The denitrification capacity of sediment from a hypereutrophic lake

SUMMARY.

• 1 In sediment from Wintergreen Lake, Michigan, denitrification was not detectable by the acetylene inhibition method at in situ nitrate concentrations. When nitrate was added to sediment slurries, denitrification capacities up to 18.8μg N g^-1 h^-1 were measured. The denitrification capacities decreased with increasing sediment depth and distance from shore.
• 2 The high denitrification capacities in these sediments which under natural conditions had no supply of nitrate and oxygen suggested that denitrifies with alternative mechanisms for anaerobic energy conversion were present. Nitrous oxide was a significant portion of the N-gas produced immediately after the nitrate addition. Small amounts (4–5% of the total N-gas production) of nitric oxide accumulated in the early phase of nitrate reduction. Presumably after depletion of nitrate and nitrite both N₂O and NO were further reduced to N₂.
• 3 About 70%r of the added nitrate was denitrified, and the remainder was assumed to have been reduced to ammonium.

     

Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans

Summary A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5–8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO ₄ ⁼ /l, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2 g NO ₂ ^– /l. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: _max=0.11 h^–1, K _S=0.2 mg NO ₃ ^– /l, maximum denitrification rate=0.78 g NO ₃ ^– /g cells·h, g cells/g NO ₃ ^– , g cells/g S₂O ₃ ⁼ . Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N₂. The concentrations of N₂O and NO were below 1 ppm.     

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

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

Linkages between organic matter mineralization and denitrification in eight riparian wetlands

Denitrification (N₂ production) and oxygen consumption rates were measured at ambient field nitrate concentrations during summer in sediments from eight wetlands (mixed hardwood swamps, cedar swamps, heath dominated shrub wetland, herbaceous peatland, and a wetland lacking live vegetation) and two streams. The study sites included wetlands in undisturbed watersheds and in watersheds with considerable agricultural and/or sewage treatment effluent input. Denitrification rates measured in intact cores of water-saturated sediment ranged from 20 to 260 mol N m^-2 h^-1 among the three undisturbed wetlands and were less variable (180 to 260 mol N M^-2 h^-1) among the four disturbed wetlands. Denitrification rates increased when nitrate concentrations in the overlying water were increased experimentally (1 up to 770 M), indicating that nitrate was an important factor controlling denitrification rates. However, rates of nitrate uptake from the overlying water were not a good predictor of denitrification rates because nitrification in the sediments also supplied nitrate for denitrification. Regardless of the dominant vegetation, pH, or degree of disturbance, denitrification rates were best correlated with sediment oxygen consumption rates (r ² = 0.912) indicating a relationship between denitrification and organic matter mineralization and/or sediment nitrification rates. Rates of denitrification in the wetland sediments were similar to those in adjacent stream sediments. Rates of denitrification in these wetlands were within the range of rates previously reported for water-saturated wetland sediments and flooded soils using whole core¹⁵N techniques that quantify coupled nitrification/denitrification, and were higher than rates reported from aerobic (non-saturated) wetland sediments using acetylene block methods.     

Benthic organic carbon influences denitrification in streams with high nitrate concentration

1. Anthropogenic activities have increased reactive nitrogen availability, and now many streams carry large nitrate loads to coastal ecosystems. Denitrification is potentially an important nitrogen sink, but few studies have investigated the influence of benthic organic carbon on denitrification in nitrate‐rich streams. 2. Using the acetylene‐block assay, we measured denitrification rates associated with benthic substrata having different proportions of organic matter in agricultural streams in two states in the mid‐west of the U.S.A., Illinois and Michigan. 3. In Illinois, benthic organic matter varied little between seasons (5.9–7.0% of stream sediment), but nitrate concentrations were high in summer (>10 mg N L⁻¹) and low (<0.5 mg N L⁻¹) in autumn. Across all seasons and streams, the rate of denitrification ranged from 0.01 to 4.77 μg N g⁻¹ DM h⁻¹ and was positively related to stream‐water nitrate concentration. Within each stream, denitrification was positively related to benthic organic matter only when nitrate concentration exceeded published half‐saturation constants. 4. In Michigan, streams had high nitrate concentrations and diverse benthic substrata which varied from 0.7 to 72.7% organic matter. Denitrification rate ranged from 0.12 to 11.06 μg N g⁻¹ DM h⁻¹ and was positively related to the proportion of organic matter in each substratum. 5. Taken together, these results indicate that benthic organic carbon may play an important role in stream nitrogen cycling by stimulating denitrification when nitrate concentrations are high.     

The Fate of 15N-Nitrate in Healthy and Declining Phragmites australis Stands

Abstract The dissimilatory nitrate-reducing processes, denitrification, and dissimilatory nitrate-reduction to ammonium were studied in freshwater lake sediments within healthy and degrading Phragmites australis (reed) stands. The samples from the healthy vegetation site contained roots and rhizomes. Cores were supplied with 1.9–5.2 μg ¹⁵N-NO₃ ⁻ g⁻¹ dry sediment in the laboratory and subsequently incubated for 8 h at 20°C, in the dark. The ¹⁵N compounds were determined before (natural percentage of ¹⁵N) and after 1 and 8 h of incubation. The uptake of ¹⁵N by the roots and rhizomes in the healthy vegetation was 61%. Nitrogen losses, interpreted as denitrification, accounted for 25 and 84% of the added ¹⁵N-NO₃ ⁻ in sediment from the healthy and degrading vegetation sites, respectively. The percentages of nitrate reduced to ammonium were 4 and 9% in sediment from the healthy vegetation and degrading vegetation sites, respectively. The percentage of ¹⁵N–total N in the sediment of the healthy vegetation site was 10%, whereas for the degrading vegetation site this percentage was 7%. The percentage of nitrate reduced to ammonium could be potentially underestimated by the percentage of ¹⁵N measured in the sediment. In this case, in healthy and degenerating P. australis stands, the percentage of produced ammonium accounted for 14–16%. The nitrate reduction rates were calculated based on an incubation period of one hour. The denitrification rate in sediment from the degrading vegetation site was higher than from the healthy vegetation site. The rate of dissimilatory nitrate reduction to ammonium was almost tenfold higher in sediment from the degrading vegetation site compared to sediment from the healthy vegetation site. The significantly lower percentages of dissimilatory nitrate reduction to ammonium and denitrification in the healthy stand compared to the degrading stand was probably due to the presence of roots and rhizomes. In the sediments of healthy and degrading P. australis stands, denitrification was the main nitrate-reducing process. Received: 24 July 1996; Accepted: 5 December 1996     

Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

Autotrophic denitrification by Thiobacillus denitrificans in a packed bed reactor

Summary An upflow packed bed reactor with lava stones as support for the microbial growth proved to be very useful for the denitrification of industrial waste water by Thiobacillus denitrificans. The application of the plug flow principle allowed higher concentrations of nitrate to be employed than in a stirred tank reactor because inhibitory concentrations of sulfate from thiosulfate oxidation built up only in the upper part of the column — if at all. In experiments with synthetic media nitrate solutions of different strength (NO ₃ ^– g/l: 1.8; 3.0; 4.3; 6.1) were tested, each at 5 different residence times (5; 3.3; 2.5; 2.0; 1.7 h). The combination of the two parameters which still allowed 95% denitrification was 3 g NO ₃ ^- /l and 2.5 h residence time; this corresponded to a volumetric nitrate loading of about 25 kg/m³·d. Higher nitrate loadings led to incomplete denitrification coupled with the occurence of nitrite in the outflow. Below the critical loading rate nitrite accumulated only in the lower part of the column and was then gradually reduced. Experiments with simulated middle active waste from processing nuclear fuel which contained numerous heavy metals yielded similar results. — Although pure inorganic media were fed into the reactor the microflora developing as a dense layer covering the lava stones consisted not only of T. denitrificans but also of heterotrophic denitrifiers, mainly Pseudomonas aeruginosa.     

The Synergistic Effects of High Nitrate Concentrations on Sediment Bioreduction

Groundwaters at nuclear sites can be characterized by low pH and high nitrate concentrations (10–100 mM). These conditions are challenging for bioremediation, often inhibiting microbial Fe(III)-reduction which can limit radionuclide migration. Here, sediment microcosms representative of the UK Sellafield site were used to study the influence of variable pH and nitrate concentrations on microbially-mediated TEAP (terminal electron accepting processes) progression. The rate of reduction through the terminal electron accepting cascade NO^? ₃ > NO^? ₂ > Mn(IV)/Fe(III) > SO^2? ₄ at low pH (～5.5) was slower than that in bicarbonate buffered systems (pH ～ 7.0), but in the low pH systems, denitrification and associated pH buffering resulted in conditioning of the sediments for subsequent Fe(III) and sulfate-reduction. Under very high nitrate conditions (100 mM), bicarbonate buffering (pH ～ 7.0) was necessary for TEAP progression beyond denitrification and the reduction of 100 mM nitrate created alkaline conditions (pH 9.5). 16S rRNA gene analysis showed that close relatives of known nitrate reducers Bacillus niacini and Ochrobactrum grignonense dominated the microbial communities in this reduced sediment. In Fe(III)-reducing enrichment cultures from the 100 mM nitrate system, close relatives of the Fe(III)-reducing species Alkaliphilus crotonatoxidans and Serratia liquifaciens were observed. These results highlight that under certain conditions and contrary to expectations, denitrification may support bioreduction via pH conditioning for optimal metal reduction and radionuclide immobilization.     

硝态氮异化还原机制及其主导因素研究进展

硝态氮(NO_3~-)异化还原过程通常包含反硝化和异化还原为铵(DNRA)两个方面,是土壤氮素转化的重要途径,其强度大小直接影响着硝态氮的利用和环境效应(如淋溶和氮氧化物气体排放)。反硝化和DNRA过程在反应条件、产物和影响因素等方面常会呈现出协同与竞争的交互作用机制。综述了反硝化和DNRA过程的研究进展及其二者协同竞争的作用机理,并阐述了在NO_3~-、pH、有效C、氧化还原电位(Eh)等环境条件和土壤微生物对其发生强度和产物的影响,提出了今后应在产生机理、土壤环境因素、微生物学过程以及与其他氮素转化过程耦联作用等方面亟需深入研究,以期增进对氮素循环过程的认识以及为加强氮素管理利用提供依据。     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Denitrification of nitrate and nitric acid with methanol as carbon source

Summary A methanol/nitrate-medium and anaerobic conditions yielded an enrichment culture which consisted ofHyphomicrobium andParacoccus. This mixed culture proved to be very effective in denitrification of solutions containing high concentrations of nitrate and free nitric acid when grown in a chemostat (D=0.04 h^-1). With 0.1 mol/l nitric acid solution as feed medium the pH in the culture vessel adjusted itself to 5.8. For the reduction of 1 g NO₃–N 2.6 g methanol were consumed and 0.56 g cells were produced.     

Effect of hydraulic residence time on microbial sulfide production in an upflow sludge blanket denitrification reactor fed with methanol

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water anion exchange resins are regenerated in a closed circuit by way of an upflow sludge blanket denitrification reactor. The regenerant (a concentrated sodium bicarbonate solution) is recirculated through the ion exchanger in the r generation mode and the denitrification reactor. In the closed system sulfate accumulates to very high concentrations. For that reason it was examined under what process conditions sulfate reduction occurs in an upflow sludge blanket denitrification reactor, when the influent contains high sulfate concentrations (5.45 g SO ₄ ^2- /l) and high sodium bicarbonate concentrations (19.8 g NaHCO₃/l) in addition to nitrate and methanol. It appeared that at a hydraulic residence time of 5 h sulfide production started, when the nitrate loading rate was 20% of the denitrification reactor capacity and methanol was added in excess. The excess of methanol was converted into acetate after nitrate was depleted. Conversion of methanol into acetate was a function of the hydraulic residence time. At hydraulic residence times above 8 h this conversion was complete. Also in batch experiments it was observed that excess of methanol was converted into acetate, and that sulfate reduction started when nitrate was depleted. From all experiments it is clear that, provided that methanol is added in good relation to the quantity of nitrate that has to be denitrified, acetate will not be produced and sulfate reduction will not occur in the denitrification reactor, even in the presence of very high sulfate concentrations.     

Response of denitrification rate associated with wetting and drying cycles in a littoral wetland area of Lake Biwa,Japan

To clarify the relationship between denitrification activity and dry–wet levels in the littoral wetland sediments of Lake Biwa, Japan, denitrification rates and their regulating parameters (degree of dryness, redox potential, nitrate concentration) were measured on different moisture sediments. Redox potential in sediments was higher in the exposed region in contact with atmosphere than the flooded region covered with water. The nitrate concentration in interstitial waters was undetectable in the flooded region. On the other hand, concentration in the exposed region increased with increase in the degree of sediment dryness. The denitrification rate ranged from <0.001 to 0.88 μg N cm⁻³ h⁻¹ in the exposed region and increased with the increase in the degree of dryness. In the flooded region, on the other hand, no detectable rate (<0.001 μg N cm⁻³ h⁻¹) was observed. This indicates that the rates in the exposed region were mainly influenced by nitrate concentration in the interstitial waters accumulated by desiccation of sediments, whereas rates in the flooded region were strongly limited by no accumulation of nitrate in the anaerobic conditions. The potential denitrification rate, under the application condition of nitrate, ranged from 0.13 to 0.26 μg N cm⁻³ h⁻¹ in the flooded region and from 0.77 to 1.5 μg N cm⁻³ h⁻¹ in the exposed region. The potential rates in the flooded region had a tendency to be lower than those in the exposed region, implying that the number of denitrifying bacteria in the flooded region was low due to inactivation of aerobic respiration and denitrification in the denitrifying bacteria community. Kinetic parameters, maximum rate (V _max) and half-saturation constant (K _s) for denitrification were calculated on the experimental procedures of the wetting–drying cycles of sediments. Both parameters decreased by the wetting treatment and increased by the drying treatment. The fluctuation of V _max values with wetting–drying cycles indicated that the number of denitrifying bacteria was influenced by aerobic respiration and denitrification in the denitrifying bacteria community similar to the potential rates, and denitrifying enzyme was induced by the nitrate supplied by nitrification accelerated through the drying process. On the other hand, the fluctuation of K _s values implied that members of denitrifying bacteria were shifted to members of high nitrate affinity by wetting treatment and of low nitrate affinity by drying treatment.     

Characterization of microbial community and kinetics for spent sulfidic caustic applied autotrophic denitrification

Spent sulfidic caustic was applied to sulfur utilizing autotrophic denitrification as the simultaneous source of electron donor and alkalinity. The two experiment set-up of upflow anoxic hybrid growth reactor (UAHGR) and upflow anoxic suspended growth reactor (UASGR) was adopted and nitrate removals were similar in both reactors. Approximately 90% of the initial nitrate was denitrified at nitrate loading rate of 0.15∼0.40 kgNO₃ ⁻/m³·d. The experimental stoichiometric ratio of sulfate production to nitrate removal was ranged from 1.5 to 2.1 mgSO₄ ²⁻/mgNO₃ ⁻. During the operation period, denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments for the sludge sample of both reactors showed the change of microbial communities. Thiobacillus denitrificans-like microorganism occupied 28.5% (18 clones) of the 63 clones by cloning the PCR products from the sludge sample of UAHGR. Acidovorax avenae, which can reduce nitrate to nitrogen gas while oxidizing phenol (heterotrophic denitrifier), was also found in 7 clones (11.1%). Although an organic carbon source was not added to the medium, a microorganism (Kaistella koreensis) capable of oxidizing organic compounds was found in 7 clones (11.1%). Therefore, the microbial community of spent sulfidic caustic applied autotrophic denitrification process well corresponds to the substrate components of spent sulfidic caustic. Through the batch cultivation of microorganisms in UAHGR, the microbial kinetic coefficients of spent sulfidic caustic applied autotrophic denitrification were estimated to be μ _max = 0.097 h⁻¹, k _d = 0.0021 h⁻¹, K _s = 200 mgNO₃ ⁻/L, and Y = 0.31 mgMLVSS/mgNO₃ ⁻.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.

     

Simultaneous nitrification and denitrification in a mixed methanotrophic culture

Simultaneous nitrification and denitrification using a mixed methanotrophic culture was investigated. When both NO₃ ^–-N (108 mg l^–1) and NH₃-N (59 mg l^–1) were added into batch reactors, nitrate removal was complete within 10 h at the rate of 47 mg NO₃ ^–-N g VSS^–1 day^–1 when dissolved oxygen (DO) concentration was maintained at 2 mg DO l^–1. Ammonia removal started simultaneously with nitrate removal at a slower rate of 14 NH₃-N g VSS^–1 day^–1. No significant accumulation of nitrite or nitrate during ammonia utilization suggested the occurrence of simultaneous nitrification and denitrification.