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PubMed2000

Inhibition of denitrification by ultraviolet radiation.

It has been shown that UV-A (lambda=320-400 nm) and UV-B (lambda=280-320 nm) inhibit photosynthesis, nitrogen fixation and nitrification. The purpose of this study was to determine the effects, if any, on denitrification in a microbial community inhabiting the intertidal. The community studied is the microbial mat consisting primarily of Lyngbya that inhabits the Pacific marine intertidal, Baja California, Mexico. Rates of denitrification were determined using the acetylene blockage technique. Pseudomonas fluorescens (ATCC #17400) was used as a control organism, and treated similarly to the mat samples. Samples were incubated either beneath a PAR transparent, UV opaque screen (OP3), or a mylar screen to block UV-B, or a UV transparent screen (UVT) for 2 to 3 hours. Sets of samples were also treated with nitrapyrin to inhibit nitrification, or DCMU to inhibit photosynthesis and treated similarly. Denitrification rates were greater in the UV protected samples than in the UV exposed samples the mat samples as well as for the Ps fluorescens cultures. Killed controls exhibited no activity. In the DCMU and nitrapyrin treated samples denitrification rates were the same as in the untreated samples. These data indicate that denitrification is directly inhibited by UV radiation.

Ammonia↗

PubMed1997

Potential nitrosamine formation and its prevention during biological denitrification of red beet juice.

High nitrate intake has been shown to result in an increased risk of endogenous formation of N-nitroso compounds. Certain vegetables and vegetable juices contain high concentrations of nitrate. Biological denitrification using strains of Paracoccus denitrificans (P.d.) has been proposed as effective means to reduce nitrate contents in such vegetable juices. During this bacterial denitrification process, substantial nitrite concentrations are transiently formed. This study investigated whether N-nitrosation reactions might occur. The easily nitrosatable amine morpholine was added to red beet juice at high concentration (100 ppm) during denitrification 10 different batches of red beet juice served as raw material. Each batch was submitted to denitrification in the presence and absence of ascorbic acid. In the absence of ascorbic acid, formation of N-nitrosomorpholine (NMOR) was observed in the low ppb range (0.5-8 ppb). Addition of ascorbic acid (500 mg/litre) inhibited the formation of NMOR, except for those instances where the pH was less than 6 and/or nitrate turnover was low (< 200 mg NO3-/litre/hr). Under conditions leading to high rates of nitrate turnover (> 200 mg NO3-/litre/hr), nitrosamine formation can reliably be prevented by ascorbic acid. The results show that bacterial denitrification of red beet juice high in nitrate can be accomplished without the risk of nitrosamine formation.

Ascorbic Acid↗

PubMed2002

Enhanced nitrate removal efficiency in wetland microcosms using an episediment layer for denitrification.

Microbial denitrification is the principal removal mechanism for nitrogen in treatment wetlands. For this study, flow-through wetland microcosms were designed to testwhether variations in the macroporous structure of the denitrification zone affected overall nitrate removal. In a sediment-only treatment, carbon as cattail (Typha latifolia) litter was mixed throughout a porous sediment matrix. A second treatment contained a distinct layer of loosely aggregated litter pieces placed atop the sediment matrix to form an episediment zone. Results showed that across nine conditions (NO3- influent = 7, 36, 65 mg N L(-1) and t(res) = 2.2, 2.9, 4.4, 7.7 d; n = 48) average denitrification was 33% greater (p < 0.0001) in the episediment treatment than in the sediment-only treatment. Both sediment-only and episediment treatments followed apparent reaction kinetics close to first order, with k(epi) = 0.21 d(-1) for the episediment treatment and k(sed) = 0.12 d(-1) for the sediment-only treatment. Analysis of vertical nitrate profile data using diffusive and turbulent mixing models indicated that denitrification occurred in a partially mixed episediment layer, as well as in an underlying sediment layer. Approximately 40% of the nitrate removal that occurred in the episediment treatment was estimated to occur in the episediments themselves. We conclude that enhancementof an episediment layer can increase denitrification in treatment wetlands which receive nitrate in overlying water.

Biodegradation, Environmental↗

PubMed1997

Enzyme diversity and mosaic gene organization in denitrification.

Denitrification is a main branch of the global nitrogen cycle. In the past ten years unravelling the underlying biochemistry and genetics has proceeded at an increasing pace. Fungal denitrification has become a new field. The biochemical investigation of denitrification has culminated in the description of the crystal structures of the two types of nitrite reductases. The N2O reductase shares with cytochrome c oxidase the CuA center as a structurally novel metal site. The cytochrome b subunit of NO reductase has a striking conservation of heme-binding transmembrane segments versus the subunit I of cytochrome c oxidase. Another putative denitrification gene product shows structural relation to the subunit III of the oxidase. N2O reductase and NO reductase may be ancestors of energy-conserving enzymes of the heme-copper oxidase superfamily. More than 30 genes for denitrification are located in a > 30-kb cluster in Pseudomonas stutzeri, and comparable gene clusters have been identified in Pseudomonas aeruginosa and Paracoccus denitrificans. Genes necessary for nitrite reduction and NO reduction have a mosaic arrangement with very few conserved locations within these clusters and relative to each other.

Amino Acid Sequence↗

PubMed1997

Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH.

The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO2). Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated.

Anaerobiosis↗

PubMed2005

Membrane-bound denitrification in the Gram-positive bacterium Bacillus azotoformans.

Gram+ bacteria are capable of complete denitrification just like Gram- (Gram-negative) bacteria. However, Gram+ (Gram-positive) bacteria have a very small periplasmic-like space. This leads to the question of whether those enzymes and electron carriers involved in denitrification, which are normally located in the periplasmic space in Gram- bacteria, are located in the periplasmic-like space in Gram+ bacteria or have been modified as membrane-bound proteins. Using Bacillus azotoformans as a Gram+ bacterial model, our study demonstrates that anaerobic denitrification is catalysed by four membrane-bound enzymes and that the electron carriers are membrane-bound c-type cytochromes and menaquinol. NADH dehydrogenase is coupled with the denitrification pathway providing menaquinol. In addition, the cytochrome b(6)f complex forms part of the denitrification pathway, oxidizing menaquinol and reducing at least three different membrane-bound c-type cytochromes. We determined that the NO reductase, qCu(A)NOR (where NOR stands for nitric oxide reductase), can accept electrons from two donors, a specific cytochrome c(551) and menaquinol. Similarly, nitrite reductase, a copper enzyme, and nitrous oxide reductase may be bifunctional enzymes. Regarding the bifunctionality of qCu(A)NOR, we propose that the menaquinol-linked pathway is involved in the detoxification of NO.

Bacillus↗

PubMed2001

Autotrophic denitrification via a novel membrane-attached biofilm reactor.

AIMS: A laboratory-scale autotrophic membrane-attached biofilm reactor was developed to remove nitrate from drinking water. METHODS AND RESULTS: Hydrogen and carbon dioxide flowed together into the lumem side of a gas-permeable silicone tube. The gases diffused through the membrane wall to feed Alcaligenes eutrophus that formed a biofilm on the surface of the silicone tube for autotrophic denitrification. Hydrogen provided the energy source, and carbon dioxide, besides serving as the carbon source, was employed to neutralize the alkalinity from denitrification. The optimal carbon dioxide concentration in the silicone tube was between 20% and 50%. CONCLUSION: This study has demonstrated that a gas-permeable silicone tube is a convenient and efficient method to feed A. eutrophus for autotrophic denitrification. Supplying a suitable amount of carbon dioxide together with hydrogen into the silicone tube solved the problem that alkalinity formation caused during denitrification. The pH of the bioreactor was maintained at about 7 to avoid nitrite accumulation, and then the nitrogen removal rate was increased. A high specific nitrogen removal rate (1.6-5.4 g Nm(2)d(-1-1) of surface area of silicone tube) was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to combining the advantages of the hydrogenotrophic denitrification process and a membrane feeding substrate bioreactor (MFSB), this bioreactor achieved a high nitrogen removal rate and is simple to operate. It therefore is highly promising in drinking-water treatment.

Biofilms↗

PubMed2005

Feasibility study of nitrogen removal with the mecellulose wasted liquor as an external carbon source in the two-stage denitrification process.

The utilization of mecellulose wasted liquor (MWL) as an external carbon source was investigated to find an alternative for methanol in the two-stage denitrification pilot process. The pilot plant was supplied with the raw water from the J-Municipal Sewage Treatment Plant (J-MSTP) in Korea. The raw water of J-Municipal Sewage Treatment Plant contains low and high concentration of biodegradable organics and nitrogen source, respectively, due to the inflow of industrial wastewater and landfill leachate. Methanol was fed to provide external carbon source for high concentration of nitrogen source removal by denitrification in this J-Municipal Sewage Treatment Plant, and thus this study was performed to test effects to the effluent quality and efficiencies of nitrogen source removal with an alternative carbon source for the cost reduction. The 6.5mg 1(-1) and 5.7mg l(-1) of total nitrogen (TN) concentration in the effluent were achieved with mecellulose and methanol, whereas mecellulose and methanol were fed to give the same ratio of gCODgNO,-N(-1), respectively. The 60% of COD in mecellulose wasted liquor was used as a carbon source for denitrification and the stable denitrification rate was earned when one half of the required total carbon source for denitrification was fed to pre-anoxic tank in the pilot plant. The required gCODgNO,-N(-1) ratio with mecellulose wasted liquor was 1.4 times higher than with methanol. Mecellulose wasted liquor is feasible to be used as external carbon source for organic loading, nitrogen and phosphorus removal. If mecellulose wasted liquor is considered as an alternative external carbon source to substitute methanol 26-28m3 mecellulose wasted liquor per 1 m3 methanol will be required. However, to meet with the effluent standard (10 mg BOD l(-1)) for J-Municipal Sewage Treatment Plant, the feed concentration of mecellulose wasted liquor should be recommended to be lower than 200 mgl(-1).

Carbon↗

PubMed2002

Treatment of antibiotics wastewater utilizing successive hydrolysis, denitrification and nitrification.

A process consisting of anaerobic hydrolysis, denitrification, and oxidation/nitrification was proposed for simultaneous removal of carbon and nitrogen from terramycin crystallizing mother solution (TCMS), and its performance was investigated by treating diluted TCMS in a lab-scale continuous flow column system. Direct denitrification-nitrification of diluted TCMS produced a significant residual of nitrate and nitrite, which disappeared 44 days after startup of anaerobic hydrolysis column. The electron donors available to denitrification were increased by 6 times after diluted TCMS was treated in the hydrolysis column under an HRT of 2.5 h or longer. The reaction rates of organics decomposition, nitrification, and denitrification were also significantly increased due to reduction of terramycin and decomposition of complicated molecules to small molecules during anaerobic hydrolysis. The specific denitrification rate and nitrification rate increased from 0.033 d(-1) and 0.01 d(-1) to 0.045 d(-1) and 0.021 d(-1) respectively after diluted TCMS (dilution ratio: 1:4) was hydrolyzed in anaerobic hydrolysis column at a hydraulic retention time (HRT) of 4 h.

Anti-Bacterial Agents↗

PubMed2003

Using oxidation-reduction potential (ORP) and pH value for process control of shortcut nitrification-denitrification.

A new low cost technology for simultaneous carbon-nitrogen removal from soybean wastewater has been developed in this study. The technology is performed through shortcut nitrification-denitrification. The process operated under realtime control of aeration and mixing time. The shortcut nitrification-denitrification in sequencing batch reactor (SBR) was achieved efficiently and steadily by controlling temperature (28 +/- 0.5 degrees C) and using real-time control strategies. This enabled the prevention of nitrite oxidation, leading to lower operational costs. The feasibility of oxidation-reduction potential (ORP) and pH value as control parameter for shortcut nitrification-denitrification process was also investigated. Results showed that the average removal efficiency of ammonium was more than 95%, and nitrosation rate (NO2(-)-N/NOx(-)-N) was reached to 96%. At the same time, the variation of oxidation--reduction potential (ORP) and pH value was well related to organic matter degradation and ammonium oxidation in SBR. So that judgment on the ending of nitrification and denitrification can be based on the inflection point on the varied curve of ORP and pH throughout each SBR processing cycle, and thus reducing aeration and mixing time for saving energy source. The method saves organic energy up to 40% of chemical oxygen demand (COD) in denitrification process, which should reduce the need for an extra external source of organic carbon. Shorter hydraulic retention time should allow the volume of the reactors to diminish, and thus diminish investment costs. Lower oxygen demand of about 25% gives lower exploitation costs.

Agriculture↗

PubMed2005

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process.

AIMS: The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification. METHODS AND RESULTS: cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification. CONCLUSIONS: It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.

Bacteroides↗

PubMed2005

The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels.

The human pathogen Neisseria meningitidis is the major causative agent of bacterial meningitis. The organism is usually treated as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. We demonstrate here that although N. meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric oxide) and that this pathway supplements growth. The expression of the gene aniA, which encodes nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional regulator FNR and two-component sensor-regulator NarQ/NarP respectively. Completion of the two-step denitrification pathway requires nitric oxide (NO) reduction, which proceeds after NO has accumulated during batch growth under oxygen-limited conditions. During periods of NO accumulation both nitrite and NO reduction are observed aerobically, indicating N. meningitidis can act as an aerobic denitrifier. However, under steady-state conditions in which NO is maintained at a low concentration, oxygen respiration is favoured over denitrification. NO inhibits oxidase activity in N. meningitidis with an apparent Ki NO = 380 nM measured in intact cells. The high respiratory flux to nitrite after microaerobic growth and the finding that accumulation of the denitrification intermediate NO inhibits oxygen respiration support the view that denitrification is a pathway of major importance in N. meningitidis.

Adult↗

PubMed2001

Role of the stratospheric polar freezing belt in denitrification.

Homogeneous freezing of nitric acid hydrate particles can produce a polar freezing belt in either hemisphere that can cause denitrification. Computed denitrification profiles for one Antarctic and two Arctic cold winters are presented. The vertical range over which denitrification occurs is normally quite deep in the Antarctic but limited in the Arctic. A 4 kelvin decrease in the temperature of the Arctic stratosphere due to anthropogenic and/or natural effects can trigger the occurrence of widespread severe denitrification. Ozone loss is amplified in a denitrified stratosphere, so the effects of falling temperatures in promoting denitrification must be considered in assessment studies of ozone recovery trends.

Journal Article↗

PubMed2007

Growth yields in bacterial denitrification and nitrate ammonification.

Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.

Acetates↗

PubMed1981

Kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification.

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K(m) values for nitrate and nitrite reduction were 15 muM or less for each isolate. The K(m) value for nitrous oxide reduction by Flavobacterium sp. was 0.5 muM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from NO(3) by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.

Journal Article↗

PubMed1984

Denitrification in san francisco bay intertidal sediments.

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO(3) + NO(2) concentrations (</=26 muM) were below the apparent K(m) (50 muM) for nitrate. During the rainy season, when ambient NO(3) + NO(2) concentrations were higher (37 to 89 muM), an accurate estimate of the K(m) could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N(2)O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N(2)O in the presence of C(2)H(2) was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N(2)O loss was caused by incomplete blockage of N(2)O reductase by C(2)H(2) at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 mumol of N(2) m h (for undisturbed sediments) to 17 to 280 mumol of N(2) m h (for shaken sediment slurries).

Journal Article↗

PubMed1992

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

Carbon↗

PubMed1993

Nitrification and denitrification in lake and estuarine sediments measured by the N dilution technique and isotope pairing.

The transformation of nitrogen compounds in lake and estuarine sediments incubated in the dark was analyzed in a continuous-flowthrough system. The inflowing water contained NO(3), and by determination of the isotopic composition of the N(2), NO(3), and NH(4) pools in the outflowing water, it was possible to quantify the following reactions: total NO(3) uptake, denitrification based on NO(3) from the overlying water, nitrification, coupled nitrification-denitrification, and N mineralization. In sediment cores from both lake and estuarine environments, benthic microphytes assimilated NO(3) and NH(4) for a period of 25 to 60 h after darkening. Under steady-state conditions in the dark, denitrification of NO(3) originating from the overlying water accounted for 91 to 171 mumol m h in the lake sediments and for 131 to 182 mumol m h in the estuarine sediments, corresponding to approximately 100% of the total NO(3) uptake for both sediments. It seems that high NO(3) uptake by benthic microphytes in the initial dark period may have been misinterpreted in earlier investigations as dissimilatory reduction to ammonium. The rates of coupled nitrification-denitrification within the sediments contributed to 10% of the total denitrification at steady state in the dark, and total nitrification was only twice as high as the coupled process.

Journal Article↗