One-stage Partial Nitritation Research Articles

Activation of iron oxides through organic matter-induced dissolved oxygen penetration depth dynamics enhances iron-cycling driven ammonium oxidation in microaerobic granular sludge

The iron redox cycle can enhance anammox in treating low-strength ammonia wastewater. However, maintaining an effective iron redox cycle and suppressing nitrite-oxidizing bacteria in a one-stage partial nitritation and anammox (PN/A) process poses challenges during long-term aeration. We proposed a novel and simple strategy to achieve an efficient iron redox cycle in an iron-mediated anoxic-microaerobic (A/O) process by controlling organic matter (OM) at medium-strength levels (30–110 mg COD/L) in microaerobic granular sludge (MGS)-dominated reactor. The developed A/O process consistently achieved >90 % OM removal and >75 % nitrogen removal. Medium-strength OM varied the penetration depths of dissolved oxygen (DO) in MGS, regulating redox conditions and promoting redox reactions across MGS layers, thus activating accumulated inert iron oxides. Ammonia-oxidizing bacteria (Nitrosomonas), iron-reducing bacteria (e.g., Ignavibacterium, Geobacter), and anammox bacteria (Ca. Kuenenia) coexisted harmoniously in MGS. This coexistence ensured high anammox and Feammox rates along with a robust iron redox cycle, thereby mitigating the adverse impacts of fluctuating DO and OM on one-stage PN/A process stability. The identification of iron reduction-associated genes within Ca. Kuenenia, Ignavibacterium, and Geobacter suggests their potential roles in supporting Feammox coupled in one-stage PN/A process. This study introduces an iron-cycle-driven A/O process as an energy-efficient alternative for simultaneous carbon and nitrogen removal from low-strength wastewater.

Decrease in Oxygen Concentration for the Fast Start-Up of Partial Nitritation/Anammox without Inoculum Addition

Initiating the partial nitritation and anammox (PN/A) process without inoculation poses a significant challenge. Thus, there is a notable amount of interest in devising a straightforward strategy for the start-up of PN/A. This study demonstrates the feasibility of achieving the rapid start-up of a one-stage PN/A process within a moving-bed sequencing batch biofilm reactor (MBSBBR) by reducing the oxygen concentrations: 3.0 mg O2/L (Stage I), 2.0 mg O2/L (Stage II), and 1.0 mg O2/L (Stage III). The anammox activity was observed 15 days after a gradual decrease in the oxygen concentration and confirmed using a specific anammox activity test (5.9 mg N/gVSS∙h). During Stage III, the average total inorganic nitrogen (TIN) removal efficiency was 60.6%. The relative abundance of planctomycetes, a typical phylum representing anammox microorganisms, increased almost three times from Stage I (2.8%) to Stage III (7.1%). These results demonstrate the potential of a decrease in oxygen concentration for the fast start-up of the one-stage partial nitritation and anammox process without inoculation. The implementation of the studied DO strategy has practical implications for wastewater treatment plant operators, particularly in the start-up of the PN/A processes. Additionally, batch assays allow for the rapid assessment of treatment plant performance, providing real-time insights into its functionality and, thereby, optimizing wastewater treatment practices.

Full-scale partial nitritation and anammox (PN/A) application in removing nitrogen from industrial wastewater: Performance and life cycle assessment

Anaerobic ammonium oxidation (anammox) is an innovative nitrogen removal process that is widely used in wastewater treatment due to its energy-saving potential and ability to reduce carbon emissions. However, research on the application of anammox in full-scale industrial wastewater treatment plants (WWTPs) is limited, and its environmental impacts are yet to be fully understood. This study evaluates the performance and environmental burdens of one-stage partial nitritation and anammox (PN/A) in a full-scale WWTP treating coal chemical wastewater, with different demonstration periods. Compared with conventional nitrification–denitrification, the energy footprint and carbon footprint of the PN/A process were significantly lower and effluent quality was improved. Based on the four-quadrant clustering method, the overall performance of the studied WWTP achieved the greatest synergy during the stable operation period. Life cycle assessment has shown that the PN/A process significantly reduces the system’s contribution to environmental indicators. According to the sensitivity analysis, the fluctuations in energy consumption and chemical usage have significant environmental impacts, with differential sensitivity in the environmental impacts of two demonstration periods. Therefore, considering the specific situation of the WWTPs, optimizing the parameters/indicators most sensitive to environmental impact is essential to reducing environmental loads. Importantly, this work demonstrated that PN/A can improve the operational performance of full-scale industrial WWTPs with lower environmental burdens, providing valuable guidance for the development of sustainable wastewater treatment processes.

Ecological insight into deterioration of one-stage partial nitritation and anammox system during environmental disturbance

This study investigated the relationship between the performance and the ecological features of a one-stage partial nitritation and anammox disturbed by oxygen. The disturbance caused an irreversible deterioration of the nitrogen removal rate from 0.8 to 0.05 kg N/(m3∙d) although the anammox genera increased from 1% to 1.4%. Meanwhile, the richness and evenness reduced from 455 and 4.00 to 429 and 3.81, respectively, following a similar pattern to the community complexity. The community drifted and formed three distinct clusters during and after the disturbance. Furthermore, 234 of 634 operational taxonomic units in the community were depleted despite recovered diversity and complexity during long-term stable operation. In conclusion, the ecological fluctuation of the microbial community with decreasing resilience was the driving force that fatally collapsed the system performance. This study suggests that ecological features are conducive to the diagnosis, prediction, and optimization of a partial nitritation and anammox system.

Long-term effect of zero-valent iron on one-stage partial nitritation and anammox

Partial nitritation and anammox (PN/A) is an energy- and carbon-saving process for nitrogen removal from organic deficient wastewater. However, the PN/A effluent often requires polishing to eliminate the nitrate produced by anammox. Iron as the essential element for living organisms has been tested for nitrate reduction and phosphorus recovery in wastewater treatment processes. Here, we proposed a novel process coupling PN/A with iron-based denitrification for efficiently removing nitrogen and phosphorus in a single reactor. Two laboratory-scale bioreactors were operated under oxygen-limited conditions in parallel. Fe0 with a dosage of ∼ 27 mg Fe/L-wastewater was continuously dosed in the experimental reactor, with the other serving as a control. Throughout 300 days of operation, the results showed that experimental system always had significantly high removal efficiencies of organic carbon, nitrogen and phosphorus compared with control system. The total nitrogen removal achieved 90.1 ± 1.2 % at the rate of 0.96 ± 0.03 kg N/(m3•d) due to the improved activity of ammonia oxidizers, anammox bacteria and denitrifiers and enriched nitrogen conversion pathway (i.e., iron-based autotrophic denitrification) with Fe0 supplement. The total phosphorus removal was synchronously improved to 80.2 ± 9.1 % mainly driven by the phosphate precipitation with iron. This study provides new insights into achieving high-level nitrogen and phosphorus removal in a single reactor, and deepens our understanding of the improved nutrients removal mechanism mediated by iron.

Effective restoration of partial nitritation and anammox biofilm process by short-term hydroxylamine dosing: Mechanism and microbial interaction

The one-stage partial nitritation and anammox (PN-A) process frequently experiences deterioration from ammonium accumulation and nitrate build-up. In this study, hydroxylamine was dosed to restore the process from deterioration in a continuously aerated PN-A sequencing biofilm batch reactor, and the impact of hydroxylamine on the metabolism of PN-A process was studied. PN-A process was totally restored in 5 days via 10 mg N·L-1 hydroxylamine dosing, reducing nitrate-produced/ammonium-removed ratio from 28.5% to less than 11.0%. hydroxylamine dosing promoted biological production of nitric oxide and nitrous oxide and reduced the production of nitrate in the PN-A process. This study advanced the understanding of the metabolism versatility of hydroxylamine and nitric oxide as well as their function in interaction between aerobic ammonium oxidation bacteria and anaerobic ammonium oxidation bacteria, and proposed the potential application of hydroxylamine dosing in ammonium-contained wastewater treatment.

High nitrogen removal performance of anaerobically treated fish processing wastewater by one-stage partial nitritation and anammox process with hydroxyapatite (HAP)-based syntrophic granules and granule structure

The one-stage partial nitritation and anammox process with the hydroxyapatite (HAP)-based syntrophic granules was studied for the ammonium nitrogen removal from the effluents of a self-agitated anaerobic baffled reactor treating the fish processing wastewater. When the ammonium in the influent was 1140 mg N·L-1, a high nitrogen removal rate and nitrogen removal efficiency of 1.51 ± 0.10 kg N·m−3·d-1 and 88.2% were obtained, respectively. Anammox bacteria of Candidatus Kuenenia stuttgartiensis and ammonium oxidizing bacteria of Nitrosomonas were the two most predominant bacteria, while nitrite oxidizing bacteria activity was low and could be neglected during the treatment. The inorganic element properties of the sludge were analyzed by several methods to confirm the existence of HAP granules. Optical microscopic observation and scanning electron microscopy analysis revealed the structure of the granular sludge.This study supports the feasibility and potential of this process for high-efficiency nitrogen removal from fish processing wastewater.

Hydroxyapatite Formation in a Single-Stage Anammox-Based Batch Treatment System: Reactor Performance, Phosphorus Recovery, and Microbial Community

Simultaneous ammonium–nitrogen (NH₄⁺–N) removal and orthophosphate–phosphorus (PO₄–P) biomineralization was studied in two granular sludge sequencing batch reactors treating urban sidestream centrate through the one-stage partial nitritation–anaerobic ammonium oxidation (anammox) process. By adding an external source of calcium, concomitant conversion rates of up to 0.32 g NH₄⁺–N/(L·days) and 12 mg PO₄–P/(L·days) were measured while reaching water-phase removal efficiencies of ca. 80% of the NH₄⁺–N and 70% of the PO₄–P loaded. The mineral cores formed inside the granules were mostly composed of hydroxyapatite (HAP), which is a recoverable phosphate salt. Yet, the high mineralization of the sludge and its excessive purge from the bioreactor limited the microbial activity in the long term. In this sense, the densest granules, accumulated at the bottom of the bioreactor, were the most susceptible to being harvested because they were the richest in HAP, but at the same time, they contained the highest percentages of anammox bacteria.

Key factors governing the performance and microbial community of one-stage partial nitritation and anammox system with bio-carriers and airlift circulation

A one-stage airlift internal circulation biofilm reactor was continuously operated for 668 days to treat 50 mg/L of ammonia wastewater to pursue the long-term stability of partial nitritation and anammox (PNA) process. The operational performance and microbial community structure of the biofilm and the flocs were investigated. A nitrogen removal efficiency (NRE) of 70% was obtained successfully at a dissolved oxygen (DO) of 0.05–0.15 mg/L by regulating aeration rate. The microbial analysis indicated Candidatus Brocadia (29.5%) and Nitrosomonas (6.8%) were dominant in both biofilms and flocs. It was found that DO control and aeration rate were the key factors in performance stability, and a stable performance could be recovered and maintained under oxygen-limiting conditions. Further, the achievement of activated ammonia oxidation bacteria (AOB), dominated anammox bacteria (AMX), suppressed NOB, and controlled heterotrophic bacteria (HB) in the biofilms played a major role in the long-term stable operation.

Evaluating the effects of micro-zones of granular sludge on one-stage partial nitritation-anammox nitrogen removal.

The one-stage partial nitritation-anammox (PN-A) process is considered an efficient process for low-cost nitrogen removal. In this study, the nitrogen removal performance of different-sized granules in a one-stage PN-A reactor was studied. The total autotrophic nitrogen removal rate (TANRR) of the granular sludge increased as the granule size increased, and the TANRR of granular sludge with a radius larger than 500μm reached 0.14 kgNkgVSS-1d-1. High-throughput sequencing revealed that the abundance of ammonium-oxidizing bacteria and anaerobic ammonium-oxidizing (anammox) bacteria in granular sludge of different sizes differed, indicating that the bacterial community structure was affected by the granule size. The TANRR of different-sized granules was affected by the volumes of aerobic micro-zone and anaerobic micro-zone inside the granule. Appropriate micro-zone volumes inside the granules could be regulated by the dissolved oxygen (DO) concentration of the reactor, which are favourable for achieving a balance between partial nitritation and anammox and then satisfactory nitrogen removal. Small-volume variations in the range of micro-zones have a significant influence on the balance between partial nitritation and anammox. The proper DO concentration required for different-sized granules to achieve better nitrogen removal differed. This study provides a novel perspective for understanding the effect of micro-zones of granular sludge on one-stage PN-A nitrogen removal.

Roles of hydroxylamine and hydrazine in the in-situ recovery of one-stage partial nitritation-anammox process: Characteristics and mechanisms

Nitrate built-up is a serious operational difficulty in one-stage partial nitritation anammox (PN/A) process. To investigate an effective method for in-situ restoration, hydroxylamine (NH2OH) and hydrazine (N2H4) of 2 mgN/L were dosed in PN/A process with nitrate built-up in a comparative study. NH2OH treatment showed better performances on TN removal and nitrate reduction than N2H4 and blank control. Through 104 days' addition of NH2OH, MRNN (mole ratio of NO3−-N production to NH4+-N removal) was decreased from 70% to 19.91%; TN removal was increased from 0.01 to 0.18 kgN/(m3 d). After stopping the chemical addition, nitrate rebounded for N2H4 treatment, but the restoration effect was stable and persistent for NH2OH. NH2OH addition resulted in a low reductive potential (−250 mV) and exerted strong inhibitions on nitrite oxidizing bacteria activities. Additionally, rapid enhancement of ammonia oxidizing bacteria activities, functional gene (hao) and Nitrosomonas gave rise to the restoration of PN/A with NH2OH addition.

Successful startup of one-stage partial nitritation and anammox system through cascade oxygen supply and potential ecological network analysis

It is challenge for the stable operation of the anaerobic ammonium oxidation (Anammox) based wastewater treatment processes. The feasibility for the startup of the partial nitritation and Anammox (PNA) process was demonstrated with cascade oxygen supply by controlling anoxic/aerobic durations. Under steady state conditions, total nitrogen removal percentages of 63.2% (pulse feeding) and 88.0% (constant feeding) were obtained with the anoxic/aerobic duration of 5 min/7 min at 26 °C, and PNA was successfully achieved. The microbial community analysis revealed that Candidatus Kuenenia and Nitrosomonas were dominant genera of Anammox bacteria and ammonia oxidizing bacteria, respectively. Microbial interactions were examined through acyl-homoserine lactones-based quorum sensing (AHLs-QS) and metagenomics analyses. N-octanoyl-homoserine lactone, N-decanoyl homoserine lactone and N-dodecanoyl homoserine lactone had obvious relationships with the abundance of the Anammox bacteria. The AHLs-QS system could control microbial interactions among Anammox bacteria, nitrifiers and heterotrophs, especially for their balances in the PNA system.

In-situ restoration of one-stage partial nitritation-anammox process deteriorated by nitrate build-up via elevated substrate levels.

The one-stage partial nitritation and anammox process (PN/A) has been a promising microbial process to remove ammonia from wastewater especially with low carbon/nitrogen ratio. The main breakdown was the deterioration caused by overgrowth of nitrite oxidizing bacteria (NOB) resulting effluent nitrate build-up in the PN/A process. This study presented an in-situ restoring strategy for suppressing NOB activity in a one-stage granular PN/A system deteriorated over 2 months, using elevated concentrations of substrates (ammonia and nitrite) under limited dissolved oxygen level. The results showed that the NOB activity was successfully suppressed after 56 days of restoration, and finally the ratio of produced nitrate/consumed ammonium was reduced from 36.8% to 7%. On day 66 the nitrogen removal rate obtained as 1.2 kg N/(m3·d). The high FA level (5–40 mg/L) and low dissolved oxygen (<0.13 mg/L) were responsible for NOB suppression. From quantitative PCR (qPCR) analysis, after this restoration, anammox bacteria had a widely growth, and AOB stay stable, but Nitrospira increase and Nitrobacter declined. High amount of NOB was still persistent in the granules, which was not easy to wash-out and threaten the deammonification performance.

Applicability of one-stage partial nitritation and anammox in MBBR for anaerobically pre-treated municipal wastewater.

Energy consumption of municipal wastewater treatment plants can be reduced by the anaerobic pre-treatment of the main wastewater stream. After this pre-treatment, nitrogen can potentially be removed by partial nitritation and anammox (PN/A). Currently, the application of PN/A is limited to nitrogen-rich streams (>500mgL(-1)) and temperatures 25-35°C. But, anaerobically pretreated municipal wastewater is characterized by much lower nitrogen concentrations (20-100mgL(-1)) and lower temperatures (10-25°C). We operated PN/A under similar conditions: total ammonium nitrogen concentration 50mgL(-1) and lab temperature (22°C). PN/A was operated for 342days in a 4L moving bed biofilm reactor (MBBR). At 0.4mgO2L(-1), nitrogen removal rate 33gNm(-3)day(-1) and 80% total nitrogen removal efficiency was achieved. The capacity of the reactor was limited by low AOB activity. We observed significant anammox activity (40gNm(-3)day(-1)) even at 12°C, improving the applicability of PN/A for municipal wastewater treatment.

Fast start-up of partial nitritation as pre-treatment for anammox in membrane bioreactor

Anaerobic ammonium oxidation (anammox) is an innovative pathway for biological nitrogen removal. The raw nitrogenous wastewater is ill-suited for direct depletion by anammox due to lack of sufficient nitrite. Partial nitritation (PN) was operated in a membrane bioreactor (MBR) to generate suitable feed for anammox, and functional microbes were analysed by 16S rRNA sequence analysis. The dissolved oxygen was adopted as the key selection factor to realize partial nitritation. The PN was realized in the MBR within 19 days. From day 26 and onwards, partial nitritation was stable and operated with an effluent NO2－-N/NH4＋-N ratio of 1.15±0.09, which was a suitable mixture to feed subsequent anammox. The free ammonia overwhelmingly restrained the activity of nitrite oxidizers. High nitrite accumulation efficiency of 94.6±3.1% was achieved throughout the experiment. The optimal dissolved oxygen of the bioreactor to create suitable media was 0.8–0.9mgL−1. The molecular cloning and phylogenetic analysis revealed that Nitrosospira sp. was the predominant ammonium-oxidizing bacteria corresponding to implement nitritation in the MBR. Sequences with original name of Nitrosospira sp. YKU were deposited in GenBank. The MBR promised potential merit to implement one-stage partial nitritation and anammox.

Pilot-scale evaluation of anammox-based mainstream nitrogen removal from municipal wastewater

Autotrophic nitrogen removal in the mainstream wastewater treatment process is suggested to be a prerequisite of energy autarkic wastewater treatment plants (WWTP). Whilst the application of anammox-related technologies in the side-stream is at present state of the art, the feasibility of this energy-efficient process at mainstream conditions is still under development. Lower operating temperature and ammonium concentration, together with required high nitrogen removal efficiency, represent the main challenges to face in order to reach this appealing new frontier of the wastewater treatment field. In this study, we report the evaluation of the process in a plug-flow granular sludge-based pilot-scale reactor (4 m3) continuously fed with the actual effluent of the A-stage of the WWTP of Dokhaven, Rotterdam. The one-stage partial nitritation–anammox system was operated for more than 10 months at 19 ± 1°C. Observed average N-removal and ammonium conversion rates were comparable or higher than those of conventional N-removal systems, with 182 ± 46 and 315 ± 33 mg-N L−1 d−1, respectively. Biochemical oxygen demand was also oxidized in the system with an average removal efficiency of 90%. Heterotrophic biomass grew preferentially in flocs and was efficiently washed out of the system. Throughout the experimentation, the main bottleneck was the nitritation process that resulted in nitrite-limiting conditions for the anammox conversion. Anammox bacteria were able to grow under mainstream WWTP conditions and new granules were formed and efficiently retained in the system.

N2O and NO emissions during autotrophic nitrogen removal in a granular sludge reactor – a simulation study

This contribution deals with NO and N2O emissions during autotrophic nitrogen removal in a granular sludge reactor. Two possible model scenarios describing this emission by ammonium- oxidizing biomass have been compared in a simulation study of a granular sludge reactor for one-stage partial nitritation–Anammox. No significant difference between these two scenarios was noticed. The influence of the bulk oxygen concentration, granule size, reactor temperature and ammonium load on the NO and N2O emissions has been assessed. The simulation results indicate that emission maxima of NO and N2O coincide with the region for optimal Anammox conversion. Also, most of the NO and N2O are present in the off-gas, owing to the limited solubility of both gases. The size of granules needs to be large enough not to limit optimal Anammox activity, but not too large as this implies an elevated production of N2O. Temperature has a significant influence on N2O emission, as a higher temperature results in a better N-removal efficiency and a lowered N2O production. Statistical analysis of the results showed that there is a strong correlation between nitrite accumulation and N2O production. Further, three regions of operation can be distinguished: a region with high N2O, NO and nitrite concentration; a region with high N2 concentrations and, as such, high removal percentages; and a region with high oxygen and nitrate concentrations. There is some overlap between the first two regions, which is in line with the fact that maximum emission of NO and N2O coincides with the region for optimal Anammox conversion.

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C(12)-HSL), was identified via LC-MS/MS. Furthermore, C(12)-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C(12)-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C(12)-HSL in AnAOB-based applications.

Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.

Nitrogen Removal from Digested Black Water by One-Stage Partial Nitritation and Anammox

This study assessed the technical feasibility to treat digested black water from vacuum toilets (> 1000 mg NH4(+)-N L(-1)) in a lab-scale oxygen-limited autotrophic nitrification/denitrification (OLAND) rotating biological contactor. After an adaptation period of 2.5 months, a stable nitrogen removal rate of ca. 700 mg N L(-1) d(-1) was reached over the subsequent 5 months. Suppression of the nitrite oxidizing bacteria at free ammonia levels above 3 mg N L(-1) resulted in a nitrogen removal efficiency of 76%. The favorable ratios of both organic and inorganic carbon to nitrogen guaranteed endured anammox activity and sufficient buffering capacity, respectively. Quantitative FISH showed that aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB) made up 43 and 8% of the biofilm, respectively. Since a part of the AerAOB was probably present in anoxic biofilm zones; their specific ammonium conversion was very low, in contrast to the high specific AnAOB activity. DGGE analysis showed that the dominant AerAOB and AnAOB species were resistant to the transition from synthetic medium to digested black water. This study demonstrates high-rate nitrogen removal from digested black water by one-stage partial nitritation and anammox, which will allow a significant decrease in operational costs compared to conventional nitrification/ denitrification.