Anaerobic ammonium oxidation process in an upflow anaerobic sludge blanket reactor with granular sludge selected from an anaerobic digestor

The purpose of this work was to evaluate the development of the anammox process by the use of granular sludge selected from a digestion reactor as a potential seed source in a lab-scale UASB (upflow anaerobic sludge blanket) reactor system. The reactor was operated for approximately 11 months and was fed by synthetic wastewater. After 200 days of feeding with NH₄ ⁺ and NO₂ ⁻ as the main substrates, the biomass showed steady signs of ammonium consumption, resulting in over 60% of ammonium nitrogen removal. This report aims to present the results and to more closely examine what occurs after the onset of anammox activity, while the previous work described the start-up experiment and the presence of anammox bacteria in the enriched community using the fluorescencein situ hybridization (FISH) technique. By the last month of operation, the consumed NO₂ ⁻N/NH₄ ⁺-N ratio in the UASB reactor was close to 1.32, the stoichiometric ratio of the anammox reaction. The obtained results from the influentshutdown test suggested that nitrite concentration would be one key parameter that promotes the anammox reaction during the start-up enrichment of anammox bacteria from granular sludge. During the study period, the sludge color gradually changed from black to red-brownish.     

Development of a fixed-bed anammox reactor with high treatment potential

A plug-flow type anaerobic ammonium oxidation (anammox) reactor was developed using malt ceramics (MC) produced from carbonized spent grains as the biomass carriers for anammox sludge. Partial nitrified effluent of the filtrate from the sludge dehydrator of a brewery company was used as influent to a 20 L anammox reactor using MC. An average volumetric nitrogen removal rate (VNR) of 8.78 kg-N/m³/day was maintained stably for 76 days with 1 h of HRT. In a larger anammox reactor (400 L), an average VNR of 4.84 kg-N/m³/day could be maintained for 86 days during the treatment of low strength synthetic inorganic wastewater. As a result of bacterial community analysis for the 20 L anammox reactor, Asahi BRW1, probably originating from the wastewater collected at Asahi Breweries, was detected as the dominant anammox bacterium. These anammox reactors were characterized by a high NH₄-N removal capacity for low strength wastewater with a short hydraulic retention time.     

Importance of the operating pH in maintaining the stability of anoxic ammonium oxidation (anammox) activity in moving bed biofilm reactors

Two bench-scale parallel moving bed biofilm reactors (MBBR) were operated to assess pH-associated anammox activity changes during long term treatment of anaerobically digested sludge centrate pre-treated in a suspended growth partial nitrification reactor. The pH was maintained at 6.5 in reactor R1, while it was allowed to vary naturally between 7.5 and 8.1 in reactor R2. At high nitrogen loads reactor R2 had a 61% lower volumetric specific nitrogen removal rate than reactor R1. The low pH and the associated low free ammonia (FA) concentrations were found to be critical to stable anammox activity in the MBBR. Nitrite enhanced the nitrogen removal rate in the conditions of low pH, all the way up to the investigated level of 50 mg NO₂-N/L. At low FA levels nitrite concentrations up to 250 mg NO₂-N/L did not cause inactivation of anammox consortia over a 2-days exposure time.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO₂ as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K_m was determined to be 1.2 mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH₄-N). The results indicated that an inf.IC/inf.NH₄-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.     

Enrichment of Anammox from Activated Sludge and Its Application in the CANON Process

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH ₄ ⁺ (g biomass)⁻¹ h⁻¹ (0.58 kg total-N m⁻³ day⁻¹). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus ‘Brocadia anammoxidans’ strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m⁻³ day⁻¹) for an extended period of time.     

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation–anammox process

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m³/day under aerobic nitrogen loading rate of 3.8 kg-N/m³/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH₃/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m³/day under anaerobic nitrogen loading rate of 4.1 kg-N/m³/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation–anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.     

Biological treatment of ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium oxidation (anammox) in a pilot plant

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 would significantly reduce the nitrogen load of the activated sludge system. Some years ago, a novel biological process was discovered in which ammonium is converted to nitrogen gas under anoxic conditions with nitrite as the electron acceptor (anaerobic ammonium oxidation, anammox). Compared to conventional nitrification and denitrification, the aeration and carbon-source demand is reduced by over 50 and 100%, respectively. The combination of partial nitritation to produce nitrite in a first step and subsequent anaerobic ammonium oxidation in a second reactor was successfully tested on a pilot scale (3.6 m(3)) for over half a year. This report focuses on the feasibility of nitrogen removal from digester effluents from two different wastewater treatment plants (WWTPs) with the combined partial nitritation/anammox process. Nitritation was performed in a continuously stirred tank reactor (V=2.0 m(3)) without sludge retention. Some 58% of the ammonium in the supernatant was converted to nitrite. At 30 degrees C the maximum dilution rate D(x) was 0.85 d(-1), resulting in nitrite production of 0.35 kg NO(2)-N m(-3)(reactor) d(-1). The nitrate production was marginal. The anaerobic ammonium oxidation was carried out in a sequencing batch reactor (SBR, V=1.6 m(3)) with a nitrogen elimination rate of 2.4 kg N m(-3)(reactor) d(-1) during the nitrite-containing periods of the SBR cycle. Over 90% of the inlet nitrogen load to the anammox reactor was removed and the sludge production was negligible. The nitritation efficiency of the first reactor limited the overall maximum rate of nitrogen elimination.     

Effects of nitrite inhibition on anaerobic ammonium oxidation

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study the toxic effects of nitrite on these biochemical reactions. In this study, the effects of nitrite on anammox bacteria entrapped in gel carriers were investigated using batch and continuous feeding tests. The results showed that the nitrite concentration in a reactor must be less than 274-mg N/L in order to prevent a decrease in the anammox activity, which occurred when the gel carriers were soaked in nitrite solutions with concentrations greater than 274-mg N/L in a batch test. In a continuous feeding test, nitrite inhibition was not observed at low concentrations of nitrite. However, the anammox activity decreased to 10% when the nitrite concentration increased to 750-mg N/L over a 7-day period in the reactor. In addition, it was shown that the effects of nitrogen on the anammox reaction were reversible because the anammox activity completely recovered within 3 days when the influent nitrite concentration was decreased to less than 274-mg N/L.     

Adaptation of anaerobic ammonium-oxidising consortium to synthetic coke-ovens wastewater

A consortium with autotrophic anaerobic ammonium oxidising (AAAO) activity was developed from municipal sludge, and its ability to remove high ammonium concentrations in a toxic wastewater such as coke ovens wastewater is presented here. The enriched AAAO consortium was acclimatised to a synthetic coke ovens wastewater to establish anaerobic ammonium oxidation (AAO) activity. Phenol was the main carbon component of the synthetic wastewater whereby it was added stepwise from 50+/-10 to 550+/-10 mg l(-1) into an anammox enrichment medium. Ammonium-N removal was initially impaired; however, it gradually recovered. After 15 months of further selection and enrichment, the ammonium removal rate reached 62+/-2 mg NH(4)(+)-N l(-1) day(-1), i.e. 1.5 times the rate in the original AAAO reactor. The new consortium demonstrated higher ammonium and nitrite removal rates, even under phenol perturbation (up to 330+/-10 mg l(-1)). It is therefore concluded that the AAO activity in the consortium was resistant to high phenol and has potential for treating coke-ovens wastewater.     

Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge

The metabolic capability of denitrifying sludge to oxidize ammonium and p-cresol was evaluated in batch cultures. Ammonium oxidation was studied in presence of nitrite and/or p-cresol by 55 h. At 50 mg/L NH₄⁺-N and 76 mg/L NO₂⁻-N, the substrates were consumed at 100% and 95%, respectively, being N₂ the product. At 50 mg/L NH₄⁺-N and 133 mg/L NO₂⁻-N, the consumption efficiencies decreased to 96% and 70%, respectively. The increase in nitrite concentration affected the ammonium oxidation rate. Nonetheless, the N₂ production rate did not change. In organotrophic denitrification, the p-cresol oxidation rate was slower than ammonium oxidation. In litho-organotrophic cultures, the p-cresol and ammonium oxidation rates were affected at 133 mg/L NO₂⁻-N. Nonetheless, at 76 mg/L NO₂⁻-N the denitrifying sludge oxidized ammonium and p-cresol, but at different rate. Finally, this is the first work reporting the simultaneous oxidation of ammonium and p-cresol with the production of N₂ from denitrifying sludge.     

Global variations and controlling factors of anammox rates

Soil anammox is an environmentally friendly way to eliminate reactive nitrogen (N) without generating nitrous oxide. Nevertheless, the current earth system models have not incorporated the anammox due to the lack of parameters in anammox rates on a global scale, limiting the accurate projection for N cycling. A global synthesis with 1212 observations from 89 peer-reviewed papers showed that the average anammox rate was 1.60 ± 0.17 nmol N g⁻¹ h⁻¹ in terrestrial ecosystems, with significant variations across different ecosystems. Wetlands exhibited the highest rate (2.17 ± 0.31 nmol N g⁻¹ h⁻¹), followed by croplands at 1.02 ± 0.09 nmol N g⁻¹ h⁻¹. The lowest anammox rates were observed in forests and grasslands. The anammox rates were positively correlated with the mean annual temperature, mean annual precipitation, soil moisture, organic carbon (C), total N, as well as nitrite and ammonium concentrations, but negatively with the soil C:N ratio. Structural equation models revealed that the geographical variations in anammox rates were primarily influenced by the N contents (such as nitrite and ammonium) and abundance of anammox bacteria, which collectively accounted for 42% of the observed variance. Furthermore, the abundance of anammox bacteria was well simulated by the mean annual precipitation, soil moisture, and ammonium concentrations, and 51% variance of the anammox bacteria was accounted for. The key controlling factors for soil anammox rates differed from ecosystem type, for example, organic C, total N, and ammonium contents in croplands, versus soil C:N ratio and nitrite concentrations in wetlands. The controlling factors in soil anammox rate identified by this study are useful to construct an accurate anammox module for N cycling in earth system models.     

Anammox反应器启动过程中颗粒污泥性状变化特性

以厌氧颗粒污泥作为接种物,通过185 d的运行,成功启动了上流式厌氧氨氧化污泥床(Upflow anaerobic sludge blanket,UASB)反应器。反应器的进水氨氮与亚硝氮浓度分别提升至224 mg/L和255 mg/L,容积氮去除速率提升至3.76 kg/(m3·d)。采用红外光谱、扫描电镜和透射电镜等对厌氧氨氧化颗粒污泥的性状进行观察,发现颗粒污泥在启动过程中经历了污泥颗粒裂解到污泥颗粒重组的过程,且厌氧氨氧化颗粒污泥表面含有丰富的官能团,说明厌氧氨氧化颗粒污泥可能具有良好的吸附性能。采用宏基因组测序的方法对启动前后颗粒污泥的生态结构进行分析,发现原接种污泥优势菌群(变形菌门、厚壁菌门、拟杆菌门)丰度大幅减少,厌氧氨氧化菌所属的浮霉状菌门丰度则由1.59%提升到23.24%。     

Simultaneous energy recovery and autotrophic nitrogen removal from sewage at moderately low temperatures

This study assessed the technical feasibility of treating sewage with a combination of direct anaerobic treatment and autotrophic nitrogen removal, while simultaneously achieving energy recovery and nitrogen removal under moderately low temperatures. The concentrations of ammonia, nitrite, and COD in effluent were below 1, 0.1, and 30 mg/L, respectively. In the up-flow, anaerobic sludge fixed-bed, there was no obvious change observed in the total methane production at temperatures of 35?±?1 °C, 28?±?1 °C, 24?±?3 °C, and 17?±?3 °C, with the accumulation of volatile fatty acids occurring with decreasing temperatures. The control strategy employed in this study achieved a stable effluent with equimolar concentrations of nitrite and ammonium, coupled with high nitrite accumulation (>97 %) in the partial nitrification sequencing batch reactor system at moderately low temperatures. In the anaerobic ammonium oxidation (anammox) reactor, a short hydraulic retention time of 0.96 h, with a nitrogen removal rate of 0.83 kgN/(m³/day) was achieved at 12–15 °C. At low temperatures, the corresponding fluorescence in situ hybridization image revealed a high amount of anammox bacteria. This study demonstrates that efficient nitrogen removal and energy recovery from sewage at moderately low temperatures can be achieved by utilizing a combined system. Additionally, this system has the potential to become energy-neutral or even energy-producing.     

Detection of Anaerobic Ammonium-Oxidizing Bacteria in Ago Bay Sediments

We identified 16S rRNA gene sequences in sediment samples from Ago Bay in Japan, forming a new branch of the anammox group or closely related to anaerobic ammonium oxidizing (anammox) bacterial sequences. Anammox activity in the sediment samples was detected by ¹⁵N tracer assays. These results, along with the results of fluorescence in situ hybridization (FISH) analysis, suggest the presence of anammox bacteria in the marine sediments.     

Microbial and Physicochemical Characteristics of Compact Anaerobic Ammonium-Oxidizing Granules in an Upflow Anaerobic Sludge Blanket Reactor

Anaerobic ammonium oxidation (anammox) is a promising new process to treat high-strength nitrogenous wastewater. Due to the low growth rate of anaerobic ammonium-oxidizing bacteria, efficient biomass retention is essential for reactor operation. Therefore, we studied the settling ability and community composition of the anaerobic ammonium-oxidizing granules, which were cultivated in an upflow anaerobic sludge blanket (UASB) reactor seeded with aerobic granules. With this seed, the start-up period was less than 160 days at a NH₄⁺-N removal efficiency of 94% and a loading rate of 0.064 kg N per kg volatile suspended solids per day. The formed granules were bright red and had a high settling velocity (41 to 79 m h⁻¹). Cells and extracellular polymeric substances were evenly distributed over the anaerobic ammonium-oxidizing granules. The high percentage of anaerobic ammonium-oxidizing bacteria in the granules could be visualized by fluorescent in situ hybridization and electron microscopy. The copy numbers of 16S rRNA genes of anaerobic ammonium-oxidizing bacteria in the granules were determined to be 4.6 × 10⁸ copies ml⁻¹. The results of this study could be used for a better design, shorter start-up time, and more stable operation of anammox systems for the treatment of nitrogen-rich wastewaters.The anaerobic ammonia oxidation (anammox) process is a recently discovered biological nitrogen removal technology in which ammonia is oxidized to nitrogen gas with nitrite as the electron acceptor (5, 29, 32). In contrast to heterotrophic denitrification (6, 26), the anammox process does not require external electron donors (e.g., methanol) due to their chemolithoautotrophic lifestyle. Furthermore, if this process is combined with a partial nitrification step, only half of the ammonium needs to be nitrified to nitrite, which together with the remaining ammonium can subsequently be converted into nitrogen through the anammox process. This reduces the oxygen demand of the system and leads to further reduction in operational costs (27).The anaerobic ammonium-oxidizing bacteria (anammox bacteria) have a low growth rate (18), with a doubling time at best estimated as 7 to 11 days (18, 28). The yield of the anammox bacteria has been determined to be 0.066 mol C biomass mol⁻¹ ammonium consumed, and the maximum ammonium consumption rate is ∼45 nmol mg⁻¹ protein min⁻¹ (18). Given the low growth rate and low yield, very efficient biomass retention is essential to retain the anammox bacteria within the reactor systems during cultivation (19). The enrichment of anammox bacteria from a mixed inoculum requires the optimization of conditions favorable for the anammox bacteria and generally takes 200 to 300 days (5, 6, 27). Thus, conditions that would reduce the start-up time of anammox reactors would positively effect the implementation of the process. Several sources of inocula, such as activated sludge (4), nitrifying activated sludge (27), and anaerobic sludge (6), have been used for the start-up of anammox reactors with start-up times of as long as 1,000 days (27).Aerobic granules have been reported to have high microbial diversity (31) and compact structure with very good settling properties resulting in an efficient means of biomass retention. These properties, including interspecies competition and mass transfer, result in the stratification of microbial species with anoxic pockets in the interior of the granules that may be suitable to harbor anammox bacteria. Therefore, the main objective of this study was to investigate the feasibility of start-up of the anammox process by seeding the reactor with aerobic granular sludge by using an upflow anaerobic sludge blanket (UASB) reactor. After the successful start-up and the formation of anammox granules, the structure and physicochemical properties of the anammox granules and the reactor performance were characterized. Microbial community analysis revealed that the dominant anammox species was related to a species of anammox bacteria present in anammox biofilms.     

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10⁸ copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

     

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N₂ gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.     

厌氧氨氧化细菌的研究进展

厌氧氨氧化是指微生物在无氧条件下,以NO_2~–为电子受体,将NH_4~+氧化成N_2的过程,该过程主要由浮霉菌门下的厌氧氨氧化细菌参与。厌氧氨氧化细菌广泛存在于海洋生态系统、淡水生态系统、陆地生态系统及其他一些特殊生境中,其在废水生物脱氮和地球氮循环中扮演着重要角色。本文从厌氧氨氧化细菌的发现历程、种类、特性、代谢途径、分布、检测方法及应用上进行了较为全面的总结;最后对厌氧氨氧化细菌研究前沿问题和未来发展方向进行了探讨与展望。     

Growth characteristic of anaerobic ammonium-oxidizing bacteria in an anaerobic biological filtrated reactor

The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1×10⁶ and 1.7×10⁷ cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (μ) was 0.39 day⁻¹. This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.