitory effects compared with those observed by the sum of individual effects of metals. This study elucidates chemical aspects determining the effects of heavy metals on denitrification, which is relevant to develop efficient biological processes for metallurgic effluents. © 2017 Society of Chemical Industry

Role of Glyoxylate Shunt in Oxidative Stress Response

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

Modeling simultaneous carbon and nitrogen removal (SCNR) in anaerobic/anoxic reactor treating domestic wastewater

This paper presents a mathematical model based on the Anaerobic Digestion Model No. 1 (ADM1) to simulate the effects of nitrate concentration and hydraulic retention time (HRT) on the simultaneous carbon and nitrogen removal (SCNR) in anaerobic/anoxic reactor treating domestic wastewater. The model was calibrated using previously published experimental data obtained from anaerobic batch tests for different COD/NO3− ratios. Model simulations were performed to predict the SCNR in a completely mixed reactor (CSTR) operating under mesophilic conditions (35 °C). Six different scenarios were evaluated to investigate the performance of the SCNR based on typical influent characteristics of domestic wastewater. The variables analyzed were chemical oxygen demand (COD) removal, nitrate concentration, methane production, nitrogen gas, volatile fatty acids (VFA) concentration, pH and percentage of COD used by the denitrifying and methanogenic microorganisms. The HRT was decreased stepwise from 15 to 4 h. The results indicate that Scenario (S5) with a COD/NO3− ratio equal to 10 and an HRT equal to 15 h ensures the occurrence of the stable SCNR. Furthermore, the accumulation of denitrification intermediates and a significant reduction in the biogas production when the organic matter is limited was verified.

Oxygen isotope fractionation during N<sub>2</sub>O production by soil denitrification

Abstract. The isotopic composition of soil-derived N2O can help differentiate between N2O production pathways and estimate the fraction of N2O reduced to N2. Until now, δ18O of N2O has been rarely used in the interpretation of N2O isotopic signatures because of the rather complex oxygen isotope fractionations during N2O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H2O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N2O production by soil denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the δ18O values of the produced N2O. In our soil incubation experiments Δ17O isotope tracing was applied for the first time to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found that N2O formation in static anoxic incubation experiments was typically associated with oxygen isotope exchange close to 100 % and a stable difference between the 18O ∕ 16O ratio of soil water and the N2O product of δ18O(N2O ∕ H2O)  =  (17.5 ± 1.2) ‰. However, flow-through experiments gave lower oxygen isotope exchange down to 56 % and a higher δ18O(N2O ∕ H2O) of up to 37 ‰. The extent of isotope exchange and δ18O(N2O ∕ H2O) showed a significant correlation (R2 = 0.70, p <  0.00001). We hypothesize that this observation was due to the contribution of N2O from another production process, most probably fungal denitrification. An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification, isotope exchange occurs prior to isotope branching and that this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0 ± 0.9) ‰, in contrast to fungal denitrification where higher values of up to 30 ‰ have been reported previously. This suggests that δ18O might be used as a tracer for differentiation between bacterial and fungal denitrification, due to their different magnitudes of branching isotope effects.

Evaluating two concepts for the modelling of intermediates accumulation during biological denitrification in wastewater treatment

The accumulation of the denitrification intermediates in wastewater treatment systems is highly undesirable, since both nitrite and nitric oxide (NO) are known to be toxic to bacteria, and nitrous oxide (N2O) is a potent greenhouse gas and an ozone depleting substance. To date, two distinct concepts for the modelling of denitrification have been proposed, which are represented by the Activated Sludge Model for Nitrogen (ASMN) and the Activated Sludge Model with Indirect Coupling of Electrons (ASM-ICE), respectively. The two models are fundamentally different in describing the electron allocation among different steps of denitrification. In this study, the two models were examined and compared in their ability to predict the accumulation of denitrification intermediates reported in four different experimental datasets in literature. The N-oxide accumulation predicted by the ASM-ICE model was in good agreement with values measured in all four cases, while the ASMN model was only able to reproduce one of the four cases. The better performance of the ASM-ICE model is due to that it adopts an “indirect coupling” modelling concept through electron carriers to link the carbon oxidation and the nitrogen reduction processes, which describes the electron competition well. The ASMN model, on the other hand, is inherently limited by its structural deficiency in assuming that carbon oxidation is always able to meet the electron demand by all denitrification steps, therefore discounting electron competition among these steps. ASM-ICE therefore offers a better tool for predicting and understanding intermediates accumulation in biological denitrification.

Inhibition of nitrate and the accumulated denitrification intermediate (nitrite) on perchlorate bioreduction

Bioreduction of perchlorate and nitrate by perchlorate-reducing microorganisms (PRMs) is an environmentally friendly, economic, and efficient technology to treat mixed plumes composed of these substances. The influence of perchlorate, nitrate, and denitrification intermediates on PRM activity is a critical factor, which may affect the efficiency of treatment technology. This study investigated the inhibition of nitrate and the intermediate (nitrite) accumulated during the denitrification process on perchlorate bioreduction via a batch-type experiment. From the experiment, it was found that perchlorate had no effect on the denitrification process and that the reduction rate of perchlorate could be improved when NO3−-N/ClO4− ≤1.2. However, a negative effect of nitrate on perchlorate reduction was observed when NO3−-N/ClO4− >1.2 with an accumulation of 18.0 mg NO2−-N /L. This negative effect increased with the concentration of nitrate. Moreover, nitrite from the denitrification process had a similar negative effect on perchlorate reduction. Bioreduction of perchlorate was not started until nitrite was totally reduced, and a 2–13 day lag period was observed for perchlorate reduction after nitrite depletion.

Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification--a pure culture study.

The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil has not yet been sufficiently investigated. The intramolecular (15)N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under denitrifying conditions, with either NO3(-) or NO2(-) as the electron acceptor, and measured the isotopic signature (δ(18)O, δ(15)Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO2(-) only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of (15)Nbulk were associated with larger N2O production. The δ(18)O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO2(-) and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ(18)O values of the N2O produced.

Feeding strategies for groundwater enhanced biodenitrification in an alluvial aquifer: Chemical, microbial and isotope assessment of a 1D flow-through experiment

Nitrate-removal through enhanced in situ biodenitrification (EISB) is an existing alternative for the recovery of groundwater quality, and is often suggested for use in exploitation wells pumping at small flow-rates. Innovative approaches focus on wider-scale applications, coupling EISB with water-management practices and new monitoring tools. However, before this approach can be used, some water-quality issues such as the accumulation of denitrification intermediates and/or of reduced compounds from other anaerobic processes must be addressed. With such a goal, a flow-through experiment using 100mg-nitrate/L groundwater was built to simulate an EISB for an alluvial aquifer. Heterotrophic denitrification was induced through the periodic addition of a C source (ethanol), with four different C addition strategies being evaluated to improve the quality of the denitrified water. Chemical, microbial and isotope analyses of the water were performed. Biodenitrification was successfully stimulated by the daily addition of ethanol, easily achieving drinking water standards for both nitrate and nitrite, and showing an expected linear trend for nitrogen and oxygen isotope fractionation, with a εN/εO value of 1.1. Nitrate reduction to ammonium was never detected. Water quality in terms of remaining C, microbial counts, and denitrification intermediates was found to vary with the experimental time, and some secondary microbial respiration processes, mainly manganese reduction, were suspected to occur. Carbon isotope composition from the remaining ethanol also changed, from an initial enrichment in 13C-ethanol compared to the value of the injected ethanol (−30.6‰), to a later depletion, achieving δ13C values well below the initial isotope composition (to a minimum of −46.7‰). This depletion in the heavy C isotope follows the trend of an inverse fractionation. Overall, our results indicated that most undesired effects on water quality may be controlled through the optimization of the C/N ratio determined from the amounts of injected ethanol vs. the amount of nitrate in groundwater, with a smaller C/N ratio causing a lower level of undesired impurities. Furthermore, the authors suggest that the biofilm life-time has a direct effect on microbial population and hence affects biodenitrification performance, influencing the accumulation of nitrite over time.

Nitrite accumulation and nitrous oxide emission during denitrification processes with quinoline or indole as the sole carbon source

AbstractBACKGROUNDBatch experiments were conducted to investigate nitrite accumulation and N2O emission during the denitrification processes when quinoline or indole was used as the sole carbon source. The effects of COD/NO3−‐N ratio and pH were studied.RESULTSWhile both significant transient nitrite accumulation and N2O emission occurred when quinoline was used as the carbon source, little denitrification intermediates were observed when indole was used as the carbon source. The optimal COD/NO3−‐N ratio for both complete denitrification and degradation of quinoline and indole were 6.8 and 5.1. Denitrification was inhibited under acidic conditions no matter with quinoline or indole as the carbon source. The optimal pH was 7.5 for both quinoline (or indole) degradation and denitrification. The nitrite accumulation and different carbon source further influenced N2O emission. When quinoline was used as the carbon source, N2O emission was observed at COD/NO3−‐N ≤ 5 and pH ≤ 7.5; when indole was used as the carbon source, N2O emission was observed at COD/NO3−‐N ≤ 3 and pH ≤ 6.5. Higher concentration of N2O was emitted at lower COD/NO3−‐N ratio and pH.CONCLUSIONCOD/NO3−‐N ratio, pH and the types of carbon source significantly influenced the accumulation of denitrification intermediates. © 2014 Society of Chemical Industry

Fungal oxygen exchange between denitrification intermediates and water

Fungi can contribute greatly to N2O production from denitrification. Therefore, it is important to quantify the isotopic signature of fungal N2O. The isotopic composition of N2O can be used to identify and analyze the processes of N2O production and N2O reduction. In contrast to bacteria, information about the oxygen exchange between denitrification intermediates and water during fungal denitrification is lacking, impeding the explanatory power of stable isotope methods. Six fungal species were anaerobically incubated with the electron acceptors nitrate or nitrite and (18)O-labeled water to determine the oxygen exchange between denitrification intermediates and water. After seven days of incubation, gas samples were analyzed for N2O isotopologues by isotope ratio mass spectrometry. All the fungal species produced N2O. N2O production was greater when nitrite was the sole electron acceptor (129 to 6558 nmol N2O g dw(-1)  h(-1)) than when nitrate was the electron acceptor (6 to 47 nmol N2O g dw(-1)  h(-1)). Oxygen exchange was complete with nitrate as electron acceptor in one of five fungi and with nitrite in two of six fungi. Oxygen exchange of the other fungi varied (41 to 89% with nitrite and 11 to 61% with nitrate). This is the first report on oxygen exchange with water during fungal denitrification. The exchange appears to be within the range previously reported for bacterial denitrification. This adds to the difficulty of differentiating N2O producing processes based on the origin of N2O-O. However, the large oxygen exchange repeatedly observed for bacteria and now also fungi could lead to less variability in the δ(18)O values of N2O from soils, which could facilitate the assessment of the extent of N2O reduction.

The impact of copper, nitrate and carbon status on the emission of nitrous oxide by two species of bacteria with biochemically distinct denitrification pathways

Denitrifying bacteria convert nitrate (NO(3) (-) ) to dinitrogen (N(2) ) gas through an anaerobic respiratory process in which the potent greenhouse gas nitrous oxide (N(2) O) is a free intermediate. These bacteria can be grouped into classes that synthesize a nitrite (NO(2) (-) ) reductase (Nir) that is solely dependent on haem-iron as a cofactor (e.g. Paracoccus denitrificans) or a Nir that is solely dependent on copper (Cu) as a cofactor (e.g. Achromobacter xylosoxidans). Regardless of which form of Nir these groups synthesize, they are both dependent on a Cu-containing nitrous oxide reductase (NosZ) for the conversion of N(2) O to N(2) . Agriculture makes a major contribution to N(2) O release and it is recognized that a number of agricultural lands are becoming Cu-limited but are N-rich because of fertilizer addition. Here we utilize continuous cultures to explore the denitrification phenotypes of P. denitrificans and A. xylosoxidans at a range of extracellular NO(3) (-) , organic carbon and Cu concentrations. Quite distinct phenotypes are observed between the two species. Notably, P. denitrificans emits approximately 40% of NO(3) (-) consumed as N(2) O under NO(3) (-) -rich Cu-deficient conditions, while under the same conditions A. xylosoxidans releases approximately 40% of the NO(3) (-) consumed as NO(2) (-) . However, the denitrification phenotypes are very similar under NO(3) (-) -limited conditions where denitrification intermediates do not accumulate significantly. The results have potential implications for understanding denitrification flux in a range of agricultural environments.

Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO− 3 most rapidly (Vmax = 15.8 μmol e−·min−1·mg protein−1) and had the fastest generation time (gt) on NO− 3 (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO− 2 tolerant and grew rapidly on NO− 2 (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO− 2 rapidly (Vmax = 10.6 μmol e−·min−1·mg protein−1) but was far less NO− 2 tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO− 3 more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO− 2 accumulation in high-NO− 3 groundwater during bioremediation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.

Autotrophic Denitrification in Microbial Fuel Cells Treating Low Ionic Strength Waters

The presence of elevated concentrations of nitrates in drinking water has become a serious concern worldwide. The use of autotrophic denitrification in microbial fuel cells (MFCs) for waters with low ionic strengths (i.e., 1000 μS·cm(-1)) has not been considered previously. This study evaluated the feasibility of MFC technology for water denitification and also identified and quantified potential energy losses that result from their usage. The low conductivity (<1600 μS·cm(-1)) of water limited the nitrogen removal efficiency and power production of MFCs and led to the incomplete reduction of nitrate and the nitrous oxide (N(2)O) production (between 4 and 20% of nitrogen removed). Cathodic overpotential was identified as the main energy loss factors (83-90% of total losses). That high overpotential was influenced by denitrification intermediates (NO(2)(-) and N(2)O) and the potential used by microorganisms for growth, activation, and maintenance.

Denitrifying sulfide removal with methanogenic culture

Simultaneous sulfide, nitrate and carbon removal in the effect of nitrate and/or sulfide addition using methanogenic cultures was investigated using batch cultures. The cultures supplemented with sulfide efficiently denitrified 50–500mg N/L nitrate to nitrogen gas. The mechanism proposed was denitrification occurred via autotrophic pathway first, followed by heterotrophic pathway. Nitrate reduction pathway slightly shifted to dissimilatory nitrate reduction to ammonia (DNRA) in cultures with no addition of sulfide, starting at initial nitrate concentration 250mg N/L. Microbial community probing suggests that autotrophic denitrification were carried out by epsilon proteobacterium (Thiomicrospira denitrificans). The heterotrophic pathway was carried out by Thauera sp. in both cultures with and without the addition of sulfide.With the presence of nitrate, sulfide was oxidized to elemental sulfur in 32h after the assay began. Due to the activity of sulfate reducing strains, it was later recovered back to nearly initial concentration via heterotrophic oxidation pathway. Sole addition of nitrate at concentration 50–500mg N/L resulted in delay of methanogenesis. At concentration 750–1000mg N/L, a complete suppression occurred. The addition of nitrate along with sulfide at concentration 100mg S/L decrease methane production, while at concentration 200mg S/L, severe inhibition occurred. However, when sulfide was dose alone, the inhibition was not as severe as when it was added with nitrate. Inhibition was due to denitrification intermediates, mainly by accumulation of nitrite and nitrous oxide. Denitrification occurred first while at the same time inhibited methanogenesis.