Significant Nitrite Accumulation Research Articles

Estimating biofilm activity on biofilter elements in recirculating aquaculture systems (RAS) for rearing Atlantic salmon parr (Salmo salar) during operation with ozone and peracetic acid

Chemical disinfection in a recirculating aquaculture system (RAS) may affect biofilm-associated bacteria and the nitrification performance in the biofilter units. The biofilm response to chemical disinfectants in RAS remains unclear, but it can be understood using methods to quantify biofilm activity. Here, we compared the effects of two disinfection strategies, continuous ozone at 0.06 mg/L Cl2 equivalent (Ozone group) and peracetic acid (PAA) at 1 mg/L (PAA group) to control group without disinfectant, on biofilm activity on biofilter elements from nine identical experimental RAS with Atlantic salmon parr (Salmo salar) during a four-week trial. Biofilm activities on biofilter elements from the three groups were examined by measuring oxygen consumption rates (OCR) following sequential spiking with pure tap water, and tap water spiked with nitrite or ammonium, as well as oxygen release rate (kor) following hydrogen peroxide (H2O2) addition, to estimate metabolic activities of biofilm related to endogenous respiration and substrate turnover. The results show that the applied PAA dose increased the endogenous respiration activity of biofilm by 39–133%, stimulated the rate of biofilm enzymatic decomposition of H2O2 by 135%, and partially impaired the biofilm metabolism for nitrite oxidation (decrease by 36%), resulting in a significant nitrite accumulation (rise by 59%) in the cultured water, compared to control over experimental period. Ozone treatment caused an enhanced endogenous respiration activity of biofilm at the beginning of the experiment (increase by 45–74%), but dropped to control levels at the end of the experiment. The results indicate that chronic exposure to PAA can alter the metabolic state of biofilm, which can have consequences for biofilter functions, while chronic exposure to ozone improved water clarity without compromising the metabolic status of biofilm. The investigations provided insights into biofilm response to chemical disinfectant in RAS, which would benefit the optimization of an effective and safe use of disinfectants in RAS.

Characterization of simultaneous ammonium and nitrate removal and microbial communities in airlift reactor using 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) as carbon source and biofilm carrier

As a novel trend, solid carbon sources are applied to act as electron donors and biofilm carrier in biological denitrification process. In this study, simultaneous nitrate and ammonium removal process in an airlift sequencing batch reactor using 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) as carbon source and biofilm carrier under intermittent aeration conditions was established to treat effluent of synthetic marine recirculating aquaculture system. The results showed that maximum nitrate and ammonia nitrogen removal rates of 0.45 and 0.09 kg m−3 d−1 were achieved. No significant nitrite accumulation was found during 200-day operation, while effluent dissolved organic carbon accumulation and particle size reduction significantly increased. Microbial community analysis and batch tests illuminate that the generated sludge and attached biofilm played important roles in nitrogen removal. This study demonstrates the potential mechanism for the nitrogen removal process mediated by 3-hydroxybutyrate-co-3-hydroxyvalerate and provide a new idea for the alternative solutions of solid carbon sources.

Denitrification performance and microbial communities of solid-phase denitrifying reactors using poly (butylene succinate)/bamboo powder composite

This study explored the denitrification performance of solid-phase denitrification (SPD) systems packed with poly (butylene succinate)/bamboo powder composite to treat synthetic aquaculture wastewater under different salinity conditions (0‰ Vs. 25‰). The results showed composite could achieve the maximum denitrification rates of 0.22 kg (salinity, 0‰) and 0.34 kg NO3−-N m−3 d−1 (salinity, 25‰) over 200-day operation. No significant nitrite accumulation and less dissolved organic carbon (DOC) release (<15 mg/L) were found. The morphological and spectroscopic analyses demonstrated the mixture composites degradation. Microbial community analysis showed that Acidovorax, Simplicispira, Denitromonas, SM1A02, Marinicella and Formosa were the dominant genera for denitrifying bacteria, while Aspergillus was the major genus for denitrifying fungus. The co-network analysis also indicated the interactions between bacterial and fungal community played an important role in composite degradation and denitrification. The outcomes provided a potential strategy of DOC control and cost reduction for aquaculture nitrate removal by SPD.

Enhancing Nitrate Removal from Freshwater Pond by Regulating Carbon/Nitrogen Ratio.

Nitrogen accumulation is a serious environmental problem in freshwater ponds, which can lead to massive death of fish and shrimps as well as the eutrophication. The removal of nitrate by regulating the carbon to nitrogen (C/N) ratio and the underlying mechanisms were investigated. The nitrate removal system comprised 530-mL medium containing 5 mg/L -N and 0–66.6 mg/L COD (i.e., C/N ratio of 0–13.3) and 20 g ponds sediments. When the C/N ratio was higher than 8, the nitrate removal efficiency nearly reached 100% during the incubation period and the accumulation of nitrite was negligible. When the C/N ratio was below 8, the nitrate removal efficiency was lower and significant nitrite accumulation occurred. The nitrate removal rate increased with the C/N ratio increased, which was ascribed to the increase in the absolute abundance of denitrifiers (nirS, nirK, and nosZ). Although both nirS-type and nirK-type denitrifiers were found in the sediments of freshwater pond, nirS-type denitrifiers were predominant. Dechloromonas was the major nirS-type denitrifier for nitrate removal in nirS-type with the C/N ratios above 5.33, while the majority of the nirK-type denitrifiers were unclassified. Thus, this study implied that the appropriate C/N ratio played an important role on the removal of excess nitrate from freshwater ponds.

Long-term effects of heavy metals and antibiotics on granule-based anammox process: granule property and performance evolution.

The feasibility of the anaerobic ammonium oxidation (anammox) process to treat synthetic swine wastewater containing antibiotics and heavy metals was studied in this work. Nitrogen removal performance and granule characteristics were tracked by continuous-flow monitoring to evaluate the long-term joint effects of Cu and Zn and of Cu and oxytetracycline (OTC). Cu and Zn with a joint loading rate (JLR) of 0.04 kg m(-3) day(-1) did not affect the performance, while a JLR of 0.12 kg m(-3) day(-1) caused a rapid collapse in performance. Cu and OTC addition with a JLR of 0.04 kg m(-3) day(-1) for approximately 2 weeks induced significant nitrite accumulation. Granule characteristic analysis elucidated the disparate inhibition mechanisms of heavy metals and antibiotics: the internalization of heavy metals caused metabolic disorders, whereas OTC functioned as a growth retarder. However, anammox reactors could adapt to a JLR of 0.04 kg m(-3) day(-1) via self-regulation during the acclimatization to subinhibitory concentrations, which had a stable nitrogen removal rate (>8.5 kg m(-3) day(-1)) and removal rate efficiency (>75 %) for reactors with Cu-OTC addition. Therefore, this study supports the great potential of using anammox granules to treat swine wastewater.

Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities

The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L−1 at nitrate loading rate (NLR) from 35 kgN m−3 d−1 to 55 kgN m−3 d−1. The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater.

Nitrate reductase and nitrite as additional components of defense system in pigeonpea (Cajanus cajan L.) against Helicoverpa armigera herbivory

Amylase inhibitors serve as attractive candidates of defense mechanisms against insect attack. Therefore, the impediment of Helicoverpa armigera digestion can be the effective way of controlling this pest population. Nitrite was found to be a potent mixed non-competitive competitive inhibitor of partially purified α-amylase of H. armigera gut. This observation impelled us to determine the response of nitrite and nitrate reductase (NR) towards H. armigera infestation in nine pigeonpea genotypes (four moderately resistant, three intermediate and two moderately susceptible). The significant upregulation of NR in moderately resistant genotypes after pod borer infestation suggested NR as one of the factors that determine their resistance status against insect attack. The pod borer attack caused greater reduction of nitrate and significant accumulation of nitrite in moderately resistant genotypes. The activity of nitrite reductase (NiR) was also enhanced more in moderately resistant genotypes than moderately susceptible genotypes on account of H. armigera herbivory. Expression of resistance to H. armigera was further revealed when significant negative association between NR, NiR, nitrite and percent pod damage was observed. This is the first report that suggests nitrite to be a potent inhibitor of H. armigera α-amylase and also the involvement of nitrite and NR in providing resistance against H. armigera herbivory.

Enhanced Heterotrophic Denitrification: Effect of Dairy Industry Sludge Acclimatization and Operating Conditions

Heterotrophic denitrification of drinking water was enhanced by selection of an anoxic sludge taken from a dairy industry among the sludges taken from various industries, and the effect of carbon sources was examined. Acclimatization to high nitrate concentration was then carried out in a five-stage process. Considering removals of both nitrate and nitrite, the sludge taken from anoxic unit of Tehran Pegah dairy industry was shown to be the superior microbial culture, with ethanol as carbon source as compared to acetate. To enhance the rate of denitrification, acclimatization to nitrate (at 100, 200, 400, 800, and 1,600mg N-NO3/L) was carried out in sequencing batch reactors over a 3-month period under anoxic condition, and comparisons were made between the performances of acclimated and non-acclimated sludges at each stage. It was found that acclimatization up to the fourth stage enhanced the specific denitrification rate to a high value of 29.6mg N-NO3/h/g mixed liquor suspended solids (MLSS), with no significant nitrite accumulation. Additionally, the effect of initial pH (6, 6.5, 7, and 7.5) and carbon-to-nitrogen (C/N) ratio (1, 1.5, 2, and 3) on the performance of this final acclimated sludge was assessed, where initial pH of 7 and C/N ratio of 1.5 resulted in the best performances considering both nitrate and nitrite removal.

The influence of aeration on nitrification and the nitrifier distribution in an upflow biological aerated filter for tertiary treatment of municipal sewage

A series of two pilot-scale aerobic and anaerobic upflow biofilters were constructed and operated for tertiary treatment of municipal sewage. The effects of aeration on the nitrification performance and nitrifier distribution between attached and suspended biomass of the aerobic upflow biofilter (UBAF) were studied. Process operating results revealed that, for the secondary effluent with volumetric loads of 0.31–0.65 kgCOD/m3 d and 0.14–0.34 kgNH4−N/m3 d, the UBAF showed an unstable ammonia removal rate of 60.6–72.2% with an average bulk DO concentration of 2.18 mg/L at an aeration rate of 0.6 m3/h. Partial nitrification was observed at the first 1.2 m of the filter (DO < 1.42 mg/L), and more than 38.4% nitrite accumulation ratio was detected. When the aeration rate was increased to 1.0 m3/h, the nitrification efficiency of UBAF was stabilized at the range of 90.1–93.5%with an average bulk DO concentration of 3.82 mg/L. No significant nitrite accumulation took place in the whole filter. Specific activity ...

A new method of autotrophic denitrification with spent sulfidic caustic as substrate and alkalinity source

A new method based on sulfide utilizing autotrophic denitrification was adopted to remove nitrate from wastewater and to reuse spent sulfidic caustic containing high sulfide and alkalinity levels. The experiments were performed using a bench-scale upflow anoxic hybrid growth reactor (UAHGR) and an upflow anoxic suspended growth reactor (UASGR) to characterize the stoichiometric relationship between sulfur and nitrate in the process as well as the performance of the reactors. The level of nitrate removal from the UAHGR and UASGR were maintained at over 90% at a nitrate loading rate ranging from 0.15∼0.40 kgNO3−/m3·d and no significant nitrite accumulation was observed in either reactor. Although the influent pH values were higher than the optimum range of autotrophic denitrification at 8.7∼10.1, the effluent pH was stable at 7.2∼7.9 due to the production of hydrogen ions during operation. The stoichiometric ratio of sulfate production to nitrate removal was 1.5∼2.1 mgSO42−/mgNO3− in both reactors. A comparison of the reactor performance revealed that the chemical parameters of the UAHGR operation corresponded to a plug flow like type reactor while the chemical parameters of the UASGR operation corresponded to a completely stirred tank reactor like type reactor. UAHGR did not require sludge recycling due to the packed media while UASGR required 300∼700% sludge recycling. Therefore, spent sulfidic caustic could be used in the sulfur utilizing autotrophic denitrification processes as substrate and alkalinity sources.

Batch Nitrifying Cultures in Presence of Mixtures of Benzene, Toluene, and m-Xylene

Benzene, toluene, and m-xylene compounds in individual (5.0 ± 0.5 mg C l−1) and mixed solutions (2.5 ± 0.2 mg C l−1 for each one) in nitrifying batch cultures induced a decrease in the specific rates of NH4 + consumption (81 ± 6%) and NO3 − production (39-79%). However, after 24 h, ammonium consumption efficiency and conversion of consumed NH4 +-N into NO3 −-N were close to 100% and there was no significant accumulation of nitrite in the medium. After 24 h, no aromatic intermediate was detected in the cultures and 50% of the mixed compounds was converted to acetate. The following order of biotransformation was found: m-xylene > toluene > benzene. Transformation rate of m-xylene was 0.051 ± 0.005 g C (g protein-N h)−1 in individual and mixed solutions. When m-xylene was added, benzene was oxidized at a faster rate (0.051 ± 0.005 g C (g protein-N h)−1) whereas toluene at a slower rate (0.012 ± 0.002 g C (g protein-N h)−1).

Short-cut nitrification of domestic wastewater in a pilot-scale A/O nitrogen removal plant

The feasibility of nitrite accumulation in a pilot-scale A/O (anoxic/oxic) nitrogen removal plant treating domestic wastewater was investigated at various dissolved oxygen (DO) concentrations and pH levels. The results showed that the pH was not a useful operational parameter to realize nitrite accumulation. Significant nitrite accumulation was observed at the low DO concentration range of 0.3-0.8 mg/l and the maximum nitrite accumulation ratio of about 90% occurred at a DO concentration of 0.6 mg/l. This suggests a reduction of 22% in the oxygen consumption, and therefore a considerable saving in aeration. However, the nitrite accumulation was destroyed at the high DO concentration and the resumption was very slow. In addition, the average ammonia removal efficiency reached as high as 93% at the low DO level. Moreover, experimental results indicated that nitrogen could be removed by simultaneous nitrification and denitrification (SND) via nitrite in the aerobic zones at the low DO concentration, with the efficiency of 6-12%.

Nitrogen removal in a fluidized bed bioreactor by using mixed culture under oxygen-limited conditions

Nitrogen removal involving nitrification and denitrification was investigated in a fluidized bed bioreactor by using mixed culture sludge under oxygen-limited conditions. Methane was used as a sole carbon source for denitrification. In this study, optimal nitrification and denitrification rates were examined by varying methane and oxygen gas dissolution flow rates, 90 ml/min, 400 ml/min and 650 ml/min, in each. Simultaneously nitrification and denitrification was achieved. The total nitrogen removal rate was 15-mg N/g VSS. d, 21-mg N/g VSS. d and 26.4-mg N/g VSS. d at gas dissolution flow rate 90 ml/min, 400 ml/min and 650 ml/min, respectively. No significant accumulation of nitrite was found in this experiment. Nitrogen removal rates depend on gas dissolution flow rates. DO concentration was at 0.5-2 mg/L.

Anoxic transformations of wastewater organic matter in sewers – process kinetics, model concept and wastewater treatment potential

The sewer is an integral part of the urban wastewater system: the sewer, the wastewater treatment plant and the local receiving waters. The sewer is a reactor for microbial changes of the wastewater during transport, affecting the quality of the wastewater and thereby the successive treatment processes or receiving water impacts during combined sewer overflows. This paper presents the results of studies on anoxic processes, namely denitrification, in the bulk water phase of wastewater as it occurs in sewers. Experiments conducted on 12 different wastewater samples have shown that the denitrification process in the bulk wastewater can be simplified by the reduction of nitrate to nitrogen with significant accumulation of nitrite in the water phase. Utilization of nitrate was observed not to be limited by nitrate for concentrations above 5 gNO3-N/m3. The denitrification rates, under conditions of excess substrate and electron acceptor, were found to be in the range of 0.8-2.0 g NO3-N/(m3h). A discussion on the interaction of the sewer processes and the effects on a downstream located wastewater treatment plant (WWTP) is provided.

Stimulation of Nitric Oxide Synthesis in Porcine Granulosa Cells by Phorbol Ester.

This study was designed to assess the release of nitric oxide (NO) from porcine granulosa cells. The cells obtained from medium-sized ovarian follicles (3-6 mm diameter) were cultured for 48 h with ovine FSH to be developed. A significant accumulation of nitrite was observed during development, showing an increase in NO synthesis. The mRNA of the endothelial NO synthase was detected in the granulosa cells, as revealed by RT-PCR. Furthermore, nitrite was accumulated in the culture medium after 1-h stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with 4α-phorbol, 4β-phorbol 13-monoacetate (4β-PMA) and forskolin. TPA led to an increase in the nitrite content in a dose-dependent manner. NO was measured directly in the cell suspension with an amperometric NO sensor. The NO sensor signal gradually increased to reach a plateau (5-15 pA) after exposure to TPA in the presence of L-arginine, but not D-arginine. The concentration of NO was approximately 5.5-11 nM, but no detectable change was observed after the addition of 4α-phorbol or 4β-PMA. These results suggest that the synthesis of NO is promoted at least in part through the activation of protein kinase C in granulosa cells.

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.

Optimal operational factors for nitrite accumulation in batch reactors.

The environmental factors that affected the accumulation of nitrite in nitrifying reactors were investigated using a mixed culture. A batch reactor with 50 mg-N/l of ammonia was used. The pH, temperature and dissolved oxygen concentration were varied. The concentration of unionized free ammonia also changed with the oxidation of ammonia and the variation of pH and temperature. The accumulation of nitrite was affected sensitively by pH and temperature. A higher nitrite concentration was observed at pH 8-9 or temperature around 30 degrees C. The dissolved oxygen also affected, giving the highest nitrite accumulation at around 1.5 mg/l. These were the favored conditions for nitrite production. The free ammonia concentration influenced the nitrite accumulation also, by inhibiting nitrite oxidation. The inhibition became apparent at a concentration of approximately 4 mg/l or above, but insignificant at below 1 mg/l. Thus, simultaneously high free ammonia concentration and maximum specific ammonia-oxidation rate (above 15 x 10(-3) mg-N/mg-VSS x h) were needed for a significant nitrite accumulation. When the two conditions were met, then the highest accumulation was observed when the ratio of the maximum specific oxidation rate of ammonia to the maximum specific oxidation rate of nitrite (ka/kn) was highest. Under the optimal operating conditions of pH 8, 30 degrees C and 1.5 mg/l of dissolved oxygen, as much as 77% of the removed ammonia accumulated in nitrite.

The effect of nitrite correction on the evaluation of the rate of nitrate utilization under anoxic conditions

The nitrogen utilization requirement (NUR) test is a useful respirometric procedure for the assessment of major kinetic constants in activated sludge systems designed for nitrogen removal. The electron transfer mechanism of nitrogenous compounds often involves significant nitrite accumulation which impairs the accuracy of the NUR test. Experimental results revealed no meaningful correlation between nitrite accumulation and the initial nitrate concentration or the food/microorganism (F/M) ratio. Significant impact of nitrite accumulation was observed both on the assessment of the readily biodegradable COD component, the correction factor for anoxic growth and the denitrification rates applicable to domestic sewage and a number of industrial wastewaters. © 1999 Society of Chemical Industry

Effect of 17beta-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta.

1. Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17beta-oestradiol on cytokine-induced nitric oxide production by the inducible nitric oxide synthase (iNOS). 2. Treatment of the isolated aortic rings with interleukin-1beta (IL-1beta, 20 micro ml(-1)) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM-10 microM)-evoked contraction. 3. Cycloheximide (1 microM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL-1beta on the isolated aortic rings. 17Beta-oestradiol (1 nM-10 microM) and the partial oestrogen receptor agonist 4-OH-tamoxifen (1 nM-10 microM) produced concentration-dependent inhibition of IL-1beta-induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 microM). 4. Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL-1beta-induced and 17beta-oestradiol-treated rings. Western blot analysis of rat aorta homogenates revealed that 17beta-oestradiol treatment resulted in a reduction in IL-1beta-induced iNOS protein level. 5. Incubation with tumour necrosis factor alpha (TNF alpha, 1 ng ml(-1)) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL-1beta. The effects of TNF alpha were also inhibited by co-incubation of the rings with 17beta-oestradiol and 4-OH-tamoxifen (1 microM). 6. The anti-transforming growth factor-beta1 (TGF-beta1) antibody, which inhibited TGF-beta1-induced suppression of nitrite production from IL-1beta-treated vascular rings, did not affect the inhibitory action of 17beta-oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF-beta1. 7. These results show that the ovarian sex steroid, 17beta-oestradiol is a modulator of cytokine-induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

Organic carbon supplement influencing performance of biological nitritification in a fluidized bed reactor

A three-phase draft-tube fluidized-bed bioreactor packed with granular activated carbon (GAC) was employed to start up a nitrification process for high-strength ammonium (500 mg/l as N) wastewater. Supplement of sucrose into the ammonium wastewater was expected to enhance the attachment of nitrifier biofilm on GAC medium, and also to promote a high rate of ammonium conversion to predominant nitrite, i.e. nitrification process. In this study, various concentrations of sucrose provided various COD/NH4+-N ratios (0 to 1) to the fluidized bed bioreactor with an extremely high loading of 2.0 kg NH4+-N/m3-day and a limited air supply at the dissolved oxygen level of 3.0 mg/l. The results clearly indicated that the addition of organic carbon would inhibit the bioactivity of ammonium oxidizer (Nitrosomonas sp.) and nitrite oxidizer (Nitrobacter sp.). When the COD/NH4+-N ratio was increased to 0.33, the applied sludge loading to the nitrite oxidizer was greater than the specific substrate utilization rate, so the intermediate product, nitrite was accumulated without further nitratification. While the COD/NH4+-N ratio was increased to 1.0, a significant nitrite accumulation above 95% was achieved with the high ammonium feeding concentration of 500 mg/l NH4+-N. Meantime, the addition of sucrose in the start-up stage promoted heterotrophic growth and biofilm attachment on GAC surface. The filamentous structure of heterotrophs provided a biofilm surface on which the discrete nitrifying bacteria could be easily entrapped. In this fluidized-bed reactor, a large amount of entrapped nitrifier and a high efficiency of oxygen transfer rate had contributed to a high removal loading rate of ammonium with short hydraulic retention time of 6 hours.