Inhibition of the metabolism of nitrifying bacteria by direct electric current

The nitrifying bacteria in activated sludge and biofilms consisting of the bacteria immobilized on polypropylene packing were subjected to an electric current via two electrodes. In activated sludge, the metabolism of nitrifying bacteria was inhibited when the applied current was over 2.5 A m^–2, whilst in biofilms, inhibition began when the current reached 5 A m^–2. At 15 A m^–2, the nitrification rate of NH₄ ⁺-N in a biofilm with a bacterial density of 1.62 g total solids, dry wt m^–2 decreased to about 80% of its initial value. Ninety-two % of the initial biomass on the packing was retained after 36 h.     

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.     

Screening and characterization of an aerobic nitrifying-denitrifying bacterium from activated sludge

Taking advantage of the good biocompatibility and high efficiency of nitrogen removal with microbes, nitrifying and denitrifying bacteria, are becoming increasingly more widely used for wastewater treatment and prevention of eutrophication. In this research, an aerobic nitrifying-denitrifying bacterium was successfully screened from activated sludge and identified as Pseudomonas sp. (CCTCC No M2010209) by the 16S rDNA sequence. The activity verification confirmed its nitrifying-denitrifying capability of removing ammonium, nitrate and nitrite nitrogen. The types of carbon sources and carbon-nitrogen ratio greatly influenced the removal efficiency of NH₄ ⁺-N and NO₃ ⁻-N. When the initial concentrations of NH₄ ⁺-N and NO₃ ⁻-N in synthetic wastewater were less than 70 and 50 mg/L, the nitrogen removal rates reached 94 and 90% in 9 h, respectively. Preliminary comparisons of nitrogen removal capacity between this isolate and other commercial preparations in the treatment of synthetic wastewater revealed its promising potential to be used in the actual wastewater treatment.     

Removal characteristics of high load ammonia gas by a biofilter seeded with a marine bacterium, Vibrio alginolyticus

When the cells of the newly isolated marine bacterium, Vibrio alginolyticus, were inoculated on to an inorganic packing material in biofilter, and a load of ammonia of 2.4–22.5 g-N kg^–1dry packing material was introduced continuously under non-sterile conditions, the average amount of NH₃removed exceeded 85% over 61-d operation. The maximum removal capacity and the complete removal capacity were 22.8 g-N kg^–1dry packing material dand 18.6 g-N kg^–1dry packing material d, respectively, which were about four times larger than those obtained in autotrophic nitrifying sludge inoculated on the same packing material.     

Ammonium Removal by the Oxygen-Limited Autotrophic Nitrification-Denitrification System

The present lab-scale research reveals the potential of implementation of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with normal nitrifying sludge as the biocatalyst for the removal of nitrogen from nitrogen-rich wastewater in one step. In a sequential batch reactor, synthetic wastewater containing 1 g of NH₄⁺-N liter⁻¹ and minerals was treated. Oxygen supply to the reactor was double-controlled with a pH controller and a timer. At a volumetric loading rate (B_v) of 0.13 g of NH₄⁺-N liter⁻¹ day⁻¹, about 22% of the fed NH₄⁺-N was converted to NO₂⁻-N or NO₃⁻-N, 38% remained as NH₄⁺-N, and the other 40% was removed mainly as N₂. The specific removal rate of nitrogen was on the order of 50 mg of N liter⁻¹ day⁻¹, corresponding to 16 mg of N g of volatile suspended solids⁻¹ day⁻¹. The microorganisms which catalyzed the OLAND process are assumed to be normal nitrifiers dominated by ammonium oxidizers. The loss of nitrogen in the OLAND system is presumed to occur via the oxidation of NH₄⁺ to N₂ with NO₂⁻ as the electron acceptor. Hydroxylamine stimulated the removal of NH₄⁺ and NO₂⁻. Hydroxylamine oxidoreductase (HAO) or an HAO-related enzyme might be responsible for the loss of nitrogen.     

Nitrification and Denitrification in High-Strength Ammonium by <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

Alcaligenes faecalis sp. No. 4, that has the ability of heterotrophic nitrification and aerobic denitrification in high-strength ammonium at about 1200 mg-N/l, converted about one-half of removed NH ₄⁺-N to intracellular nitrogen and nitrified only 3% of the removed NH₄⁺. From the nitrogen balance, 40–50% of removed NH₄⁺-N was estimated to be denitrified. Production of N₂ was confirmed by GC-MS and 90% of denitrified products was N₂. The maximum ammonium removal rate, 29 mg-N/l h and its denitrification rate in aerated batch experiments, were 5–40 times higher than those of other bacteria with the same ability.     

Nitrogen dynamics in a eutrophic lake sediment

Nitrogen flux from sediment of a shallow lake and subsequent utilization by water hyacinth (Eichhornia crassipes [Mart] Solms) present in the water column were evaluated using an outdoor microcosm sediment-water column. Sediment N was enriched with ¹⁵N to quantitatively determine the movement of NH₄-N from the sediment to the overlying water column. During the first 30 days. 48% of the total N uptake by water hyacinth was derived from sediment ¹⁵NH₄-N. This had decreased to 14% after 183 days. Mass balance of N indicates that about 25% sediment NH₄-N was released into the overlying water, but only 17% was assimilated by water hyacinth. NH₄-N levels in the water column were very low, with very little or no concentration gradients. NH₄-N levels in the interstitial water of the sediment were in the range of 30–35 mg L^–1 for the lower depths (> 35 cm), while in the surface 5 cm of depth NH₄-N levels decreased to 3.2 mg L^–1. Simulated results also showed similar trends for the interstitial NH₄-N concentration of the sediment. The overall estimated NH₄-N flux from the sediment to the overlying water was 4.8 µg cm^–2 day^–1, and the soluble organic N flux was 5.8 µg N cm^–2 day^–1. Total N flux was 10.6 µg N cm^–2 day^–1.     

The influence of nitrogen concentration and ammonium/nitrate ratio on N-uptake,mineral composition and yield of citrus

In short-term water culture experiments with different ¹⁵N labeled ammonium or nitrate concentrations, citrus seedlings absorbed NH₄ ⁺ at a higher rate than NO₃ ^–. Maximum NO₃ ^– uptake by the whole plant occurred at 120 mg L^–1 NO₃ ^–-N, whereas NH₄ ⁺ absorption was saturated at 240 mg L^–1 NH₄ ⁺-N. ¹⁵NH₄ ⁺ accumulated in roots and to a lesser degree in both leaves and stems. However, ¹⁵NO₃ ^– was mostly partitioned between leaves and roots.Adding increasing amounts of unlabeled NH₄ ⁺ (15–60 mg L^–1 N) to nutrient solutions containing 120 mg L^–1 N as ¹⁵N labeled nitrate reduced ¹⁵NO₃ ^– uptake. Maximum inhibition of ¹⁵NO₃ ^– uptake was about 55% at 2.14 mM NH₄ ⁺ (30 mg L^–1 NH₄ ⁺-N) and it did not increase any further at higher NH₄ ⁺ proportions.In a long-term experiment, the effects of concentration and source of added N (NO₃ ^– or NH₄ ⁺) on nutrient concentrations in leaves from plants grown in sand were evaluated. Leaf concentration of N, P, Mg, Fe and Cu were increased by NH₄ ⁺ versus NO₃ ^– nutrition, whereas the reverse was true for Ca, K, Zn and Mn.The effects of different NO₃ ^–-N:NH₄ ⁺-N ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at 120 mg L^–1 total N on leaf nutrient concentrations, fruit yield and fruit characteristics were investigated in another long-term experiment with plants grown in sand cultures. Nitrogen concentrations in leaves were highest when plants were provided with either NO₃ ^– or NH₄ ⁺ as a sole source of N. Lowest N concentration in leaves was found with a 75:25 NO₃ ^–-N/NH₄ ⁺-N ratio. With increasing proportions of NH₄ ⁺ in the N supply, leaf nutrients such as P, Mg, Fe and Cu increased, whereas Ca, K, Mn and Zn decreased. Yield in number of fruits per tree was increased significantly by supplying all N as NH₄ ⁺, although fruit weight was reduced. The number of fruits per tree was lowest with the 75:25 NO₃ ^–-N:NH₄ ⁺-N ratio, but in this treatment fruits reached their highest weight. Rind thickness, juice acidity, and colour index of fruits decreased with increasing NH₄ ⁺ in the N supply, whereas the % pulp and maturity index increased. Percent of juice in fruits and total soluble solids were only slightly affected by NO₃ ^–:NH₄ ⁺ ratio.     

Conversion of fat into yeast biomass in protein-containing waste-water

Summary Candida tropicalis S001 was grown on the lipid fraction of a protein-containing waste-water in order to (i) remove fat from the water, and (ii) produre yeast biomass for feed. The yeast cells were separated from the waste-water by sedimentation. Defatted waste-water was used for methane production and gave a yield of a 0.3 m³ methane/kg reduced chemical oxygen demand. The maximum specific growth rate (µ_max) of C. tropicalis growing on waste-water fat at pH 4.0 was 0.35 h^–1; the fat content was decreased from 8 g/l to about 0.1 g/l within 24 h. In continous culture a corresponding reduction was maintained at dilution rates up to 0.36 h^–1. The effect on growth of pH, temperature and CO₂ concentration was studied with triolein as the major carbon source. The µ_max was nearly constant (0.16 h^–1) in the pH and temperature range of 3.2–4.0 and 30°–38° C, respectively; 10% CO₂ was optimal for growth. Growth on triolein resulted in a biomass yield of 0.70 g dry weight/g fat. Offprint requests to: S. Rydin     

Methane oxidation in boreal peat soils treated with various nitrogen compounds

The potential methane consumption activity was examined in various plant communities of a boreal Sphagnum-dominated Bakchar bog of West Siberia. In aerobic laboratory incubations, the peat consumed methane with the maximal rates varied from 17 to 153 nmol CH₄ h^–1g^{– 1}.The highest oxidation took place in the peat from the cotton grass and dwarf shrub-cotton grass communities. The addition of different N-compounds inhibited CH₄-uptake and was not a simple influence of shift in ionic balance (`salt effect'). The introduction of sodium chloride resulted in significantly weaker inhibition effect than the same amount of nitrite and nitrate salts. The inhibition occurred at NH₄ ⁺-N concentrations exceeding 100 mg kg^–1, which was more than 200 times higher native N-content in peat. Communities with high CH₄-uptake activity were more sensitive to ammonium. The inhibition by ammonium was non-competitive. The inhibition by ammonium was mainly due to the toxic action of nitrite and/or nitrate produced by nitrifiers. A strong positive correlation was found between the potential nitrifying activity and inhibition of CH₄-uptake in ammonium-treated peat (R ²= 0.87). The oxidized N-compounds were more strong inhibitors than ammonium and their toxicity increased in the following range: NH₄ ⁺< NO₂ ^–< NO₃ ^–.     

Enhanced nitrite build-up in proportion to increasing alklinity/NH4+ ratio of influent in biofilm reactor

When the alkalinity/NH₄ ⁺ratio increased from 4.1 to 9.4, the ammonium removal rate increased from 45 to 90 mg NO_x-N l^–1 h^–1. An increase in alkalinity/NH₄ ⁺ratio was a major reason for higher pH and free ammonia (FA) concentration in the reactor. The high concentration of FA showed a selective inhibition for Nitrobacter, which caused enhanced nitrite build-up in a biofilm reactor.     

Nitrogen and phosphorus removal by wetland mesocosms subjected to different hydroperiods

The effect of hydroperiod on nutrient removal efficiency from simulated wastewater was investigated in replicate wetland mesocosms (area, 2 m², planted with Scirpus californicus). Alternate draining and flooding of sediments (pulsed discharge) increased nutrient removal efficiency compared to the continuous-flow “control”. Average PO₄³⁻ removal efficiency was 20–30% higher in wetland mesocosms that drained twice daily compared to the control. Inorganic N removal efficiency was less affected than phosphate removal by hydroperiod variation. At the higher NH₄⁺ loading rate (1.83 g N m⁻² day⁻¹), inorganic N removal efficiency was consistently 5–20% higher in pulsed-discharge wetland mesocosms than in the control. At the lower NH₄⁺ loading rate (0.9 g N m ⁻² day ⁻¹), pulsed-discharge hydrology had no effect on inorganic N removal efficiency. Twice-daily drainage exhibited average inorganic N removal efficiencies of 96% (lower N loading rate) and 87% (higher N loading) and average phosphate removal efficiencies of 81% (lower P loading) and 90% (higher P loading). Mass balance data from the continuous-flow treatment revealed that the aquatic macrophyte Scirpus californicus was the most important nutrient sink, assimilating 50% of the NH₄⁺ and PO₄³⁻ supply. The high plant productivity in the mesocosms (15.6 kg m⁻² year⁻¹) occurred under conditions of high light (high edge per mesocosm area) and high root contact with nutrient-rich influent (shallow, sandy substrate) and may overestimate plant uptake in larger wetlands. The addition of a nitrification-inhibitor (N-Serve) indicated that 34% of the NH₄⁺ supply was transformed to NO₃⁻ by nitrifying bacteria.     

Enhancement of ethene removal from waste gas by stimulating nitrification

The treatment of poorly water soluble waste gas compounds,such as ethene, is associated with low substrateconcentration levels in the liquid phase. This lowconcentration level might hamper the optimal development ofa microbial population. In this respect, the possible benefit ofintroducing nitrifying activity in the heterotrophic removal ofethene at moderate concentrations (< 1000 ppm) from awaste gas was investigated. Nitrifying activity is known to beassociated with (i) the production of soluble microbialproducts, which can act as (co-)substrates for heterotrophicmicro-organisms and (ii) the co-oxidation of ethene. Theused reactor configuration was a packed granular activatedcarbon biobed inoculated with the heterotrophic strain Mycobacterium E3. The nitrifying activity was introduced byregular submersion in a nitrifying medium prepared from (i)compost or (ii) activated sludge. In both cases a clearenhancement of the volumetric removal rate of ethene couldbe observed. When combined with a NH₃ dosage on adaily basis, a gradual increase of the volumetric removal rateof ethene could be observed. For a volumetric loading rateof 3 kg ethene-COD·m^-3·d^-1, the volumetric removal rate could thus be increased with a factor1.8, i.e. from 0.72 to a level of 1.26 kgethene-COD·m^-3·d^{- 1}.     

Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a novel halotolerant bacterium Pseudomonas mendocina TJPU04

Excess inorganic nitrogen in water poses a severe threat to enviroment. Removal of inorganic nitrogen by heterotrophic nitrifying–aerobic denitrifying microorganism is supposed to be a promising and applicable technology only if the removal rate can be maintained sufficiently high in real wastewater under various conditions, such as high concentration of salt and wide range of different nitrogen concentrations. Here, a new heterotrophic nitrifying–aerobic denitrifying bacterium was isolated and named as Pseudomonas mendocina TJPU04, which removes NH₄⁺-N, NO₃⁻-N and NO₂⁻-N with average rate of 4.69, 5.60, 4.99 mg/L/h, respectively. It also maintains high nitrogen removal efficiency over a wide range of nitrogen concentrations. When concentration of NH₄⁺-N, NO₃⁻-N and NO₂⁻-N was up to 150, 150 and 50 mg/L, 98%, 93%, and 100% removal efficiency could be obtained, respectively, after 30-h incubation under sterile condition. When it was applied under non-sterile condition, the ammonia removal efficiency was slightly lower than that under sterile condition. However, the nitrate and nitrite removal efficiencies under non-sterile condition were significantly higher than those under sterile condition. Strain TJPU04 also showed efficient nitrogen removal performance in the presence of high concentration of salt and nitrogen. In addition, the removal efficiencies of NH₄⁺-N, NO₃⁻-N and TN in real wastewater were 91%, 52%, and 75%, respectively. These results suggest that strain TJPU04 is a promising candidate for efficient removal of inorganic nitrogen in wastewater treatment.

     

Activity of nitrifying populations in grassland soil polluted by polychlorinated biphenyls (PCBs)

The activity of nitrification was studied in the period of 1992 – 1994 in two grassland plots from the surroundings of a municipal waste incinerator. The soil parameters were fully comparable in both plots and the soils differed in the level of polychlorinated biphenyls (PCBs). The concentration of PCBs found in Klajdovka-control plot (KL): 4.4 ng g_{dry soil} ^–1 can be regarded as a background value, while the polluted plot, Bílá Hora (BH), contained increased amount of PCBs: 14.0 ng g_{dry soil} ^–1.The following parameters of nitrifying activity were determined: field concentrations of N_inorg species, mineralization potentials, nitrifying activity during long-term laboratory incubations, and the potential activity of both ammonium and nitrite oxidizers in short-term incubations in soil slurries. Simultaneous application of all these methods appeared to be very suitable for reliable assessment of nitrifying activity in the field.In the case of the polluted plot, the abnormal accumulation of nitrite was observed both in the field (e.g. in September 1992: BH-656.8 ng NO _inf2 ^– -N g_{dry soil} ^–1; KL-208.2 ng NO _inf2 ^– -N g_{dry soil} ^–1) and in the laboratory incubations. Furthermore, the capability of the polluted plot to nitrify higher amount of ammonium nitrogen appeared to be significantly reduced due to detrimental changes in the activity of nitrite-oxidizing community. In contrast to the nitrification, the mineralization potential did not differ between the plots throughout the sampling period.     

Activity of nitrifiers in relation to nitrogen nutrition of plants in natural ecosystems

Three aspects of the nitrate production in natural ecosystems are discussed,i.e. the population biology of nitrifying bacteria, the nitrate-producing activity of these organisms and the uptake of nitrate by higher plants. It is concluded that the three methods used in enumerating the nitrifying bacteria,i.e. the Most Probable Number method, the Fluorescent Antibody technique and the Potential Nitrification Rate, all have serious drawbacks and count different segments of the nitrifying populations.From the number of nitrifying bacteria no reliable estimate of the rate production can be obtained and also estimates that are made using field-incubation and¹⁵N–NH ₄ ⁺ techniques do not yield reliable data. Possibly the best results can be obtained using Schimel's method to estimate the actual nitrification rate using¹⁵N–NO ₃ ^– , but this method has still not been tested under different sets of soil conditions.From the nitrate reductase activity and the chemical composition of the plant a picture can be obtained of the quantities of nitrate and ammonium that have been taken up. However, it is shown that nitrate and ammonium are taken up in different proportions that they are produced. It is concluded that the various parameters have to be studied simultaneously, preferably in defined systems with plants, in which the participating organisms are known.     

Organic reduction of tapioca wastewaters using modified RBC

A modified Rotating Biological Contactor (RBC) was used for the treatability studies of synthetic tapioca wastewaters. The RBC used was a four stage laboratory model and the discs were modified by attaching porous nechlon sheets to enhance biofilm area. Synthetic tapioca wastewaters were prepared with influent concentrations from 927 to 3600 mg/l of COD. Three hydraulic loads were used in the range of 0.03 to 0.09 m³·m^–2·d^–1 and the organic loads used were in the range of 28 to 306 g COD· m^–2·d^–1. The percentage COD removal were in the range from 97.4 to 68. RBC was operated at a rotating speed of 18 rpm which was found to be the optimal rotating speed. Biokinetic coefficients based on Kornegay and Hudson models were obtained using linear analysis. Also, a mathematical model was proposed using regression analysis.List of Symbols A m² total surface area of discs - d m active depth of microbial film onany rotating disc - K _s mg ·l^–1 saturation constant - P mg·m^–2·^–1 area capacity - Q l·d^–1 hydraulic flow rate - q m³·m^–2·d^–1 hydraulic loading rate - S ₀ mg·l^–1 influent substrate concentration - S _e mg·l^–1 effluent substrate concentration - w rpm rotational speed - V m³ volume of the reactor - X _f mg·l^–1 active biomass per unit volume ofattached growth - X _s mg·l^–1 active biomass per unit volume ofsuspended growth - X mg·l^–1 active biomass per unit volume - Y _s yield coefficient for attachedgrowth - Y _A yield coefficient for suspendedgrowth - Y yield coefficient, mass of biomass/mass of substrate removed Greek Symbols hr mean hydraulic detention time - (_max)_A d^–1 maximum specific growth rate forattached growth - (_max)_s d^–1 maximum specific growth rate forsuspended growth - _max d^–1 maximum specific growth rate - d^–1 specific growth rate - _v mg·l^–1·hr^–1 maximum volumetric substrateutilization rate coefficient     

Nitrogen and phosphorus removal rates using small algal turfs grown with dairy manure

Conservation and reuse of nitrogen (N) and phosphorus (P) from animalmanure is increasingly important as producers try to minimize transport ofthesenutrients from farms. An alternative to land spreading is to grow crops ofalgaeon the N and P present in the manure. The general goal of our research is toassess nutrient recovery from animal manure using attached algae. The specificobjective of this study was to evaluate the use of small subsections of algalturfs for determining N and P removal rates by attached algae under differentloading rates of dairy manure. Algae were grown in a laboratory–scalealgal turf scrubber (ATS) operated by recycling wastewater and adding manureeffluent daily. Replicate subsections (0.032 m²) ofalgal turf screens were removed and treated with five different loadings ofanaerobically digested dairy manure containing 5 to 80 mgL^–1 NH₄-N and 1 to 20 mgL^–1 PO₄-P over a 2-h incubationperiod. NH₄-N removal rates were biphasic with a fast initial ratefollowed by a slower rate. Biphasic rates were more pronounced for the lowestloading rates but less so for the higher ones. PO₄-P removal rateswere linear throughout the incubation period for all loading rates. N and Premoval rates increased with increasing loading rate and biomass. Inincubationsusing 1% dairy manure NH₄-N and PO₄-P removal ratesaveraged 0.72 and 0.33 g m^–2d^–1,respectively. These rates were approximately 5 to 8-fold lower than ratesmeasured on laboratory-scale ATS units using undisturbed turfs.     

Nitrogen supply rate in Scots pine (Pinus sylvestris L.) forests of contrasting slope aspect

We studied Nitrogen (N) transformations in Pinus sylvestris forest stands in the foothills of the SE Pre-Pyrenees (NE Spain). Plots were selected in two contrasting aspects (two plots per aspect) and N supply rate was measured by the resin-core incubation technique once every three months. N leaching through litter layers (L and F horizons) was evaluated by 5 zero-tension lysimeters in each plot. NH₄ ⁺-N, NO₃ ^--N and soluble organic-N were determined in all solutions. N supply rate showed a clear seasonal pattern. Ammonification and nitrification were segregated in space and in time. While ammonification showed a peak in spring, nitrification was higher in summer. There was evidence suggesting that nitrification occurs mostly in A1 horizon. Nitrification rates differed significantly among plots. N supply rate was 12.7–23.5 kg N·ha^-1·yr^-1 but it did not differ between aspects or plots. Inorganic-N leached through litter layers was 14–17 kg N·ha^-1·yr^-1, and represented a high proportion of N supply rate. Organic-N leached through litter layers (27.8–37.0 kg N·ha^-1·yr^-1) was higher than leached inorganic-N. However, in most cases organic-N did not represent a high proportion of changes in soluble organic-N pools in H and A1 horizons (about 240 kg N·ha^-1·yr^-1). This large decrease in soluble organic-N was much greater than the increase in inorganic-N. The possible fate of these large amounts of organic-N is discussed.     

Immobilization of nitrifying bacteria by polyethylene glycol prepolymer

The effects of immobilizing materials on the activity of nitrifying bacteria were investigated by using 11 kinds of prepolymers of polyethylene glycol. Relative respiratory activity of immobilized nitrifying bacteria with polyethylene glycol metacrylate prepolymer was higher than that of polyethylene glycol acrylate prepolymer, and there was a tendency for relative respiratory activity to be higher with a prepolymer of greater molecular weight. With the polyethylene glycol prepolymer, there was a drastic improvement over the conventional method of immobilization by acrylamide in the relative respiratory activity of the pellet. Inorganic synthetic wastewater was treated under a high loading rate of 1.14 kg-N/m³·d. Influent NH₄-N could be removed to 2 mg/l or less and the nitrogen removal was 90%.