Application of the Microbial Process of Anaerobic Ammonium ...

ISSN 00036838, Applied Biochemistry and Microbiology, 2012, Vol. 48, No. 8, pp. 667–684. © Pleiades Publishing, Inc., 2012. Original Russian Text © A.N. Nozhevnikova, M.V. Simankova, Yu.V. Litti, 2011, published in Biotekhnologiya, 2011, No. 5, pp. 8–31.

PROBLEMS, PERSPECTIVES

Application of the Microbial Process of Anaerobic Ammonium Oxidation (ANAMMOX) in Biotechnological Wastewater Treatment A. N. Nozhevnikova, M. V. Simankova, and Yu. V. Litti Vinogradskii Institute of Microbiology, Russian Academy of Sciences, Moscow, 117312 Russia email: [email protected], [email protected] Received July 6, 2011

Abstract—This review covers various aspects of the process of anaerobic ammonium oxidation by nitrite with the formation of molecular nitrogen called ANAMMOX (ANaerobic AMMonium Oxidation). Anaerobic ammonium oxidizing bacteria are briefly described, including their phylogenetics, habitat, and morphologi cal and physiological characteristics. The current views on the biochemistry of the microbial nitrite reduction by ammonium are presented. The review is focused on biotechnological wastewater treatment based on the ANAMMOX process. Various nitrogen removal technologies using this process, namely, the SHARON ANAMMOX CANON and DEAMOX BCDEAMOX, and their practical use are reviewed. Various types of reactors and set ups using the ANAMMOX process that are applied to the treatment of wastewater are ana lyzed. Processing methods for slowly growing ANAMMOX bacterial biomass accumulation aimed at subse quent inoculation in reactors are analyzed. The problems and methods for ANAMMOX bacterial biomass immobilization in reactors and on carriers are described. A description and parameters of laboratory and pilot plants utilizing various highammonia wastewater are given. Examples of the currently operating fullscale industrial setups with the ANAMMOX process implementation, including those for the complex biochemi cal treatment of domestic sewage (BCDEAMOX) constructed by EKOS (Russia) at the Olympic facilities in the Sochi region, are discussed. Keywords: ANAMMOX, ANAMMOX bacteria, anaerobic ammonium oxidation, BCDEAMOX, DEAMOX, denitrification, nitrification, wastewater decontamination, planctomycetes, CANON, SHARONANAMMOX DOI: 10.1134/S0003683812080042

In the modern world, the amount of generated wastewater is ever increasing as a result of domestic and industrial activity, which demands an efficient and cheap method of decontamination. The basis for bio logical decontamination of wastewater, which remains the most economical and environmentally friendly process, is aerobic and/or anaerobic degradation and mineralization of organic matter by microorganisms. The development and improvement of the existent methods of biological decontamination of wastewater from pollution is an extremely important task, as the wastewater that gets into the environment affects the quality of fresh water drastically. The global problem of drinking water is inseparable from the problem of Abbreviations: AOB, ammoniumoxidizing bacteria; BOD, bio logical oxygen demand; VFA, volatile fatty acids; NOB, nitrite oxidizing bacteria; IRBC, immersed rotating biological contrac tor; DM, dry matter; MSW, municipal solid waste; COD, chemi cal oxygen demand; ABF reactor, anaerobic biological filtrated reactor; CANON, completely autotrophic nitrogen removal over nitrite; DEAMOX, denitrifying ammonium oxidation; FISH, flu orescent in situ hybridization; RBC, rotating biological contac tor; SBR, sequential batch reactor; SHARON, singlereactor highactivity ammonium removal over nitrite; UASB reactor, up flow anaerobic sludge blanket reactor.

wastewater treatment and prevention of the contami nation of fresh water. Ammonium nitrogen is one of the major nutrients contained in wastewater. The decontamination of wastewater from nitrogen compounds is performed in the nitrification–denitrification process, where ammonium is first oxidized to nitrate, which is fol lowed by the assimilation of carbon dioxide (nitrifica tion) and subsequent reduction of nitrate to nitrogen gas (denitrification) [1]. The process of nitridenitrifi cation is quite expensive because of the high costs of aeration and nutrients, which are needed for the growth of denitrification microorganisms. The energy consumption for aeration, which is needed for the nitrification stage, could be as high as 80% of the total energy consumption (nitrification–denitrification) [2]. If the treated water does not contain a sufficient source of organic carbon, the addition of electron donors could be needed at the denitrification stage. Nitrification–denitrification technologies are used for the treatment of wastewater that have both an organic content and a low nitrogen content. Thus, the process of denitrification is used for urban wastewater treat ment where the initial BOC5/nitrogen ratio is not less

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than 6 [3]. In this case, during aerobic wastewater treatment, ammonium is oxidized to nitrate in aera tion tanks. Nutrients for denitrifying bacteria are added in a parent stock, which is the water after the decanting of the excess sludge and treated in a meth ane tank (methane tank return water, silt water). Nutrients could also be added in the form of an exog enous source of organic carbon, such as methanol or ethanol. It should be noted that there is no alternative to this process for largescale urban sewage water treatment. This process is used at the last stage (advanced treat ment) during conventional aerobic decontamination. It could also be combined with the recycling of treated water and preprocessing of treated water under microaerophilic or anaerobic conditions [1]. How ever, there is sewage water with a high nitrogen con tent. This includes methane tank silt water, food industry, and other industrial wastewater, filtered water of municipal solid waste (MSW), landfill disposals, and animal husbandry sewage water. Apart from a high nitrogen content, wastewater is characterized by a low content of organic matter. Thus, at urban wastewater treatment plants, under anaerobic stabilization of sludge water, it has been observed that the liquid phase of meth ane tanks has a significant amount of ammonium nitro gen in the concentration range 300–400 mg/l. The BOC5/nitrogen ratio in sledge water does not exceed 1–2 [3]. The nitrification and denitrification process is not costeffective for the treatment of wastewater with a high content of ammonia and a relatively low con tent of organic impurities. A thermodynamic calculation, performed in the late 20th century, showed that ammonium may serve as an electron donor for nitrate or nitrite reduction under anaerobic conditions [4]. A comparison of the energy yield of the nitrification–denitrification pro cess and anaerobic ammonium oxidation confirmed that the latter is energetically more favorable. After it was theoretically predicted, it was experimentally con firmed in the 1990s. It was first discovered in a pilot anaerobic denitrifying reactor, which was processing yeast industry wastewater. It was named ANNAMOX (ANaerobic AMMonium Oxidation) [5]. In this pro cess, ammonium is oxidized by nitrite ions and forms molecular nitrogen according to the aggregate equa tion +

–

NH 4 + NO 2 = N 2 + 2H 2 O.

(1)

A phylogenetic analysis of microbial sludge from this reactor showed that the dominant bacteria in the consortium of microorganisms belonged to the order Planctomycetales and was ascribed to the new species Brocadia anammoxidans [6]. Previously, there were observed some inconsisten cies in the nitrogen material balance in wastewater treatment that could be due to anammox reactions. During the next 15 years, the anammox process was a

subject of many investigations and there were a lot of developments associated with its largescale applica tion. Bacteria performing anammox reactions were also found in natural ecosystems. The aim of the current review is to give a brief description of anammox bacteria and the anammox process and also to discuss its biotechnological appli cation for the treatment of wastewater with a high nitrogen content. CHARACTERISTICS OF ANAMMOX BACTERIA Planctomyces The anammox bacterial species, which are described in this work, belong to the order Planctomy ces. Planctomyces were identified as a separate order on the basis of the 16S rRNA sequences [7] and a number of other features. The most important of those are unusually short 5S rRNA and the absence of pep tidoglycan in the cell wall. The latter has only been observed in chlamydia and mycoplasma. Plancto mycete bacteria vary in the form of cells. Most of them have flagella. Electron microscopy studies have revealed the presence of a craterlike structure on the cell surface, which is an indication that the organism in question belongs to the order Planctomycetes. Planctomycetes are characterized by an extremely slow growth rate. Thus, the reproduction period of “fastgrowing” organisms is 11–13 days, while for Planctomyces maris it exceeds 100 days [7]. Apart from anammox bacteria, all known planctomycetes grow on organic compounds and could be called heteroorgan otrophic. It was shown that planctomycetes are widely spread in nature and in artificial ecosystems. Planctomycetes have been found in various habitats, such as marine ecosystems, highly salty lagoons, meromictic salt lakes, alkaline lakes, acid swamps, freshwater systems, brackish water systems, soil, cattle manure, and sew age sludge of water treatment plants. Due to the spread of planctomycetes, they have a major function in glo bal water cycle processes [7]. Phylogeny and Characteristics of Anammox Bacteria Anammox bacteria are Grampositive organisms that belong to the Bacteria kingdom, Planctomycetes phylum, Planctomycetales order. Unlike other organ isms of the Planctomycetale order that are heteroorga notrophic, anammox bacteria are anaerobic chem olithotrophs. Anammox bacteria receive energy from the oxidation of ammonium nitrite [8] and use carbon dioxide as a carbon source for constructive metabo lism [9]. There is a number of special features attributed to anammox bacteria. Its cells have a coccoid appearance or an irregular jagged shape. The cell diameter is about

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Cell wall Cytoplasmic membrane Pariphoplasm Inner membrane Nucleoid Riboplasm Membrane of the anammoxosome Anammoxosome Fig. 1. Schematic representation of an anammox bacterial cell.

1 µm [10, 11]. As planctomycetes, anammox bacteria have craterlike structures on the surface of cells. There is no peptidoglycan in the cell wall, and there is an intracellular vacuoles “anammoxosome,” which is a unique organelle attributed to these bacteria [12]. Being planctomycetes, anammox bacteria have a pro tein cell wall and a differentiated cytoplasm (Fig. 1). Anammox bacteria have two membranes in the inner side of the cell wall. The cytoplasmic membrane is located close to the cell wall and surrounds the area of ??the cytoplasm, which does not contain RNA (it is called the pariphoplasm). The inner membrane sur rounds the pariphoplasm from the other side. Inside the inner membrane, there is the riboplasm (an area of the cytoplasm containing a large number of ribosomal particles) and a cell organelle, which is surrounded by a bilayer membrane called the anammoxosome. The anammoxosome takes about 50–70% of the cell vol ume. The riboplasm contains cellular RNA and DNA. Thus, the cytoplasm of anammox bacteria consists of three parts, which are separated by single bilayer membranes: pariphoplasm, riboplasm, and anam moxosome [13–15]. In the membrane, which sur rounds the anammoxosoma, there are “ladderane” lipids, i.e., lipids that have a special structure and high strength [16]. It was found that anammox bacteria contain hopanoids that increase the strength of the membrane [17–19]. The high density of anammoxo some membranes was shown by computer simulation [20] and confirmed experimentally [18]. At low rates of the anammox process, the nanamoxosome membrane maintains the concentration gradient, which is created during the anammox reaction; reduces the energy loss of cells; and prevents the toxic intermediate (hydrazine) from penetrating into the riboplasm [21]. The biomass of anammox bacteria has a reddish color, which is due to a high content of haem (or hemin) in molecules of hydrazine reductase and cyto chromes [22]. Genomic 16S rRNA and 23 S rRNA are localized in the same operon, where they are separated by a long section of about 450 bp, and the 16S rRNA APPLIED BIOCHEMISTRY AND MICROBIOLOGY

gene sequence contains an insertion of 20 nucleotides, which is located in helix 9 [23]. The doubling time of anammox bacteria is at least 11 days [2, 8]. That was the reason why all attempts to isolate a pure culture were unsuccessful The first high purity culture was obtained in 2003 using gradient centrifugation. It was a culture, which contained 99.7% of the Brocadia anammoxidans cells [24]. Five genera and nine species of anammox bacteria have been described. The latter are obtained in the form of highpurity enrichment cultures and could be new species of bacteria (Table 1). Anammox bacteria are chemolithoautotrophic. They use carbon dioxide as a carbon source. Nitrite does not only serve as an electron acceptor in the ammonia oxidation process, but also acts as an elec tron donor in the process of anabolic reduction of car bon dioxide. The equation describing catabolic and anabolic reactions is as follows [8]: +

–

NH 4 + 1.32NO 2 + 0.066HCO 3 +

–

+ 0.13H = 1.02N 2 + 0.26NO 3

(2)

+ 0.066CH 2 O 0.5 N 0.15 + 2.03H 2 O. Ammonium and nitrite are consumed in the 1 : 1.32 ratio. Molecular nitrogen is the main product of anammoxperformed transformation. Nitrogen (10%) is converted to nitrate. Anammox bacteria have a high susbtrate affinity. Nitrite is more toxic than ammonium for anammox bacteria [6, 31]. It has been recently established that anammox bactera can use organic substrates, for example, formi ate, acetate, and propionate, by oxidizing them to CO2. It was also shown that nitrites or nitrates could be used as electron acceptors [32]. An anammox bacte rium was also isolated, which is ascribed to a new genus and species—Anammoxoglobus propionicus— and is capable of growing on propionate [30]. Thus, it is shown that some anammox bacteria are capable of producing nitrite for anammox from nitrate in the presence of additional carbon sources in media; i.e.,

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Table 1. Candidates which could be new species of anammox bacteria Genus

Species, reference

Brocadia

Place of isolation

Kuenenia Scalindua

Brocadia anammoxidans, [25] Brocadia julgida, [26] Kuenenia stuttgartiensis, [23] Scalindua brodae, [11]

Jettenia Anammoxoglobus

Scalindua wagneri, [11] Scalindua sorokinii, [27] Scalindua arabica, [28] Jettenia asiatica, [29] Anammoxoglobus propionicus, [30]

anammox bacteria are not strictly chemolithoau totrophic. Biochemistry of the Anammox Process

–

+

NO + H 2 O.

(3)

At the second stage, nitrous oxide and ammonium interact in the presence of the hydrosine hydrolase (hh) enzyme with the formation of hydrazine (N2H4): +

+

NO + NH 4 + 2H + 3e NH+4

NO–2

nir 1e– cyt

N 2 H 4 + H 2 O.

NO

hh

N2H4

3e– cyt

hzo

N2

4e– cyt

6H+ +a bc1 –r

Q ATPase 3H+

Fig. 2. Anammox reaction scheme [17]: nir, nitrite reduc tase; hh, hydrozine hydrolase; hzo, hydrazineoxidizing enzyme; cyt, cytochrome; bcl, cytochrome complex 111; Q, quinine; +a and r, polarity inside and outside the anammoxosome.

Then, oxidation of hydrazine to molecular nitro gen takes place in the presence of a hydrozineoxidiz ing enzyme (hzo): N2 H4

Anaerobic ammonium oxidation is a multistep process. The first step is the reduction of nitrite cata lyzed by nitrite reductase (nir) to nitric oxide accord ing to the equation NO 2 + 2H + e

Activated sludge of tanhe anaerobic reactor the same ′′ Namibian shelf water, activated sludge of an anaerobic reactor Activated sludge of an anaerobic reactor Anaerobic zone of the Black Sea Marine sediments Activated sludge of an anaerobic reactor the same

(4)

+

N 2 + 4H + 4e.

(5)

A hydraxinoxidizing enzyme was purified from the KSU1 strain of anammox bacteria [33]. Previously, there was isolated an enzyme purified from cells [34] of bacteriacandidates of the Brocadia anammoxidans species which was similar to hydroxy lamine oxidoreductase (hao) from Nitrosomonas euro paea. It showed high activity in hydroxyl amine trans formation and low activity towards hydrazine. It was suggested that hydroxyl amine is an intermediate product of anaerobic oxidation of ammonium [25, 35–38]. However, this assumption was not confirmed in further studies [17, 33]. It was shown that it is NO that is an intermediate product of the anammox reac tion [39]. Four electrons that are formed during the oxidation of hydrazine are used for the reduction of nitrite anions. An electron transfer is performed via an elec tron transport chain in the anammoxosomal mem brane [40, 41]. The electron transfer is accompanied by a proton transport through the membrane. The resulting transmembrane potential is used by the ATP synthase to form ATP [41]. Anaerobic oxidation of ammonium is the only biological process which forms hydrazine [17]. A scheme of the reaction is shown in Fig. 2. It is shown that cytochromes of anammox bacteria played a major role in the electron transport chain [42]. The number of cytochromes in these bacteria was extraordinarily high. The presence of a significant number of cytochromes causes a color change from grey to brownish red in anammox cultures, performing the transformation of ammonium [22]. Thus, the anammoxosome performs the following key functions: (1) it is a place where the catabolyc anammox process takes place and, therefore, the place of the respective enzyme localization; (2) it protects

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the organism from proton diffusion and diffusion of toxic intermediates of metabolism, such as hydrazine; and (3) it is an energy generator, since the ATP synthe sis takes place in the anammoxisome membrane due to the generation of a transmembrane potential during proton transport [16]. Physiological Growth Characteristics of Anammox Bacteria The growth of anammox bacteria takes place in a temperature range from –2 to 80°C [43]. The opti mum temperature for the growth of bacteria isolated from sewage sludge is in a range of 30–35°C [44]. In lowtemperature ecosystems, the optimal growth temperature of anammox bacteria is in a range of 6– 15°C. In marine sediments near Greenland, where temperatures drop below 1°C, the optical mom tem perature for the growth of anammox bacteria does not exceed 12°C [45]. In marine sediments of the Skager rak Strait, where temperature does not rise above 4– 6°C, the optimal temperature for the growth of anam mox bacteria is 15°C [46]. For the growth of biomass enriched in anammox bacteria from Moscow River sludge, the optimum reactor temperature is 20°C [3]. The presence of anammox bacteria was confirmed in marine hot springs; however, the optimum growth temperature has not been established for these organ isms [47]. Anammox bacteria grew at pH 7.5–8.0 with an optimum pH value of 6.7–9.1. Active growth was observed at pH 8.5; however, after an increase in the pH value to 9.0, there was only 20% of the anammox activity observed at pH 8.0 [48]. Habitats of Anammox Bacteria Anammox bacteria were found not only in antro pogenic ecosystems (wastewater treatment plants), but also in natural habitats [25, 27, 49, 50]. In nature, the anammox process was first discovered in marine sedi ments [51]. It was later found in the anaerobic zone of the Black Sea [52] and on the border between the aer obic and anaerobic zones. It was also found in conti nental coastal waters and shelf seas, including shelf seas of Namibia [52]. The anammox process was also observed in freshwater ecosystems, particularly, in sed iments of the Thames River estuary [53] and in those of the Moscow River at discharge points of treated wastewater [3]. Anammox bacteria were also found in swamp ecosystems [43]. The discovery of the anammox process suggested that nitrogen gets into the atmosphere as a result of anammox bacteria’s activity, as well as that of the den itrification process, as it was previously thought. Glo bally, gaseous nitrogen formation and removal occurs in the world oceans. It is believed that 30–70% of nitrogen gas in the nitrogen cycle is formed in the anammox process [51]. It was shown that 19–35% of APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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N2 5

1

N2O

NO

6

NO–2

NH4+

4

2 NO–2

NO–3 3

Fig. 3. Microbial nitrogen cycle: (1) fixation of molecular nitrogen (nitrogenfixing bacteria), (2) nitrification (AOB, ammoniumoxidizing bacteria), (3) nitrafication (NOB, nitriteoxidizing bacteria), (4), denitrafication (nitrate reducing bacteria), (5) denitrification (nitritereducing bacteria), and (6) anammox (anammox bacteria).

the total nitrogen that is removed from coastal ocean waters is removed through the anammox process [52], while the same process in the depths of the ocean accounts for 13 to 51% of atmospheric nitrogen [54]. In Fig. 3 the anammox pathway in the biological nitro gen cycle is shown. APPLICATION OF THE ANAMMOX PROCESS IN BIOTECHNOLOGY The discovery of the anammox process became a new direction in the development of nitrogen removal technologies, which could be applied for the treatment of wastewater with high ammonium and low organic contents. The application of the anammox process for the treatment of municipal urban wastewater, landfill sewage water, and the food industry’s wastewater allowed for the exclusion of the denitrification stage and thus reduced substantially the costs of aerobic nitrification [55–58]. Incomplete nitrification allows for a reduction in costs by 25%. The exclusion of the organicmatter introduction stage allows one to reduce costs by 40%. Carbon dioxide emissions are decreased by 20% [56]. For the successful perfor mance of the anammox process, wastewater must con tain a small amount of organic compounds and high concentrations of ammonium. Another necessary requirement is the accumulation of a sufficiently large number of slowgrowing anammox bacteria, which is only possible under selective conditions. Besides, due to the anaerobic nature of the anammox process, there should be no oxygen present or there should be a low

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Table 2. Capacity and time needed to reach running mode for laboratory anammox reactors inoculated with different types of sludge Capacity, kg N/m3/dаy

Inoculum

Start up period, References days

Nitrifying sludg

2.090

105

[59]

Nitrifying sludge + sludge from а UASB reactor

1.800

250

[60]

Denitrifying sludge

0.609

100

[61]

Sludge from а methane tank

0.090

150

[62]

Sludge from а UASB reactor

0.090

150

[62]

Denitrifying sludge

6.200

392

[29]

River sludge

N/D*

290

[3]

River sludge enriched with anammox bacteria in an accumulating reactor

0.140

137

* N/D, no data

oxygen concentration in the reactor. Due to the low growth rate of anammox bacteria, which is on average 0.003 h–1, it is necessary to ensure that the working regime of the reactor excludes any loss of biomass related to its mechanical washout. Inoculation of Anammox Bacteria In order to start the anammox process rapidly, it is important to create a large population of anammox bacteria in a reactor. The sludge from a nitrification reactor, where anammox bacteria were accumulated, was used as an inoculum, which reduced the initial stage to 105 days and allowed us to reach a rate of ammonium removal of 2.09 kgN/m3/day (Table 2) [59]. If sludge from the denitrificating decanters was used for inoculation, the startup periods of the reac tor ranged from 100 to 390 days according to data from various sources, while the rate of nitrogen removal ranged from 0.61 to 6.20 kgN/m3/day respectively. If the period required to bring the reactor to the working mode is long, significant accumulation of biomass of anammox bacteria occurs, which leads to a tenfold increase in the productivity of the reactor. The reactor drive was started by inoculation with sludge from the bottom of the Moscow River, which was collected in the proximity of discharge points of treated wastewater. Biomass accumulation and enrichment of anammox bacteria were performed for a year. When the sludge was transferred from the decanter reactor into the anammox reactor, it took 137 days to reach the regime where the nitrogen removal efficiency was 91–99% [3]. The first large scale anammox reactor for industrial wastewater treat ment in Rotterdam (the Netherlands) used anammox

bacteria–enriched sludge from the nitrifying reactor. The initial rate of the anammox process was very slow. It took 3.5 years to achieve the estimated nitrogen removal rate, of 9.5 kgN/m3 [58]. Thus, the active sludge from the nitrificating and denitrificating reactors could be an effective primary source of anammox bacteria. The processes of nitrifi cation and denitrification provide a favorable environ ment for the growth of anammox bacteria: Ammo nium from wastewater can be oxidized to nitrite due to the presence of low oxygen content areas in the reac tor. Both nitrite and ammonium are substrates of anammox bacteria. Nitrite can also be formed by reduction of nitrate. There are a lot of anaerobic bac teria, which are capable of performing this transfor mation. Types of Reactors for the Anammox Process In order to optimize the conditions for the accu mulation and confinement of anammox biomass in a reactor, different types of reactors were tested. In the laboratory and pilot plant, it was shown that a sequen tial batch reactor (SBR) provides good conditions for the accumulation of anammox bacterial biomass. It is an installation, which consists of two or more tanks with a single input. While wastewater is decanted and settles in one tank, wastewater is directed and aerated in the other tank. There is a bioselector and a common input, which consists of a large number of walls and partitions that direct the flow from side to side. This allows one to mix the incoming flow of wastewater and the return activated sludge. Thus, the process of the treatment process is started before water enters the main part of the reactor. In an SBR, a high growth rate

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of anammox bacteria is achieved, and the substrates, biomass, and transformation products are distributed homogeneously. It prevents the local accumulation of nitrite, which inhibits anammox, and it allows to maintained low oxygen concentrations due to the activity of heterotrophic organisms, which consume wastewater oxygen. It usually takes at least several months for the process to achieve high nitrogenload ing rates. For example, a rate of 1 kgN/m3/day would only be achieved after 136 days of reactor operation. After a year of stable operation of the reactor, the retention of anammox bacterial biomass was 90% [8]. In the gaslifting reactor, the efficiency of nitrite removal reached 99% [63]. Inert gas is fed into the bot tom of the flask. It lifts up the biomass and the supplied wastewater, thus distributing the activated sludge and the incoming flow over the entire volume of the reac tor. However, gas bubbles can cause the flotation and washout of the biomass. To prevent these processes from happening, microorganisms were immobilized on a special brush. A gaslifting reactor, as well as an SBR, allows one to reach high rates of ammonium loading (e.g., 2 kgN/m3/day) [64]. A rotating biological contractor (RBC, also known as submerged rotating biological contactors or rotating bioreactor (SRBC)) consists of an open container and a number of discs located in parallel and fixed on a horizontal rotating shaft. The lower part of the disks is submersed in a wastewater tank by up to 0.3–0.4 of the diameter. Microorganisms, which are immobilized on the disks, were removing up to 89% of the supplied nitrogen at a rate of 8.3 kgM/m3/day. Both anaerobic and aerobic oxidation of ammonium took place in the installation, while there was almost no nitrite oxida tion [65]. An upflow anaerobic sludge blanket reactor (UASB) is also used for the anammox process. This is a reactor with an upward flow of incoming wastewater through a layer of anaerobic sludge or its modifications. Active biomass is retained in UASB reactors due to the high sedimentation ability of sludge granules, as well as due to the presence of a special gas separation device, which is located at the top of the reactor. Conducting of the anammox process in a UASB reactor yielded granular biomass, which contained 80% of anammox bacteria of the total number of microorganisms after two months of the reactor’s operation [66]. Microbial granules forming in an UASB reactor consist of spher ical biofilms. Anammox bacteria are located in the central part of granules, whereas the peripheral areas of granules have other microorganisms. Such a local ization of anammox bacteria protected the bacteria from inhibitory concentrations of nitrite due to the limited diffusion in the central part of the granules and an increase in the nitrite tolerance up to 400 mg N/l, which is twice the lower limit for homogenized biom ass in the same reactor. There were lattices for trapping granular biomass that prevented it from washing out of APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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the reactor, contributed to the successful running of the installation, and allowed to achieve a nitrogen removal rate of 14 kgN/m3/day. Japanese researchers reported that an anaerobic biological filtrated (ABF) reactor or an anaerobic filter were able to reach ammonium load rates of 0.93 kgN/m3/day on the 34th day of the reactor oper ating [68]. An ABF reactor was used for the treatment of dilute wastewater, which had a low organic content, contained suspended solids, and had a low COD/BOD ratio. The microorganisms in the biofilter are immobilized on the surface of an inert substrate and on the walls of the reactor. For the immobilization of media, substrates, such as gravel, slag, small stones, and a variety of polymeric materials with large surface areas, are used. The ABF reactor had very good parameters of anaerobic ammonium removal, which was due to the high growth rate of anammox bacteria (0.39 day–1, the doubling time was 1.8 days). This was comparable with the growth rate of ammoniumoxi dizing bacteria [68]. However, these results have not been confirmed yet. Thus, the reactor types SBR, UASB, RBC, and ABF and gaslifting reactors could be very efficient for performing the anammox process and growing anam mox bacteria. It is not only the construction of the reactor, but also the created conditions of biomass immobilization and retention, that are important fac tors in the stabilization and enhancement of the anammox process. Immobilization of Active Biomass in Anammox Reactors In addition to the problems associated with the slow accumulation of anammox bacteria in a reactor, there are also problems related to biomass flotation and its washing out of the reactor due to the intensity of molecular nitrogen formation [69]. A conventional way to deal with the problems of flotation and washout of a sludge solution from a reactor is the separation of the outgoing flow, which is followed by the return of the microbial biomass into the reactor. Some other technological solutions to this problem have been pro posed recently. To separate nitrogen bubbles from the bacterial biomass, short mixing of the reactor contents was performed, which contributed to gas desorption from the surface of sludge particles [63, 64] and allowed for a reduction in the proportion of suspended particles in the outgoing flow. The problem of washout of bacterial biomass from a reactor can also be solved by fixing the biomass to a solid support, which is to be followed by the develop ment of biofilms. A biofilm is a community of different microorganisms, which are embedded in a slimy matrix [67]. The microbial aggregates of the biofilm community include bacteria, archaea, phage, micro scopic fungi, and even protozoa. Nonstructured and nondifferentiated biofilms that contain selfimmobi

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lizing cells are called flocculi. Biofilms that develop on solid substrates are usually morphologically structured biofilms where various groups of microorganisms have different localization. Spherical biofilms could be formed in the presence of solid dispersed substrates and also in their absence in UASB reactors (and its modifications) where wasterwater is fed from the bot tom. In the presence of oxygen on the surface of gran ules or biofilms, there are aerobic (predominantly het erotrophic) microorganisms formed on substrates with a developed surface. The slowly growing anaerobic organisms, including methanogenic archaea, bacteria and anammox bacteria, and denitrifiers, are localized inside granules. Metal and polymer mesh, ruffs, plates, and beads, made of porous materials, etc., are used as rigid sub strates with a large surface area in anammox reactors. Small glass beads and sand particles were used as sub strates in reactors with stationary and pseudoliquid layers [70]. Nevertheless, the loss of biomass could not be completely prevented, especially in fixedbed reac tors, where freefloating sediment was observed, indi cating the weak binding of the biomass used in the media. The same phenomenon was observed in the gaslifting reactor during an increase in the nitrogen load [64]. The gas that forms in sludge flakes causes the floating and washout of sledge. However, the con struction of a reactor with a builtin brush from poly meric materials, where biomass adhere, made it possi ble to avoid the washout of biomass from the reactor and reached an anammox bacterial population of 1.7 × 107 cells/ml on the 21st day of the reactor operating [68]. The use of a brush is one of the most successful solutions to the problem of keeping bacterial biomass in a reactor due to the formation of bacterial biofilms on their surfaces. It was possible to start the reactor in a very short period of time: anaerobic oxidation of ammonium started on the 50th day, and the nitrogen removal rate was 26 kg N/m3/day on the 247th day [29]. Aggregates of anammox bacteria were formed during the process in the anaerobic reactor with built in brushes [71]. Thus, the biomass was immobilized not only due to the formation of a biofilm on the brush surface, but also due to the formation of anammox bacterial granules. The operational parameters of the nitrogen load and removal rates in the reactor were 1.26 and 1.05 kg N/m3/day, respectively. The degree of removal of ammonium and nitrite reached 90.9 and 95.0%, respectively [71]. A good result for the retention of microbial biomass was achieved using porous polyethylen slabes as a solid substrate [72]. When the number of plates was increased from six to eight, the nitrogen removal rate increased by 1.2 times. Reactors comprised of brushes, porous slab lattices, and various fillers from synthetic fibers are characterized by a short startup period (from two to six months) and a high rate of nitrogen removal (0.77–2.6 kg/N/m3/day) [29, 68, 71, 73].

Besides, the microbial biofilms on synthetic fibers are resistant to fluctuations in the concentration reagents. However, the operating of reactors with immobilized biomass on a solid substrate inevitably leads to an increase in the maintenance costs for washing or decontamination with chemical reagents. In order to retain anammox bacterial biomass in a reactor, inoculating material could be used, which consists of smallsize polymeric granules, where cells of microorganism are placed. The authors of [74] used polyvinylalcohol as a polymer substrate to which the microbial biomass of the activated sludge from the reactor was attached. It was shown by the FISH method that anammox bacteria constituted more than 50% of the total number of immobilized cells. During the entire time of the experiment, which lasted six months, the obtained granules retained their original form and the attached cells were active and did not wash out from the reactor. The immobilized cells of anammox bacteria removed 80% of nitrogencontain + – ing compounds (N H 4 and N O 2 ) within 48 hours of the reactor operating. It was also possible to use polyethylene glycol as a polymer material [75]. Active biomass of anammox cells was obtained by immobilization of the biomass, which was not washed out of the solution; the nitrogen conversion rate was on average 3.4 kg/N/m3/day. An indirect indication of the high content of anammox bacteria on the substrate was its characteristic red brown color, which is an indication of the accumula tion of significant amounts of cells of anammox bacte ria. The use of anammox bacterial biomass immobi lized in polyethylene glycol in granules as an inoculum allowed us to reduce the startup time of the reactor from three months to 25 days [75]. Thus, the immobilization of anammox bacteria on various structural elements in an anaerobic reactor creates conditions for reducing or preventing the washout of the target biomass. BASIC SYSTEM OF NITROGEN REMOVAL FROM WASTEWATER THAT USES THE ANAMMOX PROCESS Nitrogen is mostly present in the form of an ammo nium in sewage water. In order to perform the anam mox process, half of the ammonium nitrogen in treated water should be oxidized to nitrite; i.e., the first step is nitrification, which is carried out by ammo niumoxidizing bacteria (AOB) according to the reac tion +

–

+

NH 4 + 1.5O 2 = NO 2 + H 2 O + 2H .

(6)

Nitrification to nitrite, which is also known as “partial nitrification” among technologists or “nitrifi cation” among chemists, is the entire process of nitro gen conversion due to the relatively slow growth rate of

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nitrifying bacteria. Based on a combination of pro cesses of nitrification to nitrite and the anammox reaction, a wastewater treatment system was devel oped for wastewater with a high content of ammonium nitrogen. There are two stages of ammonium removal and the actual anammox process, which are described by the following equations: +

–

2NH 4 + 1.5O 2 = NH 4 + NO 2 + H 2 O + 2H

+

(7)

(partisl nitrification, 50%) + +

–

NH 4 + NO 2 = N 2 + 2H 2 O (Anammox) +

2NH 4 + 1.5O 2 = N 2 + 3H 2 O + 2H .

(8) (9)

There are several technological processes, which use the anammox process and anammox bacteria: SHARON–ANAMMOX (SingleReactor High Activity Ammonium Removal Over Nitrite–Anaero bic Ammonium Oxidation), CANON (Completely Autotrophic Nitrogen Removal Over Nitrite), and DEAMOX (Denitrifying Ammonium Oxidation) and its modification BC–DEAMOKS (biological and chemical treatment utilizing denitrification and anammox processes). Process Utilizing Partial Nitrification (SHARON–ANAMMOX) This technique involves two stages, each of which takes place in a separate reactor. Half of the stocks is fed into a SHARONa reactor, where ammonium is partially nitrified to nitrite. Then the output flow from the SHARON reactor is mixed with the second half of the stock in an anammox reactor, where the interac tion between ammonium results in the formation of molecular nitrogen. The first stage of the process is oxidation of ammo nium to nitrite by aerobic ammoniumoxidizing bac teria (AOB), which are firststage nitrifiers that include bacteria of the genera Nitrosomonas, Nitrosos pira, etc.. For the successful completion of the partial nitrification stage in a reactor, there should be estab lished favorable conditions for AOB development, which would suppress the growth of nitriteoxidizing bacteria (NOB) (secondstage nitrifier, which includes Nitrobacter, Nitrococcus, etc.). Selective retention of AOB in the reactor is performed using the physiological differences between AOB and NOB: for example, the optimal temperature for pure cultures of AOB and NOB is 38 and 35°C, respectively [76]. Thus, at temperatures above 35°C, it was possible to carry out nitrification to nitrite for 2 years [77]. How ever, in the temperature range 15–30°C, there was also observed a stable nitrification process carried out by AOB [78]. It was only at temperatures below 15°C where the performance of the system was dramatically APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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decreased due to the transformation of nitrites into nitrates. Another important parameter that determines the prevalent development of AOB vs. NOB is the reten tion time of activated sludge in a reactor. While the AOB doubling time is less than that of NOB, the growth rate of AOB is higher than that of NOB, hence the short retention time of bacterial biomass would favor the NOB washout from the reactor. In industrial wastewater treatment plants in Utrecht and Rotter dam (the Netherlands), the retention time of bacteria ranged from 1 to 2.5 days. This regime favored the NOB leaching out and AOB retention in a reactor [55]. However, even when the retention time of active biomass in a reactor was 5 days, in a batch reactor, favorable conditions were also created for NOB dis placement [79]. An important condition for limiting the growth of NOB is no sludge recycling in the sys tem. The optimization of the oxygen concentration in a reactor also allows one to manage nitrification. The optimization strategy is based on different oxygen demand levels of AOB and NOB. Thus, the Ks value of (constant oxygen saturation) for AOB is 0.3 mg/l, whereas for NOB it is 1.1 mg/l; hence, AOB displaces NOB under oxygen deprivation [80]. Even when the concentration of dissolved oxygen was maintained at about 1.0 mg/l, dominating growth of AOB was observed [81, 82]. The pH value is also an important parameter in the nitrification process. Firstly, it directly affects the AOB and NOB growth rate. The NOB growth rate at pH 7.0 is eightfold higher than that at pH 8.0, whereas the effect of pH on the growth of AOB is insignificant [77]. Secondly, the solubility of ammonium ions in waste water with pH 8.0 increases, while the solubility of nitrite ions in wastewater with pH 8.0 decreases, which also favors the growth of AOB over NOB [77]. Thus, the first stage of the nitrification process is recom mended to be performed in slightly alkaline condi tions. Thirdly, pH optimization allows one to control – + the N O 2 /N H 4 ratio by fixing the concentration of ammonium bicarbonate (1 mol/1 mol) [83]. Thus, subject to control of the required temperature, pH of about 8.0, and high flow rate and without biomass recy cling, it is possible to establish conditions for dominant AOB growth in a SHARON reactor. A study of the microbial population of a SHARON reactor by the FISH method showed that AOB were 60–70% [84]. Thus, the biomass retention time in a reactor, pH, temperature, and the dissolved oxygen concentration are the main parameters affecting the output of the partial nitrification process. However, as was previ ously discussed, the running of a reactor and its con figuration could also affect the output of the process. There are examples of successful launch of the nitrifi cation process at low temperatures (15°C) with an increase in the retention time of activated sludge [78].

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Table 3. Examples of partial nitrification (SHARON) processes in reactors with different designs under various physical and chemical conditions Reactor type Sequencing batch reactor (SBR) Reactor with a floating fiber load (Swimbed) Continuous stirred tank reactor (CSTR) Continuous stirred tank reactor (CSTR) Continuous stirred tank reactor (CSTR)

+

Retention time of treated water, days

t, °C

Concentration of dissolved oxygen, mg/l

1.0 0.25–0.5

N/D* 15–30

3–4 N/D

0.60 0.61

[87] [78]

1.0

30–40

N/D

1.09

[88]

1.2

30

N/D

1.10

[83]

1.0

N/D

Stage regulation

N/D

[86]

N H 4 N/NO2N after the partial Reference nitrification stage

* N/D, no data*

Also, when the process is limited by the dissolved oxy gen concentration, the partial nitrification process is stable for retention periods of up to 24 days [85]. Reac tors, e.g., periodic or continuousflow reactors, allow one to perform nitrification; however, the process is more stable in a chemostat [79]. The design of reactors could reduce reactor maintenance costs through the stepwise regulation of the dissolved oxygen concentra tion and control of pH [86]. Table 3 shows a few exam ples of management of the partial nitrification (SHARON) process. As was already mentioned, the second technologi cal step, which is the process of anaerobic ammonium oxidation, takes place under alkaline conditions [83]. CANON Process This technology uses a combination of the CANON process of partial nitrification and the anam mox process, which is carried out by AOB and anam mox bacteria in a single reactor. AOB convert ammo nium to nitrite and consume oxygen, thus creating anaerobic conditions, which favors the growth of anammox bacteria. Anammox utilizes the resulting nitrite, reducing it to molecular nitrogen in the pres ence of ammonium. The overall reaction is as follows: +

NH 4 + 0.85O 2 –

0.435N 2 +

(10)

+ 0.13NO 3 + 1.4H + 1.3H 2 O. The abovedescribed parameters, which were used to optimize the SHARON process, could also be apply for the CANON system. However, in the CANON technology, management is focused on the ammo nium load and the dissolved oxygen concentration, since these factors have the greatest influence on the microbial population in a reactor. In a sequencing batch reactor, AOB and anammox bacteria are active at the aer obic and anaerobic stages of the process, respectively. In

the same reactor operating under oxygen deprivation, AOB and anammox bacteria function simultaneously when the nitorgen removal rate reaches it maximum value [89]. In this case, the transport of oxygen is a rate limiting factor. An RBC reactor (see above) is poorly adjusted for regulating the oxygen supply to active microbial biomass [65]. The amount of the required oxygen for nitrogen removal is directly related to the thickness of the bacterial biofilm [90]. The biofilm thickness and granule size have the same effect on the bacterial composition. Large aggre gations (more than 500 µm in diameter) contain up to 68% of anammox bacteria from the total population, while small aggregates (less than 500 µm) contain only 35% of anammox bacteria [91]. Since large granules are less accessible to oxygen compared to small ones, there is a relatively large oxygenfree area that favors the growth of anaerobic bacteria. The optimization of the concentrations of ammo nium and dissolved oxygen in media allows AOB to consume oxygen until the oxygen content reaches a threshold level, where the oxygen concentration is toxic for anammox bacteria, but insufficient for the growth of NOB. Nitrite, which is produced during this stage, is an inhibitor for AOB, but it serves as an elec tron acceptor by anammox bacteria. Under these con ditions, syntrophy is observed between AOB and anammox bacteria [91, 92]. When ammonium is lim ited in a wastewater flow, it causes an increase in oxy gen. The presence of an oxygen excess causes the rapid transformation of ammonia by AOB. Accumulation of nitrite and an excessive amount of oxygen contribute to NOB growth and repress the growth of anammox bacteria. This breaks the optimal balance between these groups of bacteria and destabilizes the CANON process. For the sustainable removal of ammonium in an SBR reactor, the lower limit of the ammonium load rate should be 0.12 kg N/m3/day. At lower ammonium concentrations, the nitrogen removal efficiency

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Table 4. Nitrogen removal rate in the CANON process in reactors with different designs Reactor type

Nitrogen removal rate, kg N/m3/day

Sequencing batch reactor (SBR)

0.080 0.160 1.500 7.390 0.057

Gaslifting reactor Rotating bioreactor (RBC) Sequencing batch reactor with granular sludge (GSBR, granulated sludge bioreactor)

decreases by 35% from the theoretically possible level of consumption at a constant concentration of dis solved oxygen, which is 0.24 mg/l [93]. The maximum rate of nitrogen removal was achieved when the change in the concentration of dissolved oxygen was correlated with the ammonium load [90]. Thus, the stoichiometry of ammonium and oxygen is a key parameter in the CANON process. The CANON process has been performed in a lab oratory in the following systems: SBR, gaslifting reactor, RBC reactor, and GSBR (a sequencing batch reactor with granular sludge) (Table 4). The lowest rate of nitrogen removal was recorded in a sequencing batch reactor with granular sludge (GSBR). The long retention period of bacterial biom ass in an SBR provides good conditions for slowgrow ing anammox bacteria, but the efficiency of such a reactor is low (see Table 4). The highest rate of nitro gen removal, which was 1.5 kgN/m3/day, was obtained in a gaslifting reactor. The highest rate of nitrogen removal was reached in a rotating bioreactor with a biofilm (RBC reactor): 7.39 k N/m3/day. The CANON process allows one to reduce aera tion energy costs by 63%, and it does not require an additional carbon source (100% savings) in compari son with the conventional nitrification and denitrifica tion processes [89]. This process also has an advantage over the SHARON technology, since it does not require a second reactor and thus requires less capital ization. Nevertheless, there are difficulties associated with the regulation of the dissolved oxygen concentra tion in large reactors and incomplete nitrogen removal at high loads, which make the CANON technology unsuitable for the treatment of wastewater with a high ammonium content. These types of wastewater are better suitable for the SHARON technology [2]. DEAMOX Process DEAMOX is a recently developed process. It allows one to overcome some drawbacks of the SHARON–ANAMMOX and CANON technologies. The incoming wastewater is divided into two flows in the DEAMOX process. Conventional nitrification is APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Type of aeration Batchfed Continuous Continuous Continuous Surface aeration

State of the activated Reference sludge Suspended the same » Biofilm Granulated

[89] [92] [94] [65] [95]

carried out in the first flow. There is no aim of obtain ing nitrite, thus the process does not require any opti mization. The output flow from the first reactor is mixed in a DEAMOX reactor with the second half of wastewater, which usually contains a number of elec tron donors (sulfide or organic contaminants). Nitrite is formed due to the partial denitrification of nitrate under autotrophic or heterotrophic conditions; together with the present ammonium, it is absorbed by anammox bacteria, resulting in the formation of molecular nitrogen [96, 97]. There are autotrophic and heterotrophic versions of the DEAMOX process, which are being currently developed and optimized [98]. In both versions, nitrate, which is obtained after aerobic treatment of half of the incoming wastewater, is mildly reduced to nitrite. Sulphide is used as an electron donor in autotrophic denitrification (SDEAMOX process): –

NO 3 + 0.25HS –

–

2–

+

(11)

NO 2 + 0.25SO 4 + 0.25H . However, due to the low content of sulphide in wastewater, heterotrophic denitrification, where vola tile fatty acids serve as electron donors (ODEAMOX process), is more suitable process: –

NO 3 + 0.25CH 3 COO –

–

– +

(12)

NO 2 + 0.25HCO 3 + 0.25H . Nitrite, which is formed from nitrate during the first stage by denitrificators (denitrificating bacteria), is reduced by anammox bacteria to molecular nitro gen. The COD/N ratio should be maintained in a range of 2.0–3.5 for the stable running of a DEAMOX reactor. It is also important to maintain the concentra tion of bicarbonate at a level which would allow for the completion of the full nitrification process of the wastewater incoming into a nitrification reactor. The removal of ammonium nitrogen decreased to 70% of the theoretical possible level of its consumption by bacteria under different conditions [98]. In order to optimize the COD regime and the con tent of bicarbonate ions in wastewater, a technology

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utilizing the reverse sequence of nitrification in a DEAMOX reactor was used [98]. After treatment in a DEAMOX reactor, wastewater was treated in a nitrify ing reactor, from which twothirds was returned for additional treatment. This scheme allows for the removal of nitrogencontaining compounds up to 84% and also allows one to get rid of the drawback of the direct configuration sequence, which is the addition of an organic carbon source for performing partial deni trification and removing ammonia nitrogen (about 30% of loaded ammonia nitrogen is not transformed by directsequence reactors). Comparing the technology of the DEAMOX pro cess with that of SHARONANAMMOX, it should be noted that both of them do not require any additional reagents, which increases the efficiency of wastewater with a high ammonium content and a low COD/nitrogen ratio. One of the advantages of the DEAMOX process is the engineering simplicity. It uses standard UASB reactors, and its technology does not require any control of the nitrite concentration. The denitrification conditions in a DEAMOX reactor favor the formation of granules, which in turn stimu lates the anammox process. There is no accumulation of high nitrite concentrations that are toxic to anam mox bacteria in a DEAMOX reactor. In addition, a DEAMOX reactor does not emit into the atmosphere any dinitrogen monoxide and nitrogen monooxide, which are greenhouse gases [96]. A feasibility study of the SDEAMOX and O DEAMOX processes was performed in laboratory condi tions during processing baker’s yeast wastewater [96, 97] and the return waters of methane tanks [98, 99]. The process established operating mode within 1.5 months; the load of nitrogencontaining compounds was 207 mg N/m3/day, while the effective ammonium removal was approximately 47%. Studies with synthetic wastewater have shown that the optimal range for the – ratio of entering concentrations for COD/NN O 3 was (2.5–3.5) : 1 or Corg/N = (1–1.7) : 1 [99]. It should be noted that in the presence of an exces sive amount of electron donors, theDEAMOX process may continue; i.e., nitrite can be reduced to molecular nitrogen, creating competition for the main substrate (nitrite) in the system of nitrification and anammox processes. De facto, there are two parallel processes in DEAMOX reactors: denitrification and anammox. Both of them result in the formation of molecular nitrogen as the final product. Optimization of the external parameters of the DEAMOX process, such as temperature, pH, and the magnitude of nitrogen, showed that the domination of the anammox vs. deni trification processes is highly dependent on the envi ronmental conditions; however, the system is stable enough to produce molecular nitrogen under any con ditions [100]. The bacterial composition of activated sludge from a DEAMOX reactor determined by molecular methods revealed a broad diversity of

microorganisms, including all groups of bacteria that are necessary for nitrification, denitrification, and the anammox process. The presence of methanogenic archaea was also observed. The authors suggest that methanogens contribute to the formation of granules in DEAMOX reactors [101]. BHDEAMOX Process The DEAMOX process can be realized on a large scale using biological and chemical technology, utiliz ing denitrification and the anammox process (BC DEAMOX). The technology of municipal sewage treatment includes the following: firstly, pretreatment of wastewater with a coagulant to remove sediments, which reduce BOD by about half (the ammonia con centration does not decrease); secondly, to apply for brushloading of activated sludge; and, thirdly, for the return of treated water from an aeration tank to an anaerobic denitrificator. It is shown that the anammox process in this system accounts for 30–50% of the molecular nitrogen formed [102–105]. The applica tion simplicity of the DEAMOX technology and its modification—BCDEAMOX—make it an attractive option compared to the technologically more demanding CANON and SHARON–ANAMMOX processes. The BCDEAMOKS technology is especially promising for application in regions where there is a shortage of technological and engineering expertise in the management of hightech treatment of wastewater with a high ammonia content. APPLICATION OF THE ANAMMOX PROCESS FOR THE TREATMENT OF A REAL SAMPLE OF SEWAGE WATER WITH A HIGH AMMONIUM CONCENTRATION Laboratory Studies The anammox process allows one to reduce oper ating costs significantly, making it an attractive option for researchers. It was tested on various types of ammoniumrich sewage water soon after it was discov ered. Silt water is the liquid fraction of wastewater sed iment treated in a methane tank (excess sludge). Silt water has a low content of dissolved organic com + pounds and its N H 4 /alkali ratio is optimal for the process of partial nitrification–anammox process, which made it the first choice for testing the anammox process [88]. Initial studies were carried out using a laboratory device, for which the rate of nitrogen removal was 0.71 kg/day. It was followed by the launch of a largescale anammox process that allowed for the removal of up to 9.5 kg N/m3/day [58]. Treatment of silt water and other wastewater with a high ammonium content was performed in reactors with different designs by several research groups [106]. The wastewa

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Table 5. Application of ANAMMOX for the treatment of different types of wastewater with a high ammonium content Sewage

Time to reach the running regime, days

Type of process

Silt water

Partial nitrification–anammox

Wastewater from the coil chemical industry Digested residue after partial nitrification Wastewater from pig farms

Anammox

Slaughterhouse wastewater Wastewater of monosodium glutamate production Landfills’ filtration water

Capacity, Volume Reference kg N/m3/day of the reactor, l

110 150 137 465

0.710 2.400 0.140 0.062

10 2500 50 1

[88] [83] [3] [107]

the same

N/D*

3.500

3.5

[108]

'' Partial nitrification–anammox Nitrificationdenitrification anammox Anammox

N/D 60 N/D

0.600 1.360 0.031

1.5 1 1.6

[109] [87] [110]

71

0.460

5

[111]

Partial nitrification–anammox

N/D

0.27–1.2

11

[112]

*N/D, no data

ter types and the characteristics of the anammox reac tors are presented in Table 5. As can be seen from Table 5, the technology utiliz ing the anammox process could be applied for the treatment of wastewater of the coal chemical industry [113], which contains high concentrations of organic substances (COD = 2000–2500 mg/l), as well as toxic substances, such as phenol (300–800 mg/l), cyanide (10–90 mg/l), and thiocyanates (300–500 mg/l) [107, 113]. Because of the toxicity of wastewater compo nents, the treatment of of such wastewater was effec tive. In order to reduce the inhibitory effect of toxic compounds, wastewater was diluted and the process was performed in stages, which made it considerably more expensive. For the adaptation of microorganisms to the coal industry’s wastewater, model wastewater was used where the phenol concentration increased, gradually reaching 500 mg/l. Sludge from a reactor for processing municipal wastewater was used as an inoculum. The ammonium removal rate reached 0.062 kg N/m3/day in 15 months, which is 1.5 times higher than the figure obtained for a conventional reactor, which was not adapted to a high phenol con tent. The selected consortium of microorganisms allowed for the removal of ammonium and nitrite at high rates and was also susceptible to high phenol con centrations up to 330 mg/l. Filter water from landfills is sewage water with a high ammonium content. The possibility of applying the anammox process to treat this water has been pre viously shown [112, 114, 115]. It is well known that livestock wastewater is charac terized by a high ammonium content, which is formed owing to the decomposition protein compounds. The ratio of nitrite to ammonium varies from 1.5 to 1.8 for APPLIED BIOCHEMISTRY AND MICROBIOLOGY

such wastewater, depending on the COD values in the water, which can reach 25–26 g/l. It was shown that the decomposition of pig manure in a laboratory UASB reactor was performed by both denitrification and the anammox process [104]. The nitrite nitrogen removal rate reached 0.66 kg/m3/day. The nitrite load increase promoted both denitrification and anammox processes. According to the authors of this study, anammox bacteria did not compete with denitrifica tion bacteria for the substrate. During the experiment, in the lower part of the reactor, there was observed a change in the sedimentation color from grey to brown red and the formation of brown granules of biomass up to 1–2 mm in diameter. For the decomposition of wastewater from pig farms, the SHARONANAMMOX process was used [87]. Anaerobic ammonium oxidation took place in an anammox reactor. The ammonium loading was 1.36 kg N/m 3/day, and its removal rate was 0.72 kg N/m3/day. The nitrogen removal process from piggery wastewater was mostly performed by denitrification bacteria rather than anammox bacteria [87, 116]. According to Yamamoto et al. [78], as a result of tenfold dilution of piggery wastewater, the COD content only decreased to 1.45 g/l. Under these conditions, partial nitrification took place with the formation of nitrite, which then participated in an anammox reaction. Bird droppings, as well as piggery wastewater, are also characterized by a high ammonium content. Bird droppings were prediluted and treated aerobically and anaerobically. While bird droppings were treated by the anammox process, nitrite was added in such an amount that the ratio of ammonia to nitrite was 1 : 1. However, due to the slow growth rate of anammox

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bacteria, anaerobic bacterial oxidation of ammonia proceeded with low intensity, which gave an advantage to the denitrification process. By reducing COD to 0.13 g/l and below, the ratio of ammonium to nitrite approached 1 : 1 and thus created favorable conditions for the anammox process [117]. The anammox technology was also used for the food industry’s wastewater treatment, as well as wastewater of sodium glutamate production, which is characterized by a high content of solid suspended matter (200– + 10000 mg/l), COD (1500–60000 mg/l), NN H 4 (200– 15000 mg/l), and sulphate (3000–70000 mg/l). Con ventional treatment usually involves physicochemical and then biological treatment. After physicochemical treatment, the contents of suspended solids, COD, + and NN H 4 were reduced to 200–270, 1000–1400, and 250–350 mg/l, respectively. For further process ing, prenitrification and the anammox process were used, which allowed for the achieving ofnitrogen removal rates of 0.46 kg N/m3/day [111]. The yield of such a process was much higher than that of the con ventional nitrification–denitrification process. Examples of Utilisation of the Anammox Process in LargeScale (Industrial) Wastewater Treatment Plants A large scale anammox process was used for the treatment of ammoniumrich wastewater in plants, which were located in the Rotterdam Dokhawen region (the Netherlands). The SHARON–ANAM MOX technology was used for sewage treatment. Orig inally, only a SHARON reactor (1800 m3) was per forming partial nitrification of ammonium, resulting in the formation of nitrite [88]. The concentration of ammonia in the inflowing wastewater reached 1.5 g N/l. The efficiency of conversion of ammonia to nitrite was 53% in the first year of the reactor’s operation and increased to 80% in the third year of its operation. Methanol was fed into the reactor as an organic elec tron donor. After four years of successful operation of the SHARON reactor, an ANAMMOX reactor was included in the technological treatment scheme, which allowed for the removal of nitrite by reducing it with ammonia to nitrogen gas. The process was carried out in an SBR with granular sludge; the vol ume of the reactor was 70 m3, the time to reach oper ational mode was 3.5 years, and the nitrogen removal rate was 9.5 kg N/m3/day. The ammonium removal efficiency was 80% when the nitrogen load was 1.2 kg N/m3/day. The start of the SHARON– ANAMMOX process reduced operating costs by decreasing the consumption of oxygen and electricity, as well as excluding the feeding of organic electron donors into the reactor. It should be noted that the use of the SHARON–ANAMMOX technology also sig nificantly reduced the emissions of CO2 into the atmo

sphere. It took a long time for the ANAMMOX reactor to reach the given level of accumulation of anammox bacterial biomass; so, in order to avoid any biomass loss, the temperature in the reactor was maintained in the range 35–37°C and pH was maintained in the range 7.0–8.0. A similar plant was built at a wastewater treatment site in the city of Utrecht (the Netherlands). A largescale anammox industrial reactor with a volume of 500 m3 is operating in the city of Strass (Austria) [57]. This is the world’s first industrial plant utilizing the CANON technology and performing the process of aerobic and anaerobic ammonium oxida tion in a single reactor. Wastewater from concentrated fermented sediment, which is characterized by the + ratio NN H 4 /COD = 2.5–3, is flowing in the plant [57]. The nitrogen removal rate is 300 kg N/day. The nitrification process takes place in a sequencing batch reactor with variable aeration. The dissolved oxygen concentration is maintained at 0.3 mg/l, which allows for the incomplete nitrification of ammonium, result ing in the formation of nitrite and prevents any further oxidation of nitrite to nitrate [57]. During the aeration process, which lasts 6 h, nitrification proceeds at a higher rate during anaerobic oxidation of ammonium, leading to a decrease in pH values to a certain level where the oxygen supply is cut. The anammox process is activated as soon as the oxygen concentration drops down, which leads to an increase in the pH level of the processed wastewater. Thus, the regulation of the oxy gen supply in the reactor is performed via pH control. The amount of anammox bacteria in the total biomass after reaching its full capacity is 17–22%. The nitro gen removal rate in the reactor operating at its full capacity is 0.015 kg N/kgDM/h. The presence of red granules is observed in the reactor, which is character istic of anammoxbacteria accumulation. There is a silt water treatment plant in the territory of urban wastewater treatment in Zurich (Switzerland) (http://www.kurito.co.jp). It was built as a result of laboratory research and pilot reactor studies. [83] It consists of 400 and 500l SBRs, which are equipped with pHcontrolling devices, and performs a com bined partial nitrification–anammox process at tem peratures in the range 25–30°C. The nitrogen removal rate is 0.6 kg N/m3/day in the reactor 500 m3 in vol ume and 0.4 kg N/m3/day in the reactor 400 m3 in vol ume, and the effective nitrogen removal rate is 84 and 90%, respectively. The concentration of dissolved oxy gen at the aeration stage is maintained at a concentra tion of 0.5 mg/l. The regulation of the oxygen supply is carried out by monitoring the pH level, as it was reported for the reactor operating in the city of Strass. The water treatment costs for this reactor are 2 times lower than they would have been for the nitrification– denitrification process owing to a 58% reduction in the cost of aeration and a lack of methanol addition. There are six urban wastewater treatment plants, built by EKOS (Russia) in accordance with their own

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projects for shift workers that construct Olympic ven ues in the Sochi region in Russia. The plants’ capaci ties are 100–400 m3/day. Preliminary physicochemi cal wastewater treatment is used instead of traditional decanter due to the imposed space limitations and the proximity of residential areas. The developed technol ogy has some special features that create favorable conditions for the growth of anammox bacteria and utilization of the anammox process for the treatment of lowconcentration wastewater. Large solid particles are mechanically removed from wastewater, which are further treated with coagulants for the removal of small solid particles. As a result of these procedures, BOD is reduced by half. The ammonium concentra tion in treated wastewater does not decrease and can even increase slightly due to the processes of desalina tion and ammonification. Next, purified water under goes biological treatment, which is first performed in a bioreactor with a weak aeration and then in an aera tion tank with extensive aeration. Thus, it is also accompanied by the return of nitrite and nitrate enriched treated water from the aeration tank to the denitrificator, where it is mixed with wastewater con taining ammonium ions. During all stages of biologi cal treatment and further purification, a hard flexible load with brushes for the immobilization of activated sludge is used [102]. Special conditions are even estab lished for the growth of strictly anaerobic bacteria in microbial biofilms on the brushes in the denitrificator. It was shown that nitrogen is removed as a result of two anaerobic processes—denitrification and anam mox—when this technology is applied in a denitrifi cator. The operation of the anammox process and the growth of anammox bacteria in biofilms on brushes were shown in laboratory studies [105]. The technol ogy of EKOS actually represents largescale utilization of the DEAMOX process. It was called BC DEAMOKS. The operational simplicity of this system makes it more attractive compared to the complex technological performance required for the CANON and SHARON–ANAMMOX processes. In addition, for the first time, the important role of anammox bac teria for the combined system of physicochemical and biological wastewater treatment is shown using the BCDEAMOKS process.

tion of the anammox process is mainly limited to the treatment of several types of wastewater with a high ammonium content, for example, the food industry’s aerobically treated wastewater, proteinrich sewage water, landfill filtrates, sludge, methane tanks’ silt water, and wastewater from livestock farms. The tech nological development of the BCDEAMOKS pro cess opens up new prospects for its application for urban wastewater treatment. The widespread applica tion of the anammox process on a large scale depends on the availability of a sufficient amount of seeding material for starting new anammox reactors. Similar difficulties were observed during spreading the tech nology of anaerobic treatment of concentrated waste water leading to the formation of methane, which were overcome by using biomass from already running anaerobic reactors in new ones [118]. It is important to adapt anammoxbacteria enriched biomass for each type of organic and inorganic contamination in waste water. The isolation and study of anammox bacteria are essential for the further development and improve ment of wastewater purification from nitrogen. Psy chrophilic and psyhroactive anammox bacteria are very promising for wastewater treatment at reduced temperatures in cold regions. For the treatment of warm wastewater, it would be useful to develop inten sive treatment methods based on thermophilic anam mox bacteria, which are still unknown.

CONCLUSIONS The technological aspects of the application of anammox bacteria are well studied and documented. Anammoxbased technologies are costeffective and energyefficient and have a great potential for ammo niumrich wastewater treatment. The long period, which is needed to start anammox reactors, limits the application of this process. This can be improved by enriching inoculum sludge with anammox bacteria, using reactors with more effective biomass retention and nutrient balance adjustment, as well as through adjusting physical parameters. Currently, the applica APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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2012