Upgrading of Florence wastewater treatment plant: co-digestion and nitrogen autotrophic removal.

In recent years a completely autotrophic nitrogen removal process based on Anammox biomass has been tested in a few European countries in order to treat anaerobic supernatant and to increase the COD/N ratio in municipal wastewater. This work reports experimental results on a possible technical solution to upgrade the S. Colombano treatment plant which treats wastewater from the Florentine urban area. The idea is to use 50% of the volume of the anaerobic digester in order to treat external sewage sludge (as septic tank sludge) together with waste activated sludge and to treat the resulting effluent on a SHARON-ANAMMOX process in order to remove nitrogen from the anaerobic supernatant. Anaerobic co-digestion, tested in a 200 L pilot plant, enables low cost treatment of septic tank sludge and increases biogas production; however, it also increases the nitrogen load re-circulated to the WWTP, where nitrogen removal efficiency is already low (<50%), due to the low COD/N ratio, which limits predenitrification efficiency. Experimental results from a SHARON process tested in a lab-scale pilot plant show that nitrite oxidising bacteria are washed-out and steady nitrite production can be achieved at retention times in the range 1 - 1.5 days, at 35 degrees C. In a lab-scale SBR reactor, coupled with a nitration bioreactor, maximum specific nitrogen removal rate under nitrite-limiting conditions (with doubling time equal to about 26 days at 35 degrees C) was equal to 0.22 kgN/kgSSV/d, about 44 times the rate measured in inoculum Anammox sludge. Finally, a cost analysis of the proposed upgrade is reported.     

A one-stage system with partial nitritation and anammox processes in the moving-bed biofilm reactor.

The ability of bacterial cultures to create biofilm brings a possibility to enhance biological wastewater treatment efficiency. Moreover, the ability of Anammox and Nitrosomonas species to grow within the same biofilm layer enabled a one-stage system for nitrogen removal to be designed. Such a system, with Kaldnes rings as carriers for biofilm growth, was tested in a technical pilot plant scale (2.1 m(3)) at the Himmerfj?rden Waste Water Treatment Plant (WWTP) in the Stockholm region. The system was directly supplied with supernatant originating from dewatering of digested sludge containing high ammonium concentrations. Nearly 1-year of operational data showed that during the partial nitritation/Anammox process, alkalinity was utilised parallel to ammonium removal. The process resulted in a small pH drop, and its relationship with conductivity was found. The nitrogen removal rate for the whole period oscillated around 1.5g N m(-2)d(-1) with a maximum value equal to 1.9 g N m(-2)d(-1). Parallel to the pilot plant experiment, a series of batch tests were run to investigate the influence on removal rates of different dissolved oxygen conditions and addition of nitrite. The highest nitrogen removal rate (5.2g N m(-2)2d(-1)) in batch tests was obtained when the Anammox process was stimulated by the addition of nitrite. In the simultaneous partial nitritation and Anammox process, the partial nitritation was the rate-limiting step.     

Nutrient removal and microbial granulation in an anaerobic process treating inorganic and organic nitrogenous wastewater.

The sustainable anaerobic nitrogen removal and microbial granulation were investigated by using a laboratory anaerobic granular sludge bed reactor, treating synthetic (inorganic and organic) wastewater and piggery waste. From inorganic synthetic wastewater, lithoautotrophic ammonium oxidation to nitrite/nitrate was observed by an addition of hydroxylamine. Also, the results revealed that the Anammox intermediates (particularly, hydrazine) contents in the substrate would be one of the important parameters for success of the anaerobic nitrogen removal process. The results from organic synthetic wastewater show that if the Anammox organism were not great enough in the startup of the process, denitritation and anaerobic ammonification would be a process prior to the Anammox reaction. The anaerobic ammonium removal from the piggery waste was performed successfully, probably due to the Anammox intermediates contained in the substrate. This reactor shows a complex performance including the Anammox reaction and HAP crystallization, as well as having partial denitritation occurring simultaneously. From the activity test, the maximum specific N conversion rate was 0.1 g NH4-N/g VSS/day (0.77 g T-N/g VSS/day), indicating that potential denitritation is quite high. The NO2-N/NH4-N ratio to Anammox is 1.17. The colour of the biomass treating the piggery waste changed from black to dark red. It was also observed that the red-colored granular sludge had a diameter of 1-2 mm. The settleability assessment of the granular sludge revealed that the granular sludge had a good settleability even though it was worse than that of seed granular sludge.     

Nitrogen removal rates at a technical-scale pilot plant with the one-stage partial nitritation/Anammox process.

Traditional nitrification/denitrification is not suitable for nitrogen removal when wastewater contains high concentrations of ammonium nitrogen and low concentrations of biodegradable carbon. Recently, a deammonification process was developed and proposed as a new technology for treatment of such streams. This process relies on a stable interaction between aerobic bacteria Nitrosomonas, that accomplish partial nitritation and anaerobic bacteria Planctomycetales, which conduct the Anammox reaction. Simultaneous performance of these two processes can lead to a complete autotrophic nitrogen removal in one single reactor. The experiments where nitrogen was removed in one reactor were performed at a technical-scale moving-bed pilot plant, filled with Kaldnes rings and supplied with supernatant after dewatering of digested sludge. It was found that a nitrogen removal rate obtained at the pilot plant was 1.9 g m(-2) d(-1). Parallel to the pilot plant run, a series of batch tests were carried out under anoxic and aerobic conditions. Within the batch tests, where the pilot plant's conditions were simulated, removal rates reached up to 3 g N m(-2)d(-1). Moreover, the batch tests with inhibition of Nitrosomonas showed that only the Anammox bacteria (not anoxic removal by Nitrosomonas) are responsible for nitrogen removal.     

Anaerobic ammonium oxidation of ammonium-rich waste streams in fixed-bed reactors.

The feasibility of anaerobic ammonium oxidation (Anammox) in fixed-bed reactors was evaluated on laboratory and pilot scales. Using synthetic wastewater, the specific nitrogen removal rate was increased from 0.05-0.1 kgNm(-3)(reactor)d(-1) to 0.35-0.38 kgNm(-3)(reactor)d(-1) within a year (T= 22-27 degrees C) in all applications. However, the anammox activity was seriously and repeatedly inhibited at prolonged high nitrite concentrations (e.g. six days at 30-50 gNO2-Nm(-3)) and recovery was always a lengthy process. But even at a moderate nitrite concentration (11+/-10 gNO2-Nm(-3)), the observed specific growth rate was only 0.018 d(-1) at 26.4+/-0.8 degrees C, which corresponds to approximately 0.025 d(-1) at 30 degrees C (doubling time: 28 days). In a second experimental period for another 250 days, one of the laboratory reactors was fed with partially nitrified sludge liquors from a domestic wastewater treatment plant (WWTP). In this case, the specific elimination rate was as high as 3.5 kgNm(-3)(reactor)d(-1) at 26-27 degrees C. Independently of the feed, the average nitrogen elimination rate lay between 80-85% in all applications. An appropriate hydraulic design is essential to prevent clogging and local nitrite inhibition in fixed-bed reactors.     

Nitrogen removal from sludge digester liquids by nitrification/denitrification or partial nitritation/anammox: environmental and economical considerations.

In wastewater treatment plants with anaerobic sludge digestion, 15-20% of the nitrogen load is recirculated to the main stream with the return liquors from dewatering. Separate treatment of this ammonium-rich digester supernatant significantly reduces the nitrogen load of the activated sludge system. Two biological applications are considered for nitrogen elimination: (i) classical autotrophic nitrification/heterotrophic denitrification and (ii) partial nitritation/autotrophic anaerobic ammonium oxidation (anammox). With both applications 85-90% nitrogen removal can be achieved, but there are considerable differences in terms of sustainability and costs. The final gaseous products for heterotrophic denitrification are generally not measured and are assumed to be nitrogen gas (N2). However, significant nitrous oxide (N2O) production can occur at elevated nitrite concentrations in the reactor. Denitrification via nitrite instead of nitrate has been promoted in recent years in order to reduce the oxygen and the organic carbon requirements. Obviously this "achievement" turns out to be rather disadvantageous from an overall environmental point of view. On the other hand no unfavorable intermediates are emitted during anaerobic ammonium oxidation. A cost estimate for both applications demonstrates that partial nitritation/anammox is also more economical than classical nitrification/denitrification. Therefore autotrophic nitrogen elimination should be used in future to treat ammonium-rich sludge liquors.     

Nitrogen removal from sludge reject water by a two-stage oxygen-limited autotrophic nitrification denitrification process.

Nitrogen removal from sludge reject water was obtained by oxygen-limited partial nitritation resulting in nitrite accumulation in a first stage, followed by autotrophic denitrification of nitrite with ammonium as electron donor (similar to anaerobic ammonium oxidation) in a second stage. Two membrane-assisted bioreactors (MBRs) were used in series to operate with high sludge ages and subsequent high volumetric loading rates, achieving 1.45 kg N m(-3) day(-1) for the partial nitritation MBR and 1.1 kg N m(-3) day(-1) for the anaerobic ammonium oxidation MBR. Biomass retention in the nitritation stage ensured flexibility towards loading rate and operating temperature. Nitrite oxidisers were out-competed at low oxygen and high free ammonia concentration. Biomass retention in the second MBR prevented wash-out of the slowly growing bacteria. Nitrite and ammonium were converted to dinitrogen gas in a reaction ratio of 1.05, thereby maintaining nitrite limitation to assure process stability. The anoxic consortium catalysing the autotrophic denitrification process consisted of Nitrosomonas-like aerobic ammonium oxidizers and anaerobic ammonium oxidizing bacteria closely related to Kuenenia stuttgartiensis. The overall removal efficiency of the combined process was 82% of the incoming ammonium according to a total nitrogen removal rate of 0.55 kg N m(-3) day(-1), without adding extra carbon source.     

ANAMMOX and partial denitritation in anaerobic nitrogen removal from piggery waste.

The anaerobic ammonium removal from a piggery waste with high strength (56 g COD/L and 5 g T-N/L) was investigated using a lab-scale upflow anaerobic sludge bed reactor at a mesophilic condition. Based on the nitrogen and carbon balance in the process, the contribution of autotrophic and heterotrophic organisms was also evaluated in terms of the influent NO2-N/NH4-N ratio (1:0.8 and 1:1.2 for Phase 1 and Phase 2, respectively). The result of this research demonstrates that the anaerobic ammonium removal from the piggery waste, using the UASB reactor, can be performed successfully. Furthermore, it appears that by using granular sludge as the seed biomass, the ANAMMOX reaction can start more quickly. Average nitrogen conversion was 0.59 kg T-N/m3 reactor-day (0.06 kg T-N/kg VSS/day) and 0.66 kg T-N/m3 reactor-day (0.08 kg T-N/kg VSS/day) for Phase 1 and Phase 2. The NO2-N/NH4-N removal ratio by the ANAMMOX was 1.48 and 1.79 for Phase 1 and Phase 2. The higher nitrite contents (about 50%) in the substrate resulted in higher nitrite nitrogen removal by the partial denitritation, as well as the ANAMMOX reaction, implying higher potential of partial denitritation. However, the result reveals that the ANAMMOX reaction was influenced less by the degree of partial denitritation, and the ANAMMOX bacteria did not compete with denitritation bacteria. The colour of the biomass at the bottom of the reactor changed from dark gray to dark red, which was accompanied by an increase in cytochrome content. At the end of the experiment, red-coloured granular sludge with diameter of 1-2 mm at the lower part of the reactor was also observed.     

Combined partial nitritation/Anammox system for treatment of digester supernatant.

One-year (2004) comprehensive investigations in a semi-industrial pilot plant (5 m3) were carried out with the aim of assessing the influence of operational parameters on the partial nitritation/Anammox system performance. In the system designed as a moving-bed biofilm reactor, the influent nitrogen load to the Anammox reactor was progressively increased and a stable Anammox bacterial culture was obtained. Interaction between subsequent aerobic and anaerobic conditions in the partial nitritation and Anammox reactors, respectively, granted conditions to remove nitrogen through the nitrite route. It implies that the oxygen supply can be limited to a high extent. A control strategy for the partial nitritation step relied on concomitant adjustment of the air supply with a variable influent nitrogen load, which can be monitored by both pH and conductivity measurements. In the Anammox reactor, an influent nitrite-to-ammonium ratio plays a vital role in obtaining efficient nitrogen removal. During the 1-year experimental period, the Anammox reactor was operated steadily and average nitrogen removal efficiency was 84% with 97% as the maximum value.     

Post-aeration of anaerobically digested sewage sludge for advanced COD and nitrogen removal: results and cost-benefit analysis at large-scale.

At a large Austrian municipal wastewater treatment plant enhanced stabilisation of anaerobically digested sewage sludge was required in order to get a permit for landfill disposal of the dewatered stabilized sludge. By implementing a post-aeration treatment after anaerobic digestion the organic content of the anaerobically well digested sludge can be decreased by 16%. Investigations at this plant showed that during digested sludge post-aeration anoxic phases are needed to provide stable process conditions. In this way the pH value can be kept in a more favourable range for micro-organisms and concrete structures. Additionally, under the process conditions applied nitrite accumulation would inhibit the stabilisation process if denitrification is not adequately applied. By optimising the aeration/pause ratio approximately 45% of total nitrogen in digested sludge can be removed. NH4-removal occurs through nitrification and denitrification with an efficiency of 98%. This significantly improves nitrogen removal efficiency at the wastewater treatment plant. The costs/benefit analysis shows that post-aeration of digested sludge results in an increase of total annual costs for wastewater treatment of only 0.84%, corresponding to 0.19 Euro/pe/a. Specific costs for nitrogen removal (0.32 Euro/kgN) are comparable with other biological processes for N-removal in reject water.     

Tracking influent inorganic suspended solids through wastewater treatment plants.

From an experimental and theoretical investigation of the continuity of influent inorganic suspended solids (ISS) along the links connecting the primary settling tank (PST), fully aerobic or N removal activated sludge (AS) and anaerobic and aerobic sludge digestion unit operations, it was found that the influent wastewater (fixed) ISS concentration is conserved through primary sludge anaerobic digestion, activated sludge and aerobic digestion unit operations. However, the measured ISS flux at different stages through a series of wastewater treatment plant (WWTP) unit operations is not equal to the influent ISS flux, because the ordinary heterotrophic organisms (OHO) biomass contributes to the ISS flux by differing amounts depending on the active fraction of the VSS solids at that stage.     

Treatment of high loaded swine slurry in an aerobic granular reactor

Aerobic granular sludge grown in a sequential batch reactor was proposed as an alternative to anaerobic processes for organic matter and nitrogen removal from swine slurry. Aerobic granulation was achieved with this wastewater after few days from start-up. On day 140 of operation, the granular properties were: 5 mm of average diameter, SVI of 32 mL (g VSS)(-1) and density around 55 g VSS (L(granule))(-1). Organic matter removal efficiencies up to 87% and nitrogen removal efficiencies up to 70% were achieved during the treatment of organic and nitrogen loading rates (OLR and NLR) of 4.4 kg COD m(-3) d(-1) and of 0.83 kg N m(-3) d(-1), respectively. However, nitrogen removal processes were negatively affected when applied OLR was 7.0 kg COD m(-3) d(-1) and NLR was 1.26 kg N m(-3) d(-1). The operational cycle of the reactor was modified by reducing the volumetric exchange ratio from 50 to 6% in order to be able to treat the raw slurry without dilution.     

High rate nitrogen removal by the CANON process at ambient temperature

Completely autotrophic nitrogen removal over nitrite (CANON) is a cost-effective nitrogen removal process. Implementation of the CANON process relies on the cooperation of ammonium-oxidizing and Anammox bacteria, as well as the inhibition of nitrite-oxidizing bacteria. Strict limitations on dissolved oxygen (DO) concentration in the reactor, and the addition of sufficient inorganic carbon in the influent, were adopted as the main operational strategies. The reactor was fed with synthetic inorganic wastewater composed mainly of NH(4)(+)-N, and operated for 106 days. Stable nitrogen removal rates (NRR) of around 1.4 kg N m(-3) d(-1) were obtained at ambient temperature. Morphological characteristics and analysis of bacterial community confirmed the formation of functional outer aerobic and inner anaerobic granular sludge, providing evidence of stable nitrogen removal.     

Aeration of anaerobically digested sewage sludge for COD and nitrogen removal: optimization at large-scale.

The paper will report about the experiences at an Austrian large wastewater treatment plant of 720,000 population equivalents, where anaerobically digested sewage sludge is further stabilised under aerobic conditions. Enhanced stabilisation of the anaerobically digested sludge was required at the plant in order to get a permit for landfill disposal of the dewatered stabilized sludge. By implementing a post-aeration treatment (SRT approximately 6d; 36 degrees C) after anaerobic digestion the organic content of the anaerobically well digested sludge can be decreased by 16%. Investigations on site showed that during digested sludge post-aeration anoxic phases for denitrification are needed to provide stable process conditions. In this way the pH value can be kept in a more favourable range for micro-organisms and concrete structures. Additionally, inhibition of the biological process due to nitrite accumulation can be avoided. By optimising the aeration/pause ratio approximately 45% of total nitrogen in digested sludge can be removed. This significantly improves nitrogen removal efficiency at the wastewater treatment plant. NH(4)-removal occurs mainly through nitritation and denitritation with an efficiency of 98%. The costs/benefit analysis shows that post-aeration of digested sludge results in an increase of total annual costs for wastewater treatment of only 0.84%, corresponding to 0.19 Euro/pe/a. Result of molecular biological analyses (DGGE) indicate that all four ammonium-oxidizing bacteria species present in activated sludge can survive anaerobic digestion, but only two of them can adapt in the digested sludge post-aeration tanks. Additionally, in the post-aerated digested sludge a further ammonium-oxidizing bacteria species was identified.     

Dissolved oxygen as a factor influencing nitrogen removal rates in a one-stage system with partial nitritation and Anammox process

A biofilm system with Kaldnes biofilm carrier was used in these studies to cultivate bacteria responsible for both partial nitritation and Anammox processes. Due to co-existence of oxygen and oxygen-free zones within the biofilm depth, both processes can occur in a single reactor. Oxygen that inhibits the Anammox process is consumed in the outer layer of the biofilm and in this way Anammox bacteria are protected from oxygen. The impact of oxygen concentration on nitrogen removal rates was investigated in the pilot plant (2.1 m3), supplied with reject water from the Himmerfj?rden Waste Water Treatment Plant. The results of batch tests showed that the highest nitrogen removal rates were obtained for a dissolved oxygen (DO) concentration around 3 g O2 m(-3) At a DO concentration of 4 g O2 m(-3), an increase of nitrite and nitrate nitrogen concentrations in the batch reactor were observed. The average nitrogen removal rate in the pilot plant during a whole operating period oscillated around 1.3 g N m(-2)d(-1) (0.3 +/- 0.1 kg N m(-3)d(-1)) at the average dissolved oxygen concentration of 2.3 g O2 m(-3). The maximum value of a nitrogen removal rate amounted to 1.9 g N m(-2)d(-1) (0.47 kg N m(-3)d(-1)) and was observed for a DO concentration equal to 2.5 g O2 m(-3). It was observed that increase of biofilm thickness during the operational period, had no influence on nitrogen removal rates in the pilot plant.     

Rapid start-up of a nitrifying reactor using aerobic granular sludge as seed sludge

In this study, the effectiveness of aerobic granular sludge as seed sludge for rapid start-up of nitrifying processes was investigated using a laboratory-scale continuous stirred-tank reactor (CSTR) fed with completely inorganic wastewater which contained a high concentration of ammonia. Even when a large amount of granular biomass was inoculated in the reactor, and the characteristics of influent wastewater were abruptly changed, excess biomass washout was not observed, and biomass concentration was kept high at the start-up period due to high settling ability of the aerobic granular sludge. As a result, an ammonia removal rate immediately increased and reached more than 1.0 kg N/m(3)/d within 20 days and up to 1.8 kg N/m(3)/d on day 39. Subsequently, high rate nitritation was stably attained during 100 days. However, nitrite accumulation had been observed for 140 days before attaining complete nitrification to nitrate. Fluorescence in situ hybridization analysis revealed the increase in amount of ammonia-oxidizing bacteria which existed in the outer edge of the granular sludge during the start-up period. This microbial ecological change would make it possible to attain high rate ammonia removal.     

Partial nitrification of sludge reject water by means of aerobic granulation

Granular sludge formation was performed in a laboratory scale Sequencing Batch Reactor (SBR) fed with supernatant of anaerobic digestion of sewage sludge. This effluent was concentrated progressively in order to enhance biomass capacity without inhibiting it. During the first part of the study, ammonium nitrogen was converted to nitrate, so conventional nitrification took place. When a nitrogen load of 0.8 g N L(-1) d(-1) was treated, the effluent concentration of nitrite started to increase while the nitrate concentration decreased until it disappeared. So, partial nitrification was achieved. At the end of this study, a nitrogen load of 1.1 g N L(-1) d(-1) was treated obtaining an effluent with 50% ammonium and 50% nitrite. The volatile suspended solids concentration in the reactor reached 10 g VSS L(-1). At this point the granule morphology was quite round and no filamentous bacteria was observed. The Feret's diameter was in the range between 1 and 6 mm with an average value of 4.5 mm. Roundness value was all the time higher than 0.7. Granule density increased during the experimental period, obtaining a final value of 7.0 g L(-1).     

Model experiments on improving nitrogen removal in laboratory scale subsurface constructed wetlands by enhancing the anaerobic ammonia oxidation.

Anaerobic ammonia oxidation (Anammox) has been identified as a new general process-strategy for nitrogen removal in wastewater treatment. In order to evaluate the role and effects of the Anammox process in wetlands, laboratory-scale model experiments were performed with planted fixed bed reactors. A reactor (planted with Juncus effusus) was fed with synthetic wastewater containing 150-200 mg L(-1) NH4+ and 75-480 mg L(-1) NO2(-). Under these operating conditions, the plants were affected by the high ammonia and nitrite concentrations and the nitrogen removal rate fell within the same range of 45-49 mg N d(-1) (equivalent to 0.64-0.70 g Nm(-2)d(-1)) as already reported by other authors. In order to stimulate the rate of nitrogen conversion, the planted reactor was inoculated with Anammox biomass. As a result, the rate of nitrogen removal was increased 4-5-fold and the toxic effects on the plants also disappeared. The results show that, in principle, subsurface flow wetlands can also function as an "Anammox bioreactor". However, the design of a complete process for the treatment of waters with a high ammonia load and, in particular, the realisation of simple technical solutions for partial nitrification have still to be developed.     

Improved nitrogen removal in upflow anaerobic sludge blanket (UASB) reactors by incorporation of Anammox bacteria into the granular sludge.

Upflow anaerobic sludge blanket reactors may offer a number of advantages over conventional mixed-tank, SBR, and biofilm reactors, including high space-loading, low footprint, and resistance to shocks and toxins. In this study, we assessed the use of upflow anaerobic sludge blanket (UASB) reactor technology as applied to anaerobic ammonia removal, or Anammox. Four 200 ml UASB reactors were inoculated with 50% (by volume) anaerobic granular sludge and 50% flocular sludge from different sources (all with the potential for containing Anammox organisms). Tools used to assess the reactors included basic analyses, fluorescent in-situ hybridisation, and mathematical modelling, with statistical non-linear parameter estimation. Two of the reactors showed statistically identical Anammox activity (i.e., identical kinetic parameters), with good ammonia and nitrite removal (0.14 kgNHx m(-3) reactor day(-1), with 99% ammonia removal). The third reactor also demonstrated significant Anammox activity, but with poor identifiability of parameters. The fourth reactor had no statistical Anammox activity. Modelling indicated that poor identifiability and performance in the third and fourth reactors were related to an excess of reduced carbon, probably originating in the inoculum. Accumulation of Anammox organisms was confirmed both by a volume loading much lower than the growth rate, and response to a probe specific for organisms previously reported to mediate Anammox processes. Overall, the UASB reactors were effective as Anammox systems, and identifiability of the systems was good, and repeatable (even compared to a previous study in a rotating biological contactor). This indicates that operation, design, and analysis of Anammox UASB reactors specifically, and Anammox systems in general, are reliable and portable, and that UASB systems are an appropriate technology for this process.     

Development of single-stage nitrogen removal using anammox and partial nitritation (SNAP) and its treatment performances.

Single-stage Nitrogen removal using Anammox and Partial nitritation (SNAP) process was newly developed as an economical nitrogen removal process for ammonium rich wastewaters. The experimental studies for the evaluation of SNAP process were carried out using a novel biofilm reactor, in which hydrophilic net-type acryl fiber biomass carrier was applied. This SNAP reactor was operated under operational conditions of pH 7.5-7.7, 35 degrees C and DO 2-3 mg/L, and 60 to 80% of influent NH4-N was removed under loading rate of 0.48 kg-N/m3/d. Through the DNA analysis of the attached sludge, it was made clear that ammonium oxidizing bacteria (AOB) and anammox bacteria coexisted in the attach-immobilized sludge on the acryl fiber biomass carrier. Favorable conditions for the growth of anammox bacteria were created inside attach-immobilized nitrifying sludge. Two kinds of anammox bacteria and two kinds of AOB were detected in the SNAP sludge. Existence ratios of anammox and AOB were estimated to be 15% and 8.7%, respectively, based on the obtained clone numbers. This coexisting condition was confirmed by the FISH image of SNAP sludge and its confocal laser scanning microscope.