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Obuli P. Karthikeyan and Kurian Joseph Centre for Environmental Studies, Anna University, Chennai 600 025. Tamil Nadu, INDIA ABSTRACT

In wastewater treatment, nitrogen is being considered a one of the essential parameter as it has significant adverse impacts on the environment. Anaerobic ammonia oxidation (ANAMMOX) is a novel process in which nitrite is used as the electron acceptor in the conversion of ammonium to nitrogen gas. The ANAMMOX process offers great opportunities to remove ammonia in fully autotrophic systems with biomass retention. No organic carbon is needed in such nitrogen removal systems, since ammonia is used as electron donor for nitrite reduction. This paper reviews and summarizes recent developments in nitrogen removal, microbial aspects (occurrence, physiology, microbiology and biochemistry) of ANAMMOX, followed by a qualitative comparison of several components of ANAMMOX technology with conventional nitrogen removal systems and finally addresses the application of the ANAMMOX process for nitrogen management. KEYWORDS Wastewater, nitrogen, nitrification, and ANAMMOX

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Research Scholar and Corresponding author: Assistant Porfessor:

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INTRODUCTION An increasing population and industrialization will increase our water demand, placing even more pressure on water resources. Conventional wastewater treatment plants have not been designed for nitrogen removal, and many plants do not meet the current discharge limits (Jetten et al., 2002). Wastewater originated from many other sources such as tannery, food processing, fertilizer manufacturing, slaughter house and landfill leachate contain greater amount of nitrogen load, which should be treated before discharge into the surface water body (Table 1). Wastewater containing huge amount of nitrogen compounds is not allowed to be released to the surface water as it has ecological impacts and can affect human health (Kelter et al. 1997). Chemical, Physicochemical and biological methods are broadly used for treatment of wastewater loaded with highly concentrated NH4+-N. In considering the criterion like costbenefit analysis, requirement of energy and chemical doses, familiarity with operational procedures, and environmental sustainability, a particular treatment for a specific pollutant is usually selected (Mulder et al 2003). Still, the tradition is that depending on the concentration of nitrogen load presenting in the collected wastewater specimen, either physicochemical or biological treatment method is decided. According to Mulder (2003) three concentration ranges could be differentiated: NH4+-N concentration less than 100 mg NH4+-N/l - In this range biological N-removal is the preferred process based on cost-effectiveness. Domestic wastewater is within this range. NH4+-N concentrations in the range 100-5000 mg NH4+-N/l - A typical example is sludge liquor for which after extensive investigations biological treatment was preferred (Janus et al., 1997). Although ammonia stripping and producing MgNH4PO4 were identified as interesting alternatives for resource recovery these options were not cost-effective (Priestley et al., 1995; Janus et al., 1997). NH4+-N concentrations greater than 5000 mg NH4+-N/l - In this range physicochemical method are technically and economically feasible. A successful example is the steam stripping of a wastewater with an ammonium concentration of 1.5% followed by ammonia recovery which has been in operation on industrial scale since 1985 (Harmsen et al., 1986). Table 1. Wastewater containing high concentrations of nitrogen content
Sources of Nitrogen Landfill leachate Starch Production Wastewater from pectin industry Wastewater from slaughter house, after treatment in aerobic lagoon Wastewater from tannery Discarded water
Source; Hulle 2005

Total Nitrogen (mg/L) 500 - 2500 800 - 1100 1600 170 - 200 128 - 185 260 - 958

Reference Chung et al (2003) Abeling and Seyfried (1993) Deng Peterson et al (2003) Keller et al (1997) Marat et al (2003) Gil and Choi (2004)

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BIOLOGICAL NIROGEN REMOVAL The biological process is cheaper and is the most widely practiced approach for nitrogen control in wastewater treatment. Conventional biological nitrification and denitrification processes have received the maximum attention. In Table 2, three process options of the new system are presented and compared to a conventional nitrogen removal system based on autotrophic nitrification and heterotrophic denitrification system. AEROBIC AND ANAEROBIC AMMONIUM OXIDATION Ammonium oxidation has been observed in many bacterial species. Ammonia is oxidized by two pathways: first, ammonia is oxidized to nitrite by hydroxylamine, which is then oxidized to nitrate by hydroxylamine oxidoreduxctase; Second, ammonia and nitrite are anaerobically converted to nitrogen gas. The aerobic chemolithoautotrophic ammonia oxidizing bacteria (AOB) are specialists that can grow on ammonia and carbon dioxide (Purkhold et al., 2000) and use ammonia monooxygenase to convert ammonia into hydroxylamine. Many heterotrophic bacteria, such as P. Pantotropha and Alcaligenes faecalis strain TUD (Otte et al., 1999), can carry out the same reaction. Methanotrophs are capable of converting ammonia to hydroxylamine via the methane monooxygenase, whereas the ammonium monooxygenase can oxidize methane to carbon dioxide. The recently identified lithotrophic planctomycete possesses the ANAMMOX pathway, which is coupled to nitrite reduction (Strous et al., 1999). Table 2. Qualitative comparison of several components of the ANAMMOX technology with conventional nitrogen removal systems
System Num of reactor ber Feed Discharge Conditions Oxygen requirem ents pHcontrol Biom retention ass COD requirem ent Sludge production Reactor capacity (kg 3 N/m day) Bacteria 1 W astewater NH4+, NO2Oxic Low None None None Low 1 Aerobic NH4+ Oxidizers SHARON 1 Am onium nitrite m m ixture N2, NO3Anoxic None None Y es None Low 6-12 Planctomycetes ANAM MOX 1 W astewater N2, NO3Oxygen lim ited Low None Y es None Low 1-3 CANANON 2 W astewater N2, NO3- ; NO 2 Oxic; anoxic High Y es None Y es High 0.05-4 Conventional nitrification, denitrification

Aerobic NH4+ oxidizers Nitrifiers+ Various + Planctomycetes heterotrophs

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The ANaerobic AMMonium OXidation (ANAMMOX) process, which was discovered 10 years ago (Mulder, 1992) but already predicted to exist 30 years ago (Broda, 1977), could offer an alternative for the treatment of this return stream. Later, Van de Graff et al. (1997) and Bock et al. (1995) observed that nitrite was the preferred electron acceptor for the process. Also, other streams with high nitrogen and low carbon content such as landfill leachates and evaporator condensates could be treated. In the ANAMMOX process ammonium is oxidized under anoxic, i.e. oxygen depleted, conditions with nitrite as electron acceptor. Ammonium and nitrite are consumed on an almost equimolar basis. The ANAMMOX process should always be combined with a partial nitritation process, such as the SHARON process (van Dongen et al., 2001a&b), where half of the ammonium is oxidized to nitrite. Both autotrophic processes will increase the sustainability of wastewater treatment as the need for carbon addition (and concomitant increased sludge production) is omitted and oxygen consumption and the emission of nitrous oxide during oxidation of ammonia are largely reduced (Jetten et al., 1997). As such, the combined process (partial nitritation and ANAMMOX) was termed autotrophic nitrogen removal process (Jetten et al., 2002).



+ O2

Nitrogen Fixation Classical nitrogen removal Autotrophic nitrogen removal

Figure 1. ANAMMOX process in nitrogen cycle

OCCURANCE The existence of ANAMMOX bacteria capable of producing nitrogen gas from ammonium and nitrate/nitrite was demonstrated for the first time in denitrifying fluidized bed reactor treating sewage sludge digester effluent and ammonia laden wastewater (Mulder et al., 1995; and Van de Graff et al., 1995), in marine sediments (Thamdrup and Dalsgaard, 2002), Black sea sediments

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(Kuypers et al., 2003), anoxic waters of Golf Dulce, a 200 m deep coastal bay in Costa Rica (Dalsgaard et al., 2003) and landfill environments (Chung et al., 2003).

MICROBIOLOGY OF ANAMMOX Microbial nitrogen metabolism also plays an important role in the global nitrogen cycle. Microbial activities, such as denitrification and ANAMMOX, are the major mechanisms that convert combined nitrogen to dinitrogen gas, thereby completing the nitrogen cycle. The updated nitrogen cycle with ANAMMOX is depicted in Figure 3 (after Jetten et al., 1999). Nitrification is the aerobic oxidation of NH3 to NO3-. It consists of two sequential steps carried out by two phylogenetically unrelated groups of aerobic chemolithoautotrophic bacteria. Some heterotrophic bacteria can also oxidize ammonium to nitrate, but this is only a very small contribution to the overall ammonia oxidation (Pynaert, 2003). No single known autotrophic bacterium is capable of complete oxidation of NH3 to NO3- in a single step (Abeliovich, 1992). In view of coupling a partial nitrification unit with an Anammox unit, nitrite oxidising activity should be suppressed and TAN should only be oxidised for about 50 % to TNO2. The physiology of anaerobic ammonium oxidizing aggregates cultivated in a sequencing batch reactor was investigated by Strous et al. (1999). The maximum specific substrate conversion rate of the ANAMMOX biomass was measured as a function of temperature and pH in batch experiments. From the temperature dependency of ANAMMOX activity, the activation energy was calculated to be 70 kJ/mol. Strous et al. (1998) have also reported that the affinity constants for the substrates, ammonium and nitrite, are less than 0.1 mg N/L inhibited ANAMMOX process completely. In another study Strous et al. (1999) have shown that the ANAMMOX process was reversibly inhibited by the presence of oxygen. Bacteria capable of anaerobically oxidizing ammonium had not been known earlier and were referred as the lithotrophs missing from nature (Shivaraman and Geetha, 2003). These missing lithotrophs were discovered and identified as the new autotrophic members of the order of planctomycete, one of the major distinct division of bacteria (Strous et al., 1999a). The anaerobic ammonium oxidation reaction is carried out by two ANAMMOX bacteria that have been tentatively named as Brocardia anammoxidans (Strous et al., 1999a) and Kuenenia stuttgartiensis (Schmid et al., 2000). The high ANAMMOX activity observed for both bacteria in a pH range between 6.4 and 8.3 and temperature between 20oC and 43oC (Strous et al., 1999b; and Egli et al., 2001). The ANAMMOX bacterial activity is 25-fold higher than aerobic nitirifying bacterial oxidation of ammonium under anoxic conditions when using nitrite as the electron acceptor (Jetten et al., 1999). Acetylene, phosphate and oxygen are known to be strongly inhibiting ANAMMOX activity (Van De Graaf et al., 1996). BIOCHEMISTRY OF ANAMMOX The possible metabolic pathways for anaerobic ammonium oxidation are depicted in Figure 4. (Van de Graff et al., 1997). The ANAMMOX process is based on energy conservation from anaerobic ammonium oxidation with nitrite as electron accpetor without addition of external carbon source (Jetten et al., 1999). Hydrazine and hydroxylamine are known to be some

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intermediates of the process (Van de Graff et al., 1997; Schalk et al., 1998; and Jetten et al., 1999). Carbon dioxide is the main source for the growth of ANAMMOX bacteria (Van de Graff et al., 1997). Two possible pathways were hypothesized by van de Graaf et al. (1997) for the ANAMMOX process: Oxidation of ammonium ion to hydroxylamine, that reacts with nitrite which is further reduced to nitrogen. Hydroxylamine-formation from ammonium ion via the ammonium monooxygenase, however, seems unlikely because of the strong oxygen inhibition (van de Graaf et al., 1996; Jetten et al., 1999). Partial reduction of nitrite with the formation of hydroxylamine (NH2OH), that reacts further with ammonium to form hydrazine (N2H4). Hydrazine is further converted into nitrogen. This oxidation would give the necessary reducing equivalents for the initial reduction of nitrite.







4H+ N=N



Figure 4. Metabolic pathway in ANAMMOX bacteria N-labeling experiments showed that this second possibility is the correct one (van de Graaf et al.,1997). The addition of labelled hydroxylamine led to the formation of labelled nitrogen gas, in contrast to the addition of 15N2O. Sustained growth on hydroxylamine or hydrazine is however not possible (Schalk et al., 1998). Strous et al. (1999b) did notice that the addition of at least 50 M of these intermediates resulted in complete recovery of the ANAMMOX activity after inactivation with TNO2. Schalk et al. (2000) succeeded in purifying and characterizing the hydroxylamine Oxidoreductase/hydrazine reductase (HAO/HZO) of an ANAMMOX culture. The HAO/HZO was able to oxidize both hydroxylamine and hydrazine under anoxic conditions to respectively NO, N2O and N2. The HAO/HZO made up 9 % of the total soluble protein fraction of the ANAMMOX species Candidatus Brocadia anammoxidans. Schalk et al. (2000) also found that hydrazine strongly inhibits the oxidation of hydroxylamine. Kuenen and Jetten (2001) have suggested the most plausible hypothesis for the ANAMMOX mechanism. Nitrite reduction by a nitrite reducing enzyme leads to the formation of hydroxylamine. An unknown hydrazine hydrolase converts ammonia and hydroxylamine to hydrazine that is converted into nitrogen by HAO/HZO. This oxidation would give the necessary reducing equivalents for the initial reduction of nitrite. In the biochemical model, the

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ANAMMOX reaction establishes a proton gradient by the effective consumption of protons in the riboplasm and production of protons inside the anammoxosome, a mechanism known as separation of charges. This result in an electrochemical proton gradient directed from the anammoxosome to the riboplasm. Based on isotopic carbon analysis Schouten et al. (2004) concluded that different ANAMMOX bacteria, such as Candidatus Scalindua sorokinii and Candidatus Brocadia anammoxidans use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway. APPLICATION OF ANAMMOX IN LEACHATE TREATMENT Recent research has permitted the development of new ways of nitrogen removal, such as the partial nitrification and the anaerobic oxidation of the ammonium (ANAMMOX), which represent significant advances in the field of biological removal of the nitrogen pollution. The application of a combined partial nitrificationANAMMOX process to the treatment of high ammonia nitrogen content influents, ex. leachate, is particularly promising. It would lead to potential savings of up to 60% in oxygen generation and 100% in external carbon, besides significantly reducing the sludge generation and the net emission of CO2 (Van Dongen et al.,2001), diminishing the total treatment operating cost up to 90 % (Jetten et al., 2001). The introduction of partial nitrification/ANAMMOX to the treatment of high-strength wastewaters will lead to substantial savings of energy and resources. Such systems have been tested over prolonged periods and demonstrated stable effluent quality and compact ammonium removal without the need for process control. Given the low costs of our system, a full-scale implementation is to be expected in the near future. LIMITATIONS ANAMMOX coupled to nitrite reduction offers opportunities in the area of process development of nitrogen removal systems. One of the biggest challenges is how to accelerate the slow rate of nitrogen removal from these systems (the rate is less than half that of aerobic nitrification) (Strous et al., 1999; and Jetten et al., 1998). However, from a commercial application perspective, the more challenging issue is the extremely slow growth rate (10-14 days) of the bacteria known to carry out these reactions. Similar to aerobic nitrification, ANAMMOX is subjected to inhibition. This process requires anaerobic conditions for ammonia oxidation, but inhibition by oxygen is reversible FUTURE STUDY ANAMMOX technology has been evaluated using synthetic wastewater/sludge digester effluent from domestic WWTP. Research is necessary to know the feasibility of applying ANAMMOX process technology with other actual wastewater and leachates using appropriate reactor types and configuration. The performance of ANAMMOX process in treating actual wastewater/leachate would not only depend on ANAMMOX bacteria but also on the co-existence of other important oxygen scavenging and ammonia generating/ammonia to nitrite oxidizing bacteria. Research is needs to be carried out to work out optimal conditions for such an ecosystem to sustain in a reactor and develop methodologies to monitor the responsible microbial community in the system. Applied genomic research can be used to identify genes and

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patterns of expression that are critical to the performance of nitrogen metabolism in responses can be coupled with reporter systems for the development of online measurement systems. Coupling the advances related to bacterial nitrogen metabolism with improved monitors of macroscopic performance should lead to more robust operating strategies for wastewater bioreactors. Genomic information, in combination with traditional biochemical, genetic and ecological studies is needed to understand the inorganic nitrogen metabolism, and thus benefit their industrial applications. ACKNOWLEDGEMENT The authors wish to thank the financial support from Swedish International Development Agency (SIDA), and technical co-ordination from Asian Institute of Technology (AIT), Bangkok, Thailand. The cooperation of Chennai Corporation in sample collection from Kodungaiyur and Perungudi dumping grounds is gratefully acknowledged.

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