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ANAEROBIC AMMONIUM OXIDATION

(ANAMMOX) PROCESS FOR NITROGEN
REMOVAL - A REVIEW
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2 AUTHORS:
Obuli Karthik

Kurian Joseph

Hong Kong Baptist University

Anna University, Chennai

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ANAEROBIC AMMONIUM OXIDATION (ANAMMOX) PROCESS FOR

NITROGEN REMOVAL A REVIEW
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

-----------------------------------------------------------------------------------------------------------------------------------------------------------------*
1

Research Scholar and Corresponding author: opkens@gmail.com

Assistant Porfessor: kuttiani@vsnl.com

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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

Total Nitrogen (mg/L)

500 - 2500
800 - 1100

Reference
Chung et al (2003)
Abeling and Seyfried (1993)

1600

Deng Peterson et al (2003)

170 - 200

Keller et al (1997)

128 - 185

Marat et al (2003)

Discarded water

260 - 958

Gil and Choi (2004)

Source; Hulle 2005

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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

SHARON

ANAMMOX

Conventional
nitrification,
denitrification

CANANON

Number of reactor

Feed

Wastewater

Ammonium nitrite mixture

Wastewater

Wastewater

Discharge

NH4+, NO2-

N2, NO3-

N2, NO3-

N2, NO3- ; NO2-

Conditions

Oxic

Anoxic

Oxygen limited

Oxic; anoxic

Oxygen requirements

Low

None

Low

High

pHcontrol

None

None

None

Yes

Biomass retention

None

Yes

Yes

None

COD requirement

None

None

None

Yes

Sludge production

Low

Low

Low

High

Reactor capacity (kg

N/m3 day)

6-12

1-3

0.05-4

Bacteria

Aerobic NH4+
Oxidizers

Planctomycetes

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).

N2
N2
TA
TA

+ COD

ANAMMOX

+ O2

+ O2
TNO

TNO
TNO
+ O2

+ COD
NO3-

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.

5H+

NH2 OH

Cytoplasm

NI
4e-

H
HA

Anammoxosome

4H+

H2N=NH2

NO2-

NH3

N=N

Figure 4. Metabolic pathway in ANAMMOX bacteria

15

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

IN - 108

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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