HRT and nutrients affect bacterial communities grown on recirculation aquaculture system effluents

In a recirculation aquaculture system the drumfilter effluent can be used as substrate for heterotrophic bacterial production, which can be recycled as feed. Because the bacteria might contain pathogens, which could reduce its suitability as feed, it is important to characterize these communities. Bacteria were produced in growth reactors under different conditions: 7 h hydraulic retention time (HRT) vs. 2 h, sodium acetate vs. molasses, and ammonia vs. nitrate. The community of the drumfilter effluent was different from those found in the reactors. However, all major community components were present in the effluent and reactor broths. HRT influenced the bacteria community, resulting in a DGGE profile dominated by a band corresponding to an Acinetobacter sp.-related population at 2 h HRT compared to 7 h HRT, where bands indicative of alpha-proteobacterial populations most closely related to Rhizobium and Shinella spp. were most abundant. Molasses influenced the bacterial community. It was dominated by an Aquaspirillum serpens-related population. Providing total ammonia nitrogen (TAN) in addition to nitrate led to the occurrence of bacteria close to Sphaerotilus spp., Flavobacterium mizutaii and Jonesia spp. It was concluded from these results that a 6-7 h HRT is recommended, and that the type of substrate is less important, and results in communities with a comparably low pathogenic risk.     

Ammonia removal from prawn aquaculture water using immobilized nitrifying bacteria

Intensive prawn aquaculture in tropical regions is associated with high concentrations of total ammoniacal nitrogen (TAN) as a result of high rates of prawn excretion and feed loading. Excessive TAN can adversely effect productivity and result in adverse impacts on coastal waters. Cultures of indigenous nitrifying bacteria were enriched from intensive prawn aquaculture pond water using continuous and batch enrichment techniques. Cultures were capable of TAN removal over a wide range of initial TAN concentrations - up to 200 mg/l. Cultures were immobilized onto porous clay pellets to enhance cell density and applied to culture medium and TAN-augmented pond water under aerobic conditions to determine TAN removal proficiency. Immobilized cultures were able to achieve a high TAN removal proficiency in pond water--even at a low density of 0.1 pellet per liter. A concentration of less than 0.5 mg TAN/l could be maintained under a fed-batch condition of 3.2 mg TAN/l per day, after an initial 2-day lag phase. A simplified and effective culture enrichment process was developed for culture immobilization onto pellets using TAN-augmented pond water. Overall, pellet immobilization of indigenous nitrifying bacteria represents a potentially effective TAN control system for prawn aquaculture in low-cost, but intensive tropical prawn farms.     

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.     

Effect of hydraulic residence time on microbial sulfide production in an upflow sludge blanket denitrification reactor fed with methanol

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water anion exchange resins are regenerated in a closed circuit by way of an upflow sludge blanket denitrification reactor. The regenerant (a concentrated sodium bicarbonate solution) is recirculated through the ion exchanger in the r generation mode and the denitrification reactor. In the closed system sulfate accumulates to very high concentrations. For that reason it was examined under what process conditions sulfate reduction occurs in an upflow sludge blanket denitrification reactor, when the influent contains high sulfate concentrations (5.45 g SO ₄ ^2- /l) and high sodium bicarbonate concentrations (19.8 g NaHCO₃/l) in addition to nitrate and methanol. It appeared that at a hydraulic residence time of 5 h sulfide production started, when the nitrate loading rate was 20% of the denitrification reactor capacity and methanol was added in excess. The excess of methanol was converted into acetate after nitrate was depleted. Conversion of methanol into acetate was a function of the hydraulic residence time. At hydraulic residence times above 8 h this conversion was complete. Also in batch experiments it was observed that excess of methanol was converted into acetate, and that sulfate reduction started when nitrate was depleted. From all experiments it is clear that, provided that methanol is added in good relation to the quantity of nitrate that has to be denitrified, acetate will not be produced and sulfate reduction will not occur in the denitrification reactor, even in the presence of very high sulfate concentrations.     

Hydrogenotrophic denitrification of synthetic aquaculture wastewater using membrane bioreactor

A hydrogenotrophic denitrification system was evaluated in removing nitrate from synthetic aquaculture wastewater for recirculation purposes. Two membrane bioreactor (MBR) systems, namely, aeration–denitrification system (ADS) and denitrification–aeration system (DAS) were studied with 50 mg/L of influent concentrations for both organic matter and nitrate nitrogen. The DAS achieved better removal efficiency of 91.4% total nitrogen (T-N) and denitrification rate of 363.7 mg/L.day at a HRT of 3 h compared to ADS. Further, there was no nitrite accumulation in the DAS effluent. The nitrite accumulation in ADS effluent was lesser when CO₂ was used as buffer rather than K₂HPO₄ and KH₂PO₄. Estimation of kinetic parameters of hydrogenotrophic bacteria indicated lesser sludge production compared to heterotrophic denitrification. In the DAS, membrane fouling was nonexistent in the aeration reactor that was used to produce the recirculating effluent. On the contrary, membrane fouling was observed in the denitrification reactor that supplied hydrogen to the mixed liquor. Thus, this study demonstrated DAS capability in maintaining the acceptable water quality appropriate for aquaculture, in which a closed recirculating system is typically used.     

Mixed acid fermentation of paper fines and industrial biosludge

This paper uses countercurrent fermentation to anaerobically convert paper fines and industrial biosludge to carboxylate salts using a mixed culture of acid-forming microorganisms. Using the MixAlco process, the carboxylate salts can be thermally converted to ketones and hydrogenated into mixed alcohol fuels. Continuum particle distribution modeling (CPDM) correlated batch fermentation data to countercurrent fermentation data, allowing the prediction of product concentrations and conversions over a wide range of solid loading rates and liquid residence times. For 80% paper/20% biosludge, the predicted product concentrations agreed with the data within 7.7%. The predicted conversion agreed with the actual conversion within 27.8%. By correcting for varying selectivity, the predicted conversion agreed with the actual conversions within 15.2%. For 40% paper/60% biosludge, the predicted product concentrations agreed with the data within 9.6%. The predicted conversion agreed with the actual conversion within 28.3%. By correcting for varying selectivity, the predicted conversion agreed with the actual conversions within 15.4%. For both the 80/20 and 40/60 cases, CPDM predicts that 90% conversion is possible with a 20 g/l product concentration, 300 g/l substrate concentration, 16 day liquid residence time, and 2.5 g/(ld) solids loading rate. Before proceeding to an industrial plant, these predictions must be verified in a pilot plant.     

利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation,DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程,对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor,SBR)内接种混合物,分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d,定期检测反应器基质浓度变化、计算转化速率;利用16S rRNA基因系统发育分析研究功能微生物的多样性,利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌,未检测到DAMO古菌;以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物;3个反应器的脱氮速率经过初始低速期、快速提升期,最终达到稳定,但2号快速提升期开始时间比1、3号晚了80 d左右,达到稳定的时间更长,稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响;以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存,DAMO古菌可能是协同体系中脱氮速率的限制性因素。     

Kinetics of nitrification and denitrification reactions

Nitrification and denitrification are important microbiological reactions of nitrogen. In this work, the kinetics of these reactions have been investigated based on a Monod-type expression involving two growth limiting substrates: ammonium nitrogen and dissolved oxygen for nitrification and nitrate nitrogen and dissolved organic carbon for denitrification. The kinetic constants and yield coefficients were evaluated for both these reactions. Past experimental work was used to determine the constants for the nitrification reaction. For the denitrification reaction, experiments were performed in a stirred tank reactor under conditions such that only one substrate was growth limiting. Steady-state values of the substrate concentrations in the reactor were determined at various dilution rates. These data were analyzed to obtain the kinetic and stoichiometric constants. From these constants it was concluded that in the range of nitrate nitrogen concentrations encountered in waste water, the denitrification reaction can be considered a first-order reaction. It was also found that three times as much organic carbon is required as nitrate nitrogen for complete nitrogen removal.     

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions

The study investigated effect of high influent nitrate concentration on poly-beta-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57-0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO(X) utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.     

Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Influence of microbial growth kinetics on steady state multiplicity and stability of a two-step nitrification (SHARON) model

In this paper, the influence of microbial growth kinetics on the number and the stability of steady states for a nitrogen removal process is addressed. A two-step nitrification model is studied, in which the maximum growth rate of ammonium oxidizers is larger than the one of nitrite oxidizers. This model describes the behavior of a SHARON reactor for the treatment of wastewater streams with high ammonium concentrations. Steady states are identified through direct calculation using a canonical state space model representation, for several types of microbial kinetics. The stability of the steady states is assessed and the corresponding phase portraits are analyzed. Practical operation of a SHARON reactor aims at reaching ammonium conversion to nitrite while suppressing further conversion to nitrate. Regions in the input space are identified that result in this desired behavior, with only nitrite formation. It is demonstrated that not only the dilution rate plays a role, as is commonly known, but also the influent ammonium concentration. Besides, the type of microbial (inhibition) kinetics has a nonnegligible influence. While the results indicate that product inhibition does not affect the number of steady states of a (bio)reactor model, it is shown that substrate inhibition clearly yields additional steady states. Particular attention is devoted to the physical interpretation of these phenomena.     

Regulation of nitrogen levels for heterogeneous populations of sewage origin grown in completely mixed reactors

Heterogeneous populations of sewage origin were grown continuously at, dilution rates from 1/12 hr^?1 to dilute-out (1/1 hr^?1) using glucose (1000 mg/l) as carbon source and three concentrations of NH₃-N as the nitrogen source (COD:N = 70:1, 40:1, and 25:1). The effects of nitrogen level and growth rate (dilution rate) on substrate removal, biological solids production, cellular carbohydrate and protein, and NH-N in the effluent were examined. It was found that the optimum level of nitrogen supplementation for the synthetic nitrogen-deficient waste employed should not be based solely on the desired effluent quality with respect to COD removal but should include due consideration of reactor detention time (or dilution rate) and the allowable (or desirable) level of nitrogen leakage in the effluent.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi‐species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite‐oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia‐oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients. Biotechnol. Bioeng. 2010;105: 1115–1130. © 2009 Wiley Periodicals, Inc.     

Advanced anaerobic bioconversion of lignocellulosic waste for bioregenerative life support following thermal water treatment and biodegradation by Fibrobacter succinogenes

The feasibility of nearly-complete conversion of lignocellulosic waste (70% food crops, 20% faecal matter and 10% green algae) into biogas was investigated in the context of a life support project. The treatment comprised a series of processes, i.e., a mesophilic laboratory scale CSTR (continuously stirred tank reactor), an upflow biofilm reactor, a fiber liquefaction reactor employing the rumen bacterium Fibrobacter succinogenes and a hydrothermolysis system in near-critical water. By the one-stage CSTR, a biogas yield of 75% with a specific biogas production of 0.37 l biogas g(-1) VSS (volatile suspended solids) added at a RT (hydraulic retention time) of 20-25 d was obtained. Biogas yields could not be increased considerably at higher RT, indicating the depletion of readily available substrate after 25 d. The solids present in the CSTR-effluent were subsequently treated in two ways. Hydrothermal treatment (T approximately 310-350 degrees C, p approximately 240 bar) resulted in effective carbon liquefaction (50-60% without and 83% with carbon dioxide saturation) and complete sanitation of the residue. Application of the cellulolytic Fibrobacter succinogenes converted remaining cellulose contained in the CSTR-effluent into acetate and propionate mainly. Subsequent anaerobic digestion of the hydrothermolysis and the Fibrobacter hydrolysates allowed conversion of 48-60% and 30%, respectively. Thus, the total process yielded biogas corresponding with conversions up to 90% of the original organic matter. It appears that particularly mesophilic digestion in conjunction with hydrothermolysis at near-critical conditions offers interesting features for (nearly) complete and hygienic carbon and energy recovery from human waste in a bioregenerative life support context.     

Recent developments on biological nutrient removal processes for wastewater treatment

The need for preserving the environment is tightening regulations limiting the discharge of contaminants into water bodies. Nowadays most of the effort is done on the removal of more specific contaminants such as nutrients (N and P) and sulfurous compounds since they are becoming of great concern due to its impact on the quality of water bodies. There have been two recent discoveries of microbial conversions of nitrogenous compounds. One consisting on the capability of ammonia oxidizers of denitrify under certain conditions resulting in a new one-step method for the removal of N-compounds. The second has been named the ANAMMOX process, wherein ammonium is oxidized to dinitrogen gas with nitrite as the electron acceptor. Other developments consist of operational strategies aiming at obtaining the highest efficiency at removing nitrogen at lowest cost. One strategy consists of the partial nitrification to nitrite (only successful in the SHARON process) and subsequently either the heterotrophic denitrification of nitrites or its autotrophic reduction by ANAMMOX microorganisms. Another strategy consists of the coexistence of nitrifiers and denitrifiers in the same reactor by implementing high frequency oscillations on the oxygen level.The recent developments on biological phosphorous removal are based on the capacity of some denitrifying microorganisms to store ortho-phosphate intracellular as poly-phosphate in the presence of nitrate. These microorganisms store substrate (PHB) anaerobically which is further oxidized when nitrate is present. By extracting excess sludge from the anoxic phase, phosphate is removed from the system. Removing phosphate using nitrate instead of oxygen has the advantage of saving energy (oxygen input) and using less organic carbon.The microbial conversions of sulfurous compounds involve the metabolism of several different specific groups of bacteria such as sulfate reducing bacteria, sulfur and sulfide oxidizing bacteria, and phototrophic sulfur bacteria. Some of these microorganisms can simultaneously use nitrate, what has been reported as autotrophic denitrification by sulfur and sulfide oxidizing microorganisms. More recently, the anaerobic treatment of an industrial wastewater rich in organic matter, nitrogen and sulfate, reported a singular evolution of N and S compounds that initially was hypothesized as SURAMOX (SUlfate Reduction and AMmonia OXidation). The process could not have been verified nor reproduced and further investigations on the proposed SURAMOX mechanism have given no additional insights to those initial observations.     

Microbial production of 2-chloro-alpha-methylbenzyl alcohol and its adsorptive recovery

An adsorptive process was combined with yeast-mediated production of chiral 2-chloro-alpha-methylbenzyl alcohol (o-Cl-1-PA) for effective product recovery and reuse of the reaction medium. Low temperature was suitable for long-term reactor operation, and continuous production using a shallow-bed reactor was achieved for at least 22 days while maintaining a high conversion. The appropriate size of the adsorption column for product recovery from the reactor effluent was estimated through measurement of breakthrough curves of o-Cl-1-PA in a packed bed of the resin at various adsorbate concentrations and feed flow rates. Using the adsorption column, 98% of the product and the residual substrate were recovered from the reactor effluent, and the effluent from the adsorption column was successfully reused as the reaction medium after microfiltration to save the medium consumption.     

Treatment of hydroponic wastewater by denitrification filters using plant prunings as the organic carbon source

This study investigated the feasibility of using pre-treated plant liquors as organic carbon sources for the treatment of hydroponic wastewater containing high nitrate-N (>300 mg N/L). The waste plant material was pre-treated to extract organic carbon-rich liquors. When this plant liquor was used as an organic carbon source in denitrification filters at the organic carbon:nitrogen dose rate of 3C:N, nitrate removal efficiencies were >95% and final effluent nitrate concentrations were consistently <20mg N/L. However, at this dose rate, relatively high concentrations (>140 mg/L) of organic carbon (fBOD5) remained in the final effluents. Therefore, a 'compromise' organic carbon:nitrogen dose rate (2C:N) was trialled, at which nitrate removal efficiencies were maintained at >85%, final effluent nitrate concentrations were consistently below 45 mg N/L, and effluent fBOD5 concentrations were <25mg/L. This study has demonstrated that waste plant material is a suitable carbon source for the removal of nitrate from hydroponic wastewater in a denitrification filter.     

Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor

Abstract Until now, oxidation of ammonium has only been known to proceed under aerobic conditions. Recently, we observed that NH₄⁺ was disappearing from a denitrifying fluidized bed reactor treating effluent from a methanogenic reactor. Both nitrate and ammonium consumption increased with concomitant gas production. A maximum ammonium removal rate of 0.4 kg N · m⁻³ · d⁻¹ (1.2 mM/h) was observed. The evidence for this anaerobic ammonium oxidation was based on nitrogen and redox balances in continuous-flow experiments. It was shown that for the oxidation of 5 mol ammonium, 3 mol nitrate were required, resulting in the formation of 4 mol dinitrogen gas. Subsequent batch experiments confirmed that the NH₄⁺ conversion was nitrate dependent. It was concluded that anaerobic ammonium oxidation is a new process in which ammonium is oxidized with nitrate serving as the electron acceptor under anaerobic conditions, producing dinitrogen gas. This biological process has been given the name ‘Anammox” (anaerobic ammonium oxidation), and has been patented.