10CriticalReviews Anammox 2012

See discussions, stats, and author profiles for this publication at: https://www.researchgate.
net/publication/232710702
Critical Reviews in Environmental Science and Technology

Autotrophic Ammonia Removal Processes: Ecology to Technology
Article in Critical Reviews in Environmental Science and Technology · August 2012

DOI: 10.1080/10643389.2011.556885
CITATIONS READS
29 1,877
3 authors, including:
Samik Bagchi Rima Biswas

Ghent University National Environmental Engineering Research Institute
67 PUBLICATIONS 186 CITATIONS 18 PUBLICATIONS 222 CITATIONS
SEE PROFILE SEE PROFILE
Some of the authors of this publication are also working on these related projects:
Chemo-biochemical desulfurization of industrial emissions containing oxides of sulfur View project
REWATER View project
All content following this page was uploaded by Rima Biswas on 16 May 2014.
The user has requested enhancement of the downloaded file.

This article was downloaded by: [National Envir Engg Res Instt]
On: 11 May 2012, At: 03:21
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Critical Reviews in Environmental

Science and Technology
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/best20
Autotrophic Ammonia Removal

Processes: Ecology to Technology
a a a
Samik Bagchi , Rima Biswas & Tapas Nandy
a
Wastewater Technology Division, National Environmental
Engineering Research Institute, Council of Scientific and Industrial
Research, Nagpur, India
Available online: 30 Aug 2011
To cite this article: Samik Bagchi, Rima Biswas & Tapas Nandy (2012): Autotrophic Ammonia Removal
Processes: Ecology to Technology, Critical Reviews in Environmental Science and Technology, 42:13,
1353-1418
To link to this article: http://dx.doi.org/10.1080/10643389.2011.556885
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation
that the contents will be complete or accurate or up to date. The accuracy of any
instructions, formulae, and drug doses should be independently verified with primary
sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
demand, or costs or damages whatsoever or howsoever caused arising directly or
indirectly in connection with or arising out of the use of this material.
Critical Reviews in Environmental Science and Technology, 42:1353–1418, 2012
Copyright © Taylor & Francis Group, LLC
ISSN: 1064-3389 print / 1547-6537 online
DOI: 10.1080/10643389.2011.556885
Autotrophic Ammonia Removal Processes:

Ecology to Technology
SAMIK BAGCHI, RIMA BISWAS, and TAPAS NANDY

Wastewater Technology Division, National Environmental Engineering Research Institute,
Council of Scientific and Industrial Research, Nagpur, India
Downloaded by [National Envir Engg Res Instt] at 03:21 11 May 2012
The last decade has witnessed rapid spur in technoeconomic au-

totrophic ammonia removal technologies for wastewater treatment
such as SHARON, ANAMMOX, SNAD, CANON, OLAND, DEMON,
and BABE. These technologies have the potential to remove high
concentrations of ammonia in wastewaters. Despite their high re-
moval efficiency, the quantum of full-scale applications of these
processes is far from trivial. The issues that create a bottleneck in
the application of such processes are often overlooked. Recent dis-
coveries made in marine anaerobic niches provide some clues for
resolving the problems faced while implementing these processes
commercially. Some thoughts on the future research areas are also
presented.
KEY WORDS: ammonia, ammonia oxidation, anaerobic ecology,

microbial interaction, technology
INTRODUCTION
Ammonia removal through biological oxidation has always been a matter

of concern for environmental engineers because of its toxic effect on all
biological forms of life in receiving water bodies, and also due to its rela-
tive hindrance in stabilizing into its nontoxic product nitrogen. In almost all
wastewater treatment plants (WWTPs) ammonia is removed by nitrification
(conversion of ammonia to nitrate) and denitrification processes (conversion
of nitrate to nitrogen). In terms of process operation, the nitrification and the
Address correspondence to Rima Biswas, National Environmental Engineering Research

Institute, Council of Scientific and Industrial Research, Wastewater Technology Division,
Nehru Marg, Nagpur 440 020, India. E-mail: ra biswas@neeri.res.in
1353
1354 S. Bagchi et al.
denitrification processes are mutually opposite. On one hand, nitrification is

carried out by autotrophic and aerobic groups of microorganisms, while on
the other hand, denitrification is carried out by anaerobic and heterotrophic
groups of microorganisms, making the entire process more complicated.
The rise in the demands for environmental protection has forced the de-
velopment of more efficient nitrogen-removing technologies in wastewater
treatment. The new technologies that have been developed are mostly based
on the anaerobic ammonium oxidation (ANAMMOX) process in combina-
tion with partial nitrification. These processes are more efficient than the
conventional nitrification-denitrification processes in terms of time, space,
and cost economics. The state-of-the-art nature of these technologies has
already been reviewed extensively (Ahn, 2006; Khin and Annachhatre, 2004;
Kuenen, 2008; Schmidt et al., 2003; Sun et al., 2010; Zhang et al., 2008). The
outcome of this introspection has clearly outlined the practical utility of these
technologies. Even 15 years after its discovery, the ANAMMOX process and
the processes based on it have limited full-scale application (Lopez et al.,
2008), which is mostly due to two reasons. First, the slow growth rate of
the anaerobic ammonia-oxidizing bacteria (AOB) lead to very long start-up
period and, second, the intricate process controls needed to achieve a typical
ammonia to nitrite ratio for the ANAMMOX process is very atypical in the
wastewater (Bagchi et al., 2010b, 2009).
Thus, there are two important things to understand in the ANAMMOX
process. First, the anaerobic ANAMMOX bacteria are dependent on the avail-
ability of nitrite in the anoxic environments and, second, nitrite is provided
by aerobic AOB in the oxic environment. In oxygen minimum zones (OMZ),
AOB compete with ANAMMOX bacteria for ammonia. Regulating the fine
balance of the oxic-anoxic interphase in a manmade ecosystem appears to
be the root cause of all problems in the field application of ANAMMOX-
based technologies. Hence, it is very essential to understand the underlying
microbial mechanisms, their interrelationship and interactions to achieve sus-
tainable operation control, and consequently successful field applications of
ANAMMOX-based technologies. Recent advances in molecular genetics and
evolutionary analysis have contributed to significant progress in the role of
aerobic and anaerobic AOB in OMZ.
This article is an effort to link the microbial interactions in ecological
environments to technological process developments for the successful field
application of autotrophic ammonium oxidation processes in wastewater
renovation. Before the link is established, a brief introduction on related
microbial pathways of importance concerning biological ammonia oxidation
in global scenario is presented. A short description of various pathways
on microbial ammonia oxidation and the biological processes based on it
is also addressed. Finally, the challenges faced during scaling up of the
technologies, and gap of knowledge between the technologies and science
behind the existing ecology are discussed.
Autotrophic Ammonia Removal Processes 1355
AMMONIA-OXIDIZING MICROORGANISMS
The pioneering studies carried out by Beijerinck and Kluyver, founders of the
Delft School of Microbiology, on the bacterial inorganic nitrogen conversion
are true to date (Jetten et al., 1997b). Nitrification is basically considered to
be an obligatively aerobic and lithotrophic process. In the last 1.5 decades,
a range of new microbial principles on inorganic nitrogen conversion mech-
anism has been discovered (Fiencke et al., 2005; Konneke et al., 2005; Poth,
1986; Schmidt and Bock, 1997; Strous et al., 1999b). Besides lithotrophic nitri-
fication by bacteria, various fungi, algae, heterotrophic bacteria and Archaea
have also been found to be involved in the process of nitrification (Fiencke
et al., 2005; Hayatsu et al., 2008). The anoxic metabolism by the aerobic Nitro-
somonas species and the discovery of novel pathway of anaerobic ammonia

oxidation by the group Planctomycetes has challenged the classical concepts
of ammonia oxidation. Studies through molecular ecological techniques and
genome analysis have revealed greater diversity of microorganisms involved
in nitrogen cycle that are contributing to development of autotrophic nitro-
gen removal processes. For example, the relative contribution of fungi and
bacteria in release of N2 O in soil ecosystems has been found to be 70%
and 20%, respectively (Hayatsu et al., 2008). Underwater filled pore spaces
of soil, around 20% of the nitrogen loss is contributed by aerobic denitri-
fiers (Bateman and Baggs, 2005). Similarly, in marine environments with low
organic contents, ANAMMOX contributes to up to 20–50% of nitrogen pro-
duction through ammonia oxidation (Strous and Jetten, 2004), while in the
soil ecosystem archeal nitrification is now evolving as the major player for
autotrophic ammonia oxidation (Francis et al., 2007). Detailed reviews on
the metabolic pathways of microorganisms capable of autotrophic ammonia
oxidation under aerobic, anoxic, and anaerobic conditions have been dis-
cussed extensively elsewhere (Ahn, 2006; Arp et al., 2002; Arp and Stein,
2003; Jetten et al., 1997b, 1999; Schmidt et al., 2002; Wrage et al., 2001).
This section focuses briefly on the microbial principles in context to their
application in wastewater treatment.
Ammonia Oxidation by Nitrosomonas Species

Most of the AOB fall within one taxonomic group in the b subclass of the
Proteobacteria. Nitrosomonas europaea and Nitrosomonas eutropha are the
most studied autotrophic ammonia oxidizers to date. Nitrosomonas species
are a group of chemolithotrophic AOB that derive energy from ammonia
oxidation and assimilate carbon from atmospheric CO2 . Through advances
in molecular technology, the high through-put whole-genome sequencing
has tremendously increased the knowledge on microbial physiology of au-
totrophic ammonia oxidizers and reconstructed the molecular evolutionary
history of these physiologies (Arp et al., 2007; Klotz and Stein, 2008).
Autotrophism was considered to be the only mode of growth for such

β-proteobacteriace group of bacteria. Although chemolithoheterotrophic
growth of N. europaea with fructose or pyruvate as the carbon source (Arp
et al., 2007; Hommes et al., 2003) and chemoorganoheterotrophic growth
of Nitrosomonas species with variety of organic carbon sources have also
been reported (Schimdt, 2009). Until recently, O2 -dependent aerobic oxida-
tion of ammonia was thought to be the only pathway for ammonia oxidation
by this autotrophic ammonia-oxidizing bacteria (AOB). Schmidt and Bock
(1997), first demonstrated the anaerobic ammonia-oxidizing capacity of Ni-
trosomonas eutropha.
These microorganisms can not only oxidize ammonia to nitrite (NO− 2)
under anaerobic condition (anaerobic ammonia oxidation) and but can also
reduce the nitrite generated from both aerobic and anaerobic ammonia ox-
idation pathways to nitric oxide (NO−), nitrous oxide (N2 O−) and finally
to molecular nitrogen (N2 ) through, nitrifiers denitrification pathway. Be-
cause of its metabolic versatility this organism is suitable for adaptation un-
der extreme conditions encountered during wastewater treatment (Park and
Noguera, 2007; Schmidt and Bock 1998; Schmidt et al., 2001a; Schmidt et al.,
2001b; Zart et al., 2000). The metabolic scheme as presented in Figure 1
depicts the relationship between the three modes of energy generation in
Nitrosomonas species (i.e., aerobic ammonia oxidation, anaerobic ammo-
nia oxidation and nitrifier denitrification). The aerobic ammonia oxidation
FIGURE 1. The three energy-evolving pathways in Nitrosomonas species. Figure modified,

with permission, from Bagchi et al. (2009) (Color figure available online).
pathway has been worked out extensively and reviewed by various authors
(Arp et al., 2002; Arp and Stein, 2003; Hooper et al., 1978; Hooper et al.,
1997). Hence, this section is not discussed in this paper; instead the other
two metabolic pathways are discussed in view of their eco-physiological role
in wastewater treatment.
NITRIFIER DENITRIFICATION
The term nitrifier denitrification has been coined to avoid confusion be-
tween denitrification carried out by autotrophs and those by heterotrophs
(Wrage et al., 2001). As the name suggests, heterotrophic denitrification is
carried out by heterotrophic anaerobic bacteria and is in contrast to nitrifier
denitrification carried out by autotrophic aerobic bacteria.
In nitrification, N2 O is produced as a by-product during ammonia oxida-

tion by AOB. The unstable intermediate (HNO−) formed during oxidation of
NH2 OH to NO− −
2 is spontaneously decomposed to N2 O (Hooper and Terry,
1979). N2 O− is also released as a by-product of the denitrification pathway.
The relative contribution of nitrification and denitrification to N2 O− produc-
tion can be determined using the stable isotope technique (Sutka et al., 2006)
or a selective inhibitor such as acetylene (Wrage et al., 2004).
Production of nitrous oxide as an intermediate step during the nitri-
fication pathway has been known since 1960s, but its ecological impact
by the soil ecosystem was first realized in 1978 (Bremner and Blackmer,
1978; Lipschultz et al., 1981). Previous workers attributed the nitrification
pathway to the cause of N2 O release to the atmosphere, both in soil and
marine sediments. This conclusion was derived from the observations that
(a) N2 O emissions have a correlation with ammonia concentrations, (b) the
amount produced was the function of soil moisture (i.e., anaerobicity), and
(c) N2 O emissions were completely stopped by the application of nitrifi-
cation inhibitors, namely, acetylene and nitropyrene. Through a series of
15
N isotope tracer experiments, Poth and Fotch (1985) reported that Nitro-
somonas europaea produces N2 O only under oxygen-limiting conditions and
that the labeled N from nitrite, but not nitrate, is incorporated into N2 O, in-
dicating the presence of denitrifying enzyme, nitrite reductase. They further
observed that no labeled nitrite was reduced to ammonium and concluded
that N. europaea is a denitrifier, which under oxygen stress utilizes nitrite
as terminal electron acceptor and produces nitrous oxide. Poth (1986) also
demonstrated denitrification of nitrite to nitrogen in Nitrosomonas isolate. He
attributed denitrification as an energy-generating mode under oxygen stress
but could not feature any electron donor for this pathway. Bock (1995) re-
solved Poth’s dilemma and stated that hydrogen was used as electron donor
and the energy-generating nitrifier denitrification pathway in N. eutropha
is probably meant for reducing nitrite accumulation under oxygen-limiting
conditions. However, during the whole genome sequencing of Nitrosomonas
species, genes that may allow utilization of inorganic energy sources such
as H2 , CO, Fe2+ and S2− were not identified except in N. multiformis (Arp
et al., 2007).
The two enzymes responsible for nitrifier denitrification have been iden-
tified so far in the genome of N. europaea ATCC 19718 as nitrite reductases
(nir) and nitric oxide reductases (nor) (Beaumont et al., 2004a; Beaumont
et al., 2004b; Schmidt et al., 2004a). Nitrous oxide reductase (N2 O reduc-
tase) has not been detected yet in Nitrosomonas species (Schmidt et al.,
2004a). This is in contrast to detection of nitrogen from nitrite reduction in
Nitrosomonas species isolated by Poth (1986) from natural sources. But, Ni-
trosomonas europaea ATCC 19718 cannot produce molecular nitrogen (Poth,
1986). A recent study using a mutant deficient for denitrifying enzyme, Nir
K or Nor B has shown that nitrifier denitrification was the major source of
N2 O− produced by N. europaea ATCC 19718 (Schmidt et al., 2004a). N2 O− is
often detected as the final product during nitrifier denitrification. This implies
that not all species of autotrophic ammonia oxidizers can produce molecular
nitrogen through nitrifier denitrification pathway.
Nitrifier denitrification pathway was first elucidated in N. europaea. But,
N. europaea is not the dominating AOB in the soil. Nitrospira sp. dominate
in soil ecosystem where it is one of the major contributors to global N2 O−
emissions. Shaw et al. (2006) showed production of N2 O− in seven strains
of cultured Nitrospira lineage through nitrifier denitrification pathway. It is
imperative from this study that denitrifying ability is a wide spread trait in
ammonia oxidizers, if not ubiquitous.
The ecological significance of the nitrifier denitrification pathway is be-
ginning to be understood. There are many views regarding its ecological
significance. One view suggested reduction of nitrite as a strategy to reduce
competition for oxygen by nitrite oxidizers by removing their substrate (i.e.,
nitrite). Another view is that nitrite is reduced to conserve oxygen and to pro-
duce energy in low oxygen environments (Poth and Focht, 1985; Schmidt and
Bock, 1997) as in classical heterotrophic nitrification. Zumft (1997) opined
that if the role of ammonia oxidizers in denitrifying NO2 is same as in the
heterotrophs, the pathway should be regulated by the concentration of NO2
and oxygen available. However, NorB and nirK are expressed aerobically
and nirK is also expressed in response to increasing concentration of nitrite
(Beaumont et al., 2004a). The later findings also suggest that the role of
nitrifier denitrification is to protect the cells from the toxic concentrations of
nitrite, produced during nitrification (Beaumont et al., 2002, 2004a; Stein and
Arp, 2003).
Even though NorB and nirK are expressed in aerobic conditions, their
expression is increased several folds (approximately 700 times) under anaer-
obic conditions (Beaumont et al., 2004b; Schmidt et al., 2004b). This ob-
servation was correlated with AOB found in environments with high con-
centrations of ammonia and high availability of carbon dioxide. With the
availability of high substrate concentrations, AOB has to compete with other
heterotrophic microorganisms for iron and oxygen. Hence such AOB are
equipped with higher detoxification capacities and ability to use terminal
oxidases that can reduce electron acceptors other than oxygen (Arp et al.,
2007).
Genomic analysis has revealed that the nirK gene clusters of Nitro-
somonas and Nitrobacter genera are upregulated by a transcriptional regu-
lator NsrR (a member of the Rrf2 family of transcriptional regulators; Beau-
mont et al., 2004a; Cantera and Stein, 2007). Although putative nsrR genes
are also identified in the genomes of Nitrosopira multiformis and Nitroso-
coccus oceani, they are not near the nirK genes, nor are the nirK genes
are preceded by the NsrR binding motif. Thus, it appears that NsrR-specific
regulation of nirK occurs only in nitrifiers adapted to high ammonia con-
centration (Cantera and Stein, 2007).

High ammonia concentrations lead to elevated NOx production, in-
cluding production of toxic NO−. To reduce toxicity due to NOx, nitrite
reductases such as NorB, P460 , and C554 reduce NO− − −
2 to NO and N2 O , re-
− −
spectively. NsrR can sense NO2 and NO concentrations directly through its
2fe-2S clusters and it regulates many NOx-dependent operons such as nirK
gene clusters in nitrosomonads and it regulates many NOx-dependent indi-
rect regulation of several regulons of SoxR, OxyR, metR, FNR, and Fur. The
nirK gene cluster is involved in biotin synthesis, iron uptake, and synthesis
of coxAB. Recently it has been proposed that the CoxAB complex found in
AOB and in certain bacterial sulfur oxidizers is basically a NO− reductase
(Stein et al., 2007). Upon sensing increased concentration of NO− −
2 /NO , the
nirK mutant cell likely attempts to repair the iron-sulfur centers in NsrR, up-
regulate Fur-dependent iron uptake genes, and increase transcription of nirK
genes cluster and abate the threat. Hence, it is not surprising that NorB or
nirK mutant cells can still maintain a functional NOx metabolism (Beaumont
et al., 2002; Schmidt et al., 2004a).
The unfolding of nitrifier genome has also thrown light on the evo-
lutionary significance of the nitrifier denitrification pathway. The incom-
plete denitrification pathway or the nitrifiers’ denitrification has more prim-
itive evolutionary history than the complete denitrification pathway. In the
very early anoxic atmosphere, N2 was abundant and there was no evo-
lutionary pressure on the biotic nitrogen cycle to return the fixed nitro-
gen (NH3 or NO3 ) back to atmosphere. Hence, the denitrification pathway
was incomplete and was carried out by the autotrophic bacteria. The com-
plete denitrification pathway by the heterotrophic denitrifiers evolved af-
ter the earth’s crust became abundant with metals such as copper, which
formed the cofactors for the denitrification (Klotz and Stein, 2008). The
significance of nitrifier denitrification in biological nitrogen removal can-
not be underestimated. NO2 is a potent greenhouse gas (GHG) and its
emission is directly related to prevalence of anoxic conditions in nitrifying
reactors.
ANAEROBIC AMMONIA OXIDATION

After the discovery of denitrification in Nitrosomonas, it was known that Ni-
trosomonas eutropha, an obligatively lithoautrophic bacterium, was able to
nitrify and denitrify simultaneously when gaseous nitrogen dioxide was sup-
plemented to the atmosphere (Schmidt and Bock, 1997). The first evidence
of complete conversion of ammonia to dinitrogen under anoxic conditions
by Nitrosomonas europaea was given by Shrestha et al. (2001, 2002).
Through studies with cell free extract, Schmidt and Bock (1998) showed
that ammonia was oxidized to hydroxylamine and NO− under an oxygen-free
environment using NO− −
2 /N2 O4 as an oxidant. Interestingly, extremely low
ammonia oxidation activities in cell-free extracts under oxic environments
was observed and it was conferred that oxygen is inhibitory to ammonia
oxidation in cell-free extracts. Nitrite was found to be essential for stimu-

lating ammonia oxidation both under oxic and anoxic conditions (Schmidt
et al., 2001a, 2001b) and was observed to retrieve ammonia oxidation activ-
ities in cells that have either lost ammonia oxidation potential (Schmidt and
Bock, 1998) or are starved for long time under ammonia deficient conditions
(Laanbroek et al., 2002). Further studies have shown that NO rather than
NO2 was essentially required for anaerobic ammonia oxidation by Nitro-
somonas and that ammonia oxidation was distinctly inhibited when gaseous
NO was removed from the cultures either by means of intensive aeration,
chemical binding with DMPS (2,3-Dimercapto-1-propane-sulfonic acid), or
by detoxifying activity of Pseudomonas PS 88 (Zart et al., 2000). Until then,
formation of NO was interpreted as formation of by-product of nitrification
for ammonia metabolism.
As shown by Schmidt and Bock (1997, 1998), NO2 was able to act as an
oxygen donor in anaerobic oxidation of ammonia by N. europaea (Figure 1).
Hence, NO2 might be involved in the conversion of ammonia to hydroxy-
lamine even in an oxic condition. Under an anaerobic condition, in a similar
mechanism as O2 -dependent aerobic oxidation, one molecule of NH3 is ox-
idized by one molecule of N2 O4 to form two molecules of NO and one
molecule hydroxylamine (Equation 1). Hydroxylamine is further oxidized to
HNO2 , while NO remains in the system (Equation 2). This oxidation is cat-
alyzed by hydroxylamine oxidoreductase (HAO) and releases four electrons,
two of which are required for first reaction and the remaining two electrons
are utilized for assimilation and energy generation (Schmidt et al., 2002). The
second step of oxidation of hydroxylamine to nitrite by HAO does not re-
quire molecular oxygen, but rather oxygen from water is used for oxidation
(Anderson and Hooper, 1998; Hooper et al., 1984). The nitrite produced acts
as an electron acceptor for the reducing equivalents leading to the formation
of dinitrogen gas, as per Equation 3 (Schmidt and Bock, 1998).
NH3 + N2 O− + −
4 + 2H + 2e → NH2 OH + H2 O + 2NO
−
(1)
NH2 OH + H2 O → HNO2 + 4H+ + 4e− (2)
NH3 + N2 O− − +
4 → HNO2 + 2NO + 2H + 2e
−
(Net reaction) (3)
HNO2 + 3H+ + 3e− → 0.5N2 + 2H2 O (4)
Oxidation of ammonia to hydroxylamine is catalyzed by ammonia

monooxygenase (AMO). Because it was known that AMO oxidizes ammonia
along with other compounds such as methanol, formaldehyde, methyl fluo-
ride, and dimethyl ether (Hyman et al., 1994; Voysey and Wood, 1987), it was
hypothesized that AMO has relaxed active site for oxygen and hence can use
N2 O4 in place of O2 under anoxic conditions for oxidation of ammonia to
hydroxylamine. Although AMO is assumed to catalyze oxidation of ammo-
nia with NO2 /N2 O4 as an electron acceptor under an anoxic condition, this
reaction was not inhibited in presence of acetylene (Schmidt et al., 2001a).

This study clearly demonstrated difference between the O2 -dependent and
NO2 -dependent/O2 -independent oxidation of ammonia by AMO.
The three energy-generating pathways in Nitrosomonas make it very a
adaptable microorganism under various environmental conditions. Hence,
these species have managed to stay active for long periods under most un-
favorable conditions. The key factor that aids in switching from one mode
of pathway to another has been speculated to be oxygen and nitrite. As dis-
cussed, under an anoxic environment, low dissolved oxygen concentration
and presence of nitrite triggers the metabolic enzymes of the nitrifier denitri-
fication pathway. Similarly, absence of oxygen forces these microorganisms
to use nitrite as oxidizing agent for oxidation of ammonia to hydroxylamine.
Therefore, anoxic oxidation of ammonia depends on the transport of NO− 2
from an oxic environment to the anoxic environment. When external sup-
ply of NO− −
2 is not available, cells generate NO during anaerobic ammonia
−
oxidation and reoxidize to NO2 in presence of O2 (Schmidt et al., 2002;
Zart et al., 2000; Figure 1). The mechanism of producing NO2 in biological
systems under aqueous environment from oxidation of NO was assumed to
be similar to the reaction proposed by Bodenstein (1918) under gas phase
(Zart et al., 2000 and the references therein). Hence, anaerobic ammonia
oxidation can effectively occur at oxic/anoxic interphase. However, the rate
of ammonia oxidation with NO− 2 as the sole oxidant was found to be 10%
of the rate of aerobic ammonia oxidation (Schimidt et al., 2001a).
ANAEROBIC AMMONIA OXIDATION BY ANAMMOX BACTERIA

The ANAMMOX process is a lithotrophic biological conversion of ammonia
to nitrogen under an anaerobic condition. The process is mediated by Planc-
tomycetes bacteria, also called the ANAMMOX bacteria. Although ammonia-
oxidizing Nitrosomonas species also have the ability of oxidizing ammonium
anaerobically along with its normal aerobic ammonium oxidation (Fux and
Siegrist, 2004; Schmidt and Bock, 1997; Zart and Bock, 1998), the ANAMMOX
reaction is exclusively carried out for nitrogen removal by Planctomycetes
bacteria as given in equation 5 (Jetten et al., 2001; Strous et al., 1998).
−
NH+ +
4 + 1.32NO2 + 0.066HCO3 + 0.13H →
1.02N2 + 0.26NO−
3 + 0.066CH2 O0.5 N0.15 + 2.03H2 O (5)
After the discovery of ANAMMOX bacteria by Strous et al. (1998; Ap-

pendix A), extensive research has been carried out to understand the micro-
biological and physiological properties of these groups of microorganisms
(Dapena-Mora et al., 2004a; Dapena-Mora et al., 2004b; Egli et al., 2001;
Schmid et al., 2000; Sinninghe Damste et al., 2002a; Sinninghe Damste et al.,
2002b; Strous et al., 1999a; Strous et al., 2002; Strous et al., 1999b). Several
reviews are reported describing the physiological, metabolic, and genomic

properties of these microorganisms (Jetten et al., 2003; Jetten et al., 2001;
Kartal et al., 2004; Kuenen, 2008).
Because ANAMMOX bacteria cannot be cultured by classical microbio-
logical practices, a molecular approach is the only alternative for detection,
isolation, and cultivation on a large scale (Bagchi et al., 2010a; Kuenen,
2008; Strous, 2000). Dominant ANAMMOX bacteria could be obtained phys-
ically from enrichment cultures through percoll density gradient centrifuga-
tion (Strous et al., 1999b). Table 1 enlists the various species identified so
far from three genera (Candidatus Brocardia, Scalindua, and Kunenia) in
the phylum Planctomycetes. Molecular analysis has revealed that significant
populations of ANAMMOX bacteria exist in almost all types of wastewater
treatment plants and in aquatic ecosystems. Marine sediments are stratified
habitats.
The biochemistry of ANAMMOX has been partly elucidated. Recently,
the 4.3 Mbp genome of Cancidatus Kunenia stuttgartiensis has been assem-
bled and annotated (Strous et al., 2006). Around 200 genes were found in
this species that was directly involved in catabolism and respiration. This
number of catabolic genes detected in Cancidatus Kunenia stuttgartiensis
TABLE 1. Microbial diversity of ANAMMOX bacteria discovered so far
Species Source Reference
Candidatus “Brocadia anammoxidans” Denitrifying reactor Strous et al., 1999a

Candidatus “Kuenenia stuttgartiensis” Wastewater Penton et al., 2006
Candidatus “Scalindua brodae” Wastewater Schmid et al., 2003
Candidatus “Scalindua wagneri” Wastewater Schmid et al., 2003
Candidatus “Scalindua sorokinii” Seawater Schmid et al., 2003
Candidatus “Brocadia fulgida” Wastewater Kartal et al., 2004
Candidatus “Jettenia asiatica” Not reported Tsushima et al., 2007
Candidatus “Anammoxoglobus propionicus” Synthetic water Kartal et al., 2007
Candidatus “Scalindua arabica” Seawater Woebken et al., 2008
exceeds the number possessed by most of the other bacteria and also in-
dicates the versatility of ANAMMOX bacteria in respiring different energy
sources with different electron acceptors. The ANAMMOX bacteria also en-
codes nine HAO-like proteins and two enzymes active in denitrification,
namely narGH and nirS. The two enzymes that are unique to ANAMMOX
bacteria are hydrazine hydrolase (HH; the enzyme that produces hydrazine
from nitric oxide and ammonium) and hydrazine dehydrogenase (HD; the
one that transfers electron from hydrazine to ferridoxin). Experiments with
purified HD using immunogold electronmicroscopy detected the enzyme in
a membrane-bound organelle (the anammoxosome) that made up more than
30% of the cell volume. In a follow-up study, it was found that this organelle
was composed of unique ladderane lipids—another characteristic feature of
these bacteria (van Niftrik et al., 2004). Studies have indicated evolution-
ary relationship between ANAMMOX bacteria belonging to Planctomycetes
and Chlamydiae through detection of a gene encoding a outer membrane
protein characteristic of Chlamydiae in all Planctomycetes members. Both
Planctomycetes and Chlamydiae are also characterized by peptidoglycan
less cell envelopes (Chopra et al., 1998; Fuerst, 2005; Strous et al., 2006).
Thus genomic analysis have revealed that the ANAMMOX bacteria, though
slow and specialized, they are the global players in the biological nitrogen
cycle and are generalists in their lifestyle adaptability in both natural and
artificial environment (Meyer et al., 2005; Penton et al., 2006; Schimid et al.,
2007; Schubert et al., 2006; Strous et al., 2006; Tal et al., 2005; Toh et al.,
2002).
In wastewater treatment the ANAMMOX by ANAMMOX bacteria is de-
limited by its difficulty in cultivating in large numbers. To sustain the activity
of ANAMMOX bacteria, favorable growth conditions are very essential for
maintaining the necessary population. Control of pH and temperature is one
of the important ways of controlling other competing anaerobic microorgan-
isms in a mixed culture system. ANAMMOX bacteria can sustain wide range
of pH (6.4–8.3) and temperature (20–43◦ C). An alkaline pH in the range
7.5–8.0 and temperature between 30 and 35◦ C are optimum for ANAMMOX
activity (Egli et al., 2001; Strous et al., 1999b). The highest ANAMMOX activ-
ity by Candidatus “Brocadia anammoxidans” has been reported to be 55 nM
N2 per milligram of protein per min at pH 8.0 and 40◦ C (Jetten et al., 1999).
Candidatus “Kuenenia stuttgartiensis” has similar pH and temperature op-
tima, which makes them suitable for application in tropical conditions (Egli
et al., 2001). ANAMMOX bacteria are very sensitive to oxygen and nitrite.
Oxygen concentration as low as 2 µM and NO− 2 concentrations between 5
and 10 mM inhibit the ANAMMOX activity completely but reversibly (Jetten
el al., 2001). In addition, ANAMMOX bacteria are also severely inhibited by
very low methanol and ethanol concentrations (Guven et al., 2005; Guven
et al., 2005). Under the anaerobic condition, the ammonia-oxidizing activity
of these bacteria is 25-fold higher than the anaerobic activity of Nitrosomonas
species (Jetten et al., 1999). Though it is 7 times slower than aerobic am-
monia oxidation, but it is still effective in removing high concentrations of
ammonia, particularly for wastewaters having low C/N ratio (Strous et al.,
1999b).
Archeal Nitrification
Until recently, ammonia oxidation was considered to be performed largely
by autotrophic AOB that form two distinct monophyletic groups within g
and β proteobacteria. After more than 100 years of this dogmatic view, our
understanding of nitrification has been upended twice in the last 15 years,
first by the discovery of anaerobic ammonium oxidation and more recently
by the discovery of aerobic ammonium oxidation within the domain of

Archaea.
For years microbiologists characterized Archaea as obligate ex-
tremophiles that thrive in harsh environments and they are metabolically spe-
cialized to survive under such conditions. Crenarchaeota have been known
as sulfur-metabolizing thermophiles until cultivation independent methods
uncovered vast numbers of Crenarchaeota in cold oxic ocean waters capa-
ble of aerobically oxidizing ammonia to nitrite under autotrophic conditions
(Konneke et al., 2005). Because of this initial discovery, crenarchaeal se-
quences have been recovered from all major moderate environment ecosys-
tems (Nicol and Schleper, 2006 and the references therein).
The ubiquitous Crenarchaeota are also abundant in the ocean (up to
98% of the total 16s rDNA copies) in the suboxic layers with oxygen levels
as low as 1 mM, including layers where previously ANAMMOX bacteria were
described (Coolen et al., 2007). Sinninghe Damste et al. (2002a; Sinninghe
Damste et al., 2002b) reported the presence of crenarchaeol in the OMZ of
the Arabian Sea where the oxygen level is less than 5 mM. This provided
indirect evidence of marine Crenarchaeota capable of surviving at low oxy-
gen levels. This is in contrast to most of the studies where abundance of an
archeal group was detected in oxic open ocean water from a wide variety of
locations (Bano et al., 2004; Francis et al., 2005; Herndl et al., 2005; Ingalls
et al., 2006; Karner et al., 2001; Massana et al., 2000).
Correlation between detection of crenarchaeol in the suboxic to OMZ
layers of the oceans and abundance of Crenarchaeota was often challenged,
as crenarchaeol is a recalcitrant compound and its presence in the suboxic
zones may derive from suspended or sinking dead cell debris. Studies carried
out by Coolen et al. (2007) using 16s rDNA and amoA provided stronger
indication of presence of living microorganisms in the suboxic waters of
stratified Black Sea.
Few metagenomic studies of seawater and soil revealed putative amoA
genes derived from uncultured Crenarchaeota (Coolen et al., 2007; Treusch
et al., 2005; Venter et al., 2004). Consequent to these findings many workers
have identified an amo homologue of A, B, and C genes on an archaeal asso-

ciated scaffold in various ecosystems (Coolen et al., 2007; Francis et al., 2007;
Nicol and Schleper, 2006). The definitive link between the novel amo genes
and the archaeal ammonia oxidation was recently established by cultivation
of an ammonia-oxidizing crenarchaeon—Nitrosopumilus maritimus (Kon-
neke et al., 2005). With this discovery, further studies revealed that many
marine Crenarchaeota might be capable of ammonia oxidation and have
been putatively identified at ammonia-oxidizing archaea (AOA; Francis et al.,
2005). Another putative amo gene cluster was isolated from C. symbiosum,
which was quasi-cultivated in laboratory aquaria at only 10◦ C as a symbiont
of the marine sponge Axinella mexicana (Hallam et al., 2006). The iden-
tification of genes encoding potential ammonia permease, urease and urea
transport system, and putative nitrite reductase and nitric oxide reductase
accessory proteins in C. symbiosum all indicated its chemoautotrophic am-
monia oxidation properties. However, homologues of the second enzymatic
step of bacterial ammonia oxidation—hydroxylamine oxidoreductase and
cytochromes C554 and C552 —were not identified, indicating that C. symbio-
sum in fact employs some other metabolism of ammonia oxidation (Hallam
et al., 2006). Besides genes related to chemoautotrophic ammonia oxidation,
many genes pertaining to modified 3-hydroxy propionate cycle and nearly
complete oxidative tricarboxylic acid cycle was also identified suggesting
that C. symbiosum has the potential to assimilate carbon both autotrophi-
cally and heterotrophically. Thus it is not surprising that Crenarchaeota are
ubiquitous in nature.
Phylogenetic analysis of both bacterial and archaeal amo genes show
that crenarchaeol genes are relatively distant to bacterial homologues.
However, around 25% sequence identity and 45% sequence similarity can
be found at the protein level between archaeal and bacterial variants that
coordinate potential metal binding centers indicating that these enzymes
share evolutionary history (Nichol and Schelper, 2006). Another study has
shown that amoA gene copies of Crenarchaeota were up to 3,000-fold more
abundant than those of AOB in 12 pristine and agricultural soils from three
climatic zones (Leininger et al., 2006). This indicated that Crenarchaeota is
a major contributor is both soil and oceanic ammonia oxidation. However,
studies that have compared laboratory growth kinetics of AOB to that of
the natural environment do not indicate the presence of a missing nitrifier
group. However, inhibition experiments have also indicated that AOB
are not the major contributors to nitrification processes in certain habitats
(Jordan et al., 2005). Thus studies are needed combining metagenomics and
cultivation to explore the evolutionary and ecological impacts of the archaeal
nitrification and to know if the phylogeny of nitrification originated from the
hot environments of oceanic sediments. Further, the role of Crenarchaeota
in autotrophic anaerobic ammonium oxidation in wastewater is yet to be
explored.
Heterotrophic Nitrification and Aerobic Denitrification

In addition to lithotrophic nitrification, various heterotrophic bacteria are ca-
pable of oxidizing ammonia to nitrate in presence of oxygen (Killham, 1986;
Verstraete, 1975). In contrast to lithotrophic nitrification, heterotrophic nitrifi-
cation is not coupled to energy generation. Consequently, the growth of het-
erotrophic nitrifiers is dependent on the oxidation of organic substrates and is
a sort of cometabolism. During heterotrophic nitrification, either ammonia or
reduced nitrogen from organic compounds is co-oxidized. The final product
of heterotrophic nitrification is often nitrite (Castignetti and Gunner, 1980).
The rate of heterotrophic nitrification is much slower than that accomplished
by the chemolithotrophic nitrifying bacteria and hence was considered of
lesser ecological significance (Robertson et al., 1989a and the references
therein). This conclusion was derived from the observation that most het-
erotrophic nitrifiers accumulated very less nitrite or nitrate as compared with
lithoautotrophic nitrifiers. Soon it was shown that a heterotrophic nitrifier,
Thiosphaera panotropha (now called Paracoccus denitrificans), is capable
of simultaneous nitrification and denitrification (Robertson, 1988; Robertson
and Kuenen, 1988). It was found that under fully aerobic conditions this
bacterium is capable of nitrifying ammonia to nitrite and immediately re-
duces nitrite to nitrogen (Robertson et al., 1989a; Robertson and Kuenen,
1983, 1988). Paracoccus denitrificans is an unusual chain-forming coccus
having not only simultaneous nitrification-denitrification capacity but also
has ability to oxidize reduced sulfur compounds (Robertson and Kuenen,
1990). This is a facultative anaerobic organism and can grow autotrophically
as well as heterotrophically on a wide range of substrates. P. denitrificans
can use oxygen, nitrate, nitrite, or a nitrogen oxide as the terminal electron
acceptor (Gupta, 1997). It can also use carbon dioxide as a sole carbon
source when heterotrophic carbon is not present in the medium. This was
not the first evidence of existence of aerobic denitrifier, but it was of course
first evidence of a bacterium having both heterotrophic nitrifying and aer-
obic denitrifying properties. Soon it was found many of the heterotrophic
nitrifiers are capable of aerobic denitrification in the presence of organic
matter, leading to complete elimination of dissolved nitrogen compounds
with formation of gaseous nitrogen oxides and/or nitrogen gas (Anderson
and Levine, 1986; Castignetti and Hollocher, 1984; Robertson et al., 1989b;
van Niel et al., 1987). Consequently, with this began a new era of rediscov-
ering new bacterial metabolisms having similar metabolic functions. Readers
may like to read a review on aerobic denitrification in various heterotrophic
nitrifiers (Robertson et al., 1989a).
During 1980s, there was lot of debate on the existence of aerobic deni-
trifiers. In general sense, a denitrifying bacteria functions in absence of oxy-
gen, under anaerobic conditions. Some specialized strains such as Thiomi-
crospira denitrificans can tolerate trace concentration of oxygen. Another
group of microorganisms, however, can utilize oxygen and can denitrify si-
multaneously and they are classified as aerobic denitrifiers. However, not all
microorganisms falling under the second group can tolerate similar concen-
trations of oxygen while denitrifying. Some of them is inhibited by relatively
low amounts of oxygen (e.g., Hypomicrobium X; Meiberg et al., 1980),
whereas others can denitrify even at oxygen saturation (e.g., Paracoccus
denitrificans).
Because heterotrophic nitrifiers accumulate very small quantity of nitrite
or nitrate, it was dismissed for a long time as of little ecological significance.
However, the activity of heterotrophic nitrifiers such as Paracoccus deni-
trificans, which can simultaneously denitrify the nitrite they produce, can
be seriously under estimated unless complete nitrogen balances are made.
Similar to other heterotrophic nitrifiers, Paracoccus denitrificans does not

gain any energy from ammonia oxidation. Hence, heterotrophic nitrification
in Paracoccus denitrificans cannot be treated as mixotrophy (the metabolism
of an organic substrate and an inorganic energy supplement). It uses the am-
monia oxidation step for dumping excess reducing power possibly because
of a rate-limiting step in its electron transport chain (Robertson and Kuenen,
1990).
Besides Paracoccus denitrificans, many other microbial strains such as
nonflocculating strains of Zooglea ramifera, Ps. aeruginosa, Ps. stutzeri, and
Propionibacterium theonii have exhibited denitrifying property even in the
presence of oxygen. The obligatively aerobic species such as Nitrosomonas
(Poth, 1986) and Aquaspirullum magnetotactum (Bazylinski and Blakemore,
1983) have also been classified by aerobic denitrifiers by earlier workers
owing to its property to denitrify albeit at oxygen stress. The other reviews
on this topic may be referred (Kuenen and Roberston, 1994; Robertrson and
Kuenen, 1992).
The ecological significance of such versatile microorganisms has of-
ten been speculated. Heterotrophic nitrification is assumed to occur under
conditions unfavorable for lithoautotrophic nitrifiers (e.g., in acidic environ-
ments; Killham 1986). But according to the recent reports, even in acidic
environments, heterotrophic nitrification contributes to overall nitrate pro-
duction only to a minor extent (Barraclough and Puri, 1995). It is known
that oxygen saturates terminal electron oxidase at concentrations in the or-
der of 1–2% air saturation (2–4 µL) and if denitrification occurs at oxygen
concentrations higher than 1–2% air saturation then it is called aerobic den-
itrification (Robertson and Kuenen, 1990). At a glance, corespiring oxygen
and nitrate in microorganisms may result in lower biomass yield. This seems
to be an ecological disadvantage and would act as evolutionary pressure
against the organism to maintain such a trait. The only assumed reason for
development of such behavioral pattern could be exposure of the organ-
ism to a frequently fluctuating aerobic-anaerobic environmental condition
(Robertson and Kuenen, 1990). Through several electron acceptor utiliza-

tion experiments, Robertson and Kuenen (1990) concluded that a specialist
denitrifier such as Paracoccus denitrificans can always have an ecological
advantage over generalized denitrifiers in environments where a long period
of anaerobiosis is interspaced with short period of aerobiosis. In such condi-
tions, Paracoccus denitrificans prevents rapid deactivation of its denitrifying
capacity upon exposure to short aerobic period and gains advantage by ex-
tracting extra energy using available oxygen while simultaneously utilizing
nitrite/nitrate as electron acceptor.
Even after two decades of research on heterotrophic nitrifiers and aero-
bic denitrifiers, their ecophysiological role is still baffling in the perspective
of their feeble contribution to the global nitrogen cycle (Bock and Wag-
ner, 2006). Nevertheless, the unique ability of heterotrophic nitrification and

aerobic denitrification makes bacterium such as Paracoccus denitrificans a
potential microorganism in treatment of ammonia-containing wastewater in
single sludge reactor system. Although Paracoccus denitrificans has 10–103
times lower nitrifying activity than autotrophs, a higher growth rate, and
therefore more effective ammonia oxidation can be obtained under het-
erotrophic condition (Gupta and Gupta, 2001).
TECHNOLOGICAL ADVANCEMENTS IN AUTOTROPHIC AMMONIA

REMOVAL PROCESSES
Based on the knowledge available on nitrogen-removing microorganisms, a

number of new cost effective technologies have been developed for efficient
removal of ammonia from wastewaters (Figure 2). A brief overview on, the
newly developed processes are presented along with their current status on
field applications.
ANAMMOX
The name ANAMMOX (for ANAerobic aMMonia OXidation) was coined by
Arnold Mulder in 1992 (Appendix A), when the reaction was first detected
in a denitrifying pilot plant from Gist Brocades Fermentation Company
(Mulder, 1992). The ANAMMOX reaction carried out by members of the
Planctomycetes need NH+ −
4 /NO2 in a ratio of 1:1.3. Generally, wastewaters
always do not have both ammonia and nitrite in the required ratio of 1:1.3.
Hence, the ANAMMOX process has been always applied in association with
other low-cost technologies that could partially convert ammonia in wastew-
ater into nitrite. The ANAMMOX process is widely accepted, as it achieved
60% savings in aeration cost and 100% savings in external organic carbon
dosing (Figure 3).
FIGURE 2. Schematic figure of microbial principles in some of the new biological nitrogen
removal processes, namely the (a) ANAMMOX process, (b) SHARON process, (c) SHARON-
ANAMMOX process, (d) CANON process, (e) OLAND process, (f) DEAMOX process, and (g)
BABE process (modified from Kalyuzhnyi and Gladchenko, 2009; Paredes et al., 2007).
SHARON
The Single reactor High activity Ammonium Removal Over Nitrite (SHARON)
process was originally developed for the removal of ammonia via the nitrite
route as represented in Figure 3 (Hellinga et al., 1997; Jetten et al., 1997a). In
principle, this process involves removal of ammonia to nitrite stage through
partial nitrification followed by denitritation of nitrite to nitrogen. Both au-
totrophic nitrification and heterotrophic denitritation take place under high
temperature (35◦ C) and pH above 7.0 with no sludge retention (Hellinga
et al., 1997). Partial nitrification of ammonium to nitrite reduces the require-
ment of aeration up to 25% and denitrification of nitrite to nitrogen also
FIGURE 3. Savings in terms of cost of aeration and cost of electron addition in the three
processes. 1. Conventional nitrification/denitrification requires 100% aeration cost and 100%
methanol cost. 2. The SAHRON Process (through the Nitrite Route) saves 25% of aeration cost
and 40% of methanol cost through nitritation and denitritation. 3. The PN-ANAMMOX process
saves 60% of aeration cost and 100% of methanol cost.
reduces the cost of addition of electron donor by 40% (Hellinga et al., 1998).
Basically the SHARON process involves a single-stage completely mixed
tank, which is intermittently aerated to accommodate sequential nitrification
and denitrification. The process can be carried out in a chemostat such as
a continuous stirred tank reactor or in a sequential batch reactor (SBR) with
suspended biomass (Fux and Siegrist, 2004; van Dongen et al., 2001). Alter-
natively, a method with two separate tanks, one for nitrification and another
for denitrification, has also been used to lower the requirement of aerator
capacity.
For achieving stable performance, the SHARON process requires par-
tial nitrification of ammonia at nitrite level. Stable partial nitrification can
be achieved by eliminating the growth of nitrite-oxidizing bacteria (NOB)
without harming the activity of AOB in the nitrifying culture (Bagchi et al.,
2009). NOB can be washed out of a nitrifying culture through controlling
temperature, pH, hydraulic retention time, substrate concentration, and dis-

solved oxygen. The mechanisms to obtain stable partial nitrification are dis-
cussed elsewhere (Sinha and Annachhatre, 2007). In the SHARON process,
NOB are washed out from the system by maintaining higher operating tem-
perature (35◦ C) and lower sludge retention time (SRT) or, to be more spe-
cific, lower aeration retention time (ART), of 1.5–2 days (Hellinga et al.,
1997). Temperature and ART forms the two major process parameters in
SHARON process. In a single-stage configuration, cyclic phases of aeration
and no aeration are provided for nitrification and denitrification to occur in-
termittently. The accumulation of nitrite, which is toxic to AOB, is prevented
by the denitrification step. Denitrification also restores alkalinity consumed
during nitrification process. For denitrification to proceed, the organic car-
bon source has to be additionally supplied. Besides, temperature (between

30 and 40◦ C) and ART, inlet ammonia concentration, system pH, dissolved
oxygen (DO) concentrations, and system nitrite concentrations are also to
be critically controlled for successful operation of the SHARON process.
In wastewater treatment, the SHARON process can substantially reduce
high ammonia concentration along with organics. The first full-scale demon-
stration of the SHARON process was carried out at WWTPs of Utrecht (the
Netherlands) and Rotterdam Dokhaven (the Netherlands; Mulder et al., 2001;
van Kempen et al., 2001). At present, six full-scale plants have been devel-
oped in the Netherlands and one has been developed in New York (USA).
The applied nitrogen loads in these plants are in the range 400–2500 kg
N/day (Table 2).
SHARON-ANAMMOX Process
In the SHARON-ANAMMOX process, partial nitrification in SHARON process
is used to deliver feed having NH+ −
4 /NO2 ratio close to 1 for the ANAM-
MOX process. The combination of SHARON and ANAMMOX processes
(SHARON-ANAMMOX process) offers several advantages over the conven-
tional nitrification-denitrification process, namely 60% lower oxygen require-
ment, no need for organic carbon, and low production of the excess biomass
(van Dongen et al., 2001). This process has been successfully demonstrated
at laboratory scale on various types of wastewaters such as anaerobic sludge
digesters (Vazquez-Padin et al., 2009a), sludge-rejected water from domestic
wastewater treatment plants, the food and agriculture industry (Fux et al.,
2002; Strous et al., 1997; van Dongen et al., 2001), coke oven effluent (Toh
and Ashbolt, 2002; Zhang et al., 1998), and animal wastewater such as slaugh-
ter house wastewater and piggery wastewater (Hwang et al., 2005; Reginatto
et al., 2005; Waki et al., 2007). The first full-scale application of this process
was demonstrated in Rotterdam, the Netherlands, in 2002 (van der Star et al.,
2007).
1372
TABLE 2. Full scale SHARON Plants at the Netherlands
Since in System Wastewater Nitrogen loading Reactor Efficiency

Site operation configuration treated (kg N/day) type (%) Reference
Utrecht 1997 Two (3000 m3 Sludge dewatering 900 Two-stage ASP 90–95 van Kempen
and 1500 m3) et al., 2001
Rotterdam 1999 Single (1800 m3) Sludge dewatering 850 Two-stage ASP 85–98 van Kempen
Dokhaven et al., 2001
Zwolle 2003 Two (900 m3 Sludge dewatering 410 ASP 85–95 van Kempen
and 450 m3) et al., 2005
Beverwijk 2003 Two (1500 m3 Sludge dewatering 1200 ASP 85–95 van Kempen
and 750 m3) et al., 2005
The Hague 2005 Single (2000 m3) Sludge dewatering 1300 ASP 85–98 van Kempen
Houtrust et al., 2005
Groningen 2005 Two (4900 m3 Sludge dewatering 2400 ASP ≥95 van Kempen
Garmerwolde and 2450 m3) et al., 2005
CANON
The Completely Autotrophic Nitrogen Removal Over Nitrite (CANON) pro-
cess is a combination of partial nitrification and ANAMMOX in a single aer-
ated reactor, employing two groups of bacteria, namely, Nitrosomonas-like
aerobic microorganisms and Planctomycete-like anaerobic bacteria (Gong
et al., 2007; Sliekers et al., 2002; Sliekers et al., 2005; Third et al., 2001).
This process is critically controlled under limited oxygen condition. AOB
consumes all available oxygen while partially oxidizing ammonia to nitrite.
This creates the oxygen-free environment needed for the ANAMMOX pro-
cess. Thus, the cooperation and coexistence of aerobic and anaerobic AOB
is necessary for successful operation of this process (Figure 2). The Fluo-
rescent In Situ Hybridization (FISH) assay indicated 40% of AOB and 60%
of ANAMMOX bacteria in CANON biomass with absence of aerobic NOB

(Sliekers et al., 2002). However, prolonged exposure to an ammonia-limited
condition results in the development of NOB in CANON biomass (Third
et al., 2001).
Virtually, CANON in an integration of SHARON-ANAMMOX process into
one single reactor. Although similar operational controls are needed for both
processes, CANON provides more sensitive operational controls in terms of
dissolved oxygen, nitrogen-surface load, biofilm thickness, and temperature
(van Loosdrecht et al., 2004). The ANAMMOX bacteria are reversible and
inhibited by low concentration of oxygen, above 0.5% air saturation (Strous
et al., 1997). So an appropriate DO concentration enables the consump-
tion of oxygen to an extent in which DO concentration is not over the
toxic threshold for ANAMMOX bacteria and inadequate for the growth of
NOB. The oxygen-limited condition (< 0.5% air saturation) provides an ade-
quate environment for stable interaction between Nitrosomonas-like aerobic
microorganisms and Planctomycete-like anaerobic bacteria (Schmidt et al.,
2002). Model stimulations indicate that the maximum nitrogen removal rate
was achieved only when the DO concentration kept place with the ammo-
nium surface load and it was found that for the CANON biofilm reactor, DO
of 1 mg O2 per liter was optimum (Hao et al., 2001).
Most CANON systems reported in the literature were operated at 30–35
∞C with a maximum nitrogen removal rate of 0.075–1.5 Kg N m−3 day−1
(Sliekers et al., 2002; Sliekers et al., 2003). At this temperature AOB grow
faster than NOB and also the growth of ANAMMOX bacteria is simulated
because this temperature range lies close to their optimal temperature.
The CANON process has been tested in different lab-scale reactors. The
SBR has been considered an effective reactor system for CANON due to its
efficient biomass retention (Zhang et al., 2008). However, in a gas-lift reactor,
higher ammonia removal rate of 1.5 Kg N/ m3.day has also been achieved
as against SBR (0.3 Kg N/ m3.day; Sliekers et al., 2003).
Few pilot- and full-scale applications of the CANON process have been
reported in literature (Joss et al., 2009; Vazquez-Padin et al., 2009b). In a
separate study, urea was used as feed for CANON system (Sliekers et al.,
2004) instead of conventional wastewater. The system adopted very quickly
and reached to full capacity within two weeks of adaptation (Sliekers et al.,
2004). This has widened the scope of application of this process to urine
waste along with other low C/N ratio wastewaters.
OLAND and DEMON

The Oxygen-Limited Autotrophic Nitrification Denitrification (OLAND) pro-
cess refers to the removal of ammonium-N under strict oxygen limitation
by autotrophic organisms (Kuai and Verstraete, 1998). Initially, the process

was considered to be dependent mainly on aerobic ammonium oxidizing
bacteria (aerAOB), but recently it has been reported that under conditions
of low oxygen supply and the absence of organic electron donors, both
aerAOB and anaerobic ammonium oxidizing bacteria (anAOB) coexist in
the OLAND process (Pynaert et al., 2003). The b subclass of proteobacteria
includes most of the aerAOB, with Nitroso species (e.g., Nitrosomonas, Ni-
trosococcus, Nitrosospira) as the principal members, while the anAOB belong
to a deep-branching lineage within the order of the Planctomycetes. Thus,
in principle the process mechanism of OLAND is similar to CANON. Table
3 shows comparative performances of the OLAND and CANON process. An
application of the OLAND process on pilot scale is reported for treatment of
digested black water using single-stage SBR (Vlaeminck et al., 2009b).
Similar to CANON, another process known as the deammonification
process or DEMON was first used when significant losses of inorganic nitro-
gen up to 90% were observed in the nitrification step of a rotating biological
contractor (RBC) treating ammonium-rich landfill leachate under low oxy-
gen concentrations (Hippen et al., 1997). Extensive nitrogen loss was also
observed in other RBCs in Switzerland and the United Kingdom (Hippen
et al., 2001; Siegrist et al., 1998). The first full-scale application of deam-
monification process in a moving bed reactor using Kaldnes carriers came
into operation in April 2001 at a WWTP of Haltigen, Germany (Cornelius
and Rosenwinkel, 2002; Jardin et al., 2001). Presently the DEMON process
has been successfully under operation at full scale at WWTP Strass (Austria)
since 2004 (Wett, 2006, 2007), at Glarnerland (Switzerland) since 2007 (Joss
et al., 2009), and at Olburgen since 2006 (Abma et al., 2007).
DEAMOX
The DEnitrifying AMmonia OXidation (DEAMOX) process was proposed by
Mulder in 2004 for the ANAMMOX reaction occurring in the autotrophic den-
itrifying plant (Kalyuzhnyi et al., 2006). DEAMOX eliminates the requirement
TABLE 3. Comparison of maximum nitrogen loading rate and nitrogen removal rate of different CANON and OLAND processes
Wastewater Reactor NLRmax NRRmax

Process treated type (kg N/m3·day) (kg N/m3·day) Reference
CANON Synthetic SBR 0.131 0.075 Sliekers et al., 2002

Synthetic SBR NR 0.08 Third et al., 2005
Synthetic SBR 0.22 0.12 Third et al., 2001
Sludge digester effluents SBR 0.46 0.36 Vazquez-Padin et al., 2009b
Digester supernatant SBR NR 0.5 Joss et al., 2009
Synthetic MABR 0.87 0.77 Gong et al., 2007
Synthetic Gas lift 5.5 1.5 Sliekers et al., 2003
OLAND Synthetic SBR 0.13 0.05 Kuai and Verstraete, 1998
Synthetic RBC 0.08307∗ 0.083∗ Pynaert et al., 2003
Digested black water RBC 0.716 0.7 Vlaeminck et al., 2009a
Note. Values are in kg N/m2.day. CSTR = continuous stirred tank reactor; MABR = membrane-aerated bioreactor; NLRmax = maximum nitrogen
loading rate; NRRmax = maximum nitrogen removal rate; RBC = rotator biological contractor; SBR = sequential batch reactor.
1375
of partial nitritation by introducing denitritation of nitrate using sulfide as an

electron donor (Equation 6).
NO− − − −
3 + 0.25HS → NO2 + 0.25SO4 + 0.25H
+
(6)
The process is applicable for sulfur-bearing nitrogenous wastewater

such as Baker’s yeast effluent (Kalyuzhnyi and Gladchenko, 2009; Kalyuzh-
nyi et al., 2006). In treatment of Baker’s yeast effluent, the whole process
operates in combination of a three-reactor system: an anaerobic reactor, a
nitrifying reactor, and a DEAMOX reactor. In the anaerobic reactor ammo-
nia and sulfide are generated, which is partially fed to nitrifying reactor
and the remainder is fed to the DEAMOX reactor (Figure 2). In the nitri-
fying reactor, ammonia is nitrified into nitrate and nitrite. The third reactor
(i.e., the DEAMOX reactor) receives effluent from both the anaerobic and
nitrifying reactors. The denitrification of nitrate takes place in presence of
sulfide (Equation 6) and the ANAMMOX bacteria coexisting in the DEAMOX
reactor along with the autotrophic denitrifying bacteria oxidize ammonia
using nitrite as an electron acceptor (Equation 5). Basically the scheme in
the DEAMOX process is similar to the autotrophic denitrifying reactor plant
in which ANAMMOX was first detected and then discovered by Mulder
(1992).
Because the standard application of this process is restricted to sulfur-
bearing wastewaters, the incorporation of heterotrophic denitratation using
volatile fatty acids (VFA) as a more widespread electron donor has been
investigated to implement the process for commonly found wastewaters
(Kalyuzhnyi et al., 2008). However, the feasibility study showed a severe
competition for nitrite between autotrophic ANAMMOX and heterotrophic
denitritation resulting in poor removal efficiency (Kalyuzhnyi et al.,
2007).
The typical sulfide driven DEAMOX processes have a maximum nitro-
gen loading rate of 1 kg N/m3.day with more than 90% removal efficiency
(Kalyuzhnyi et al., 2006). An organics-driven DEAMOX process has a sim-
ilar loading capacity (1.24 1 kg N/m3.day) with relatively lower ammonia
removal (40%), mainly due to the competition of heterotrophic denitrifiers
(Kalyuzhnyi and Gladchenko, 2009). Although the process is 10 times lower
than the ANAMMOX process, and a simple process operation is claimed to
favor the DEAMOX process over the ANAMMOX process. So far, this process
has been tested only at bench scale.
SNAD
Low-cost biological nitrogen-removing technologies such as ANAMMOX,
SHARON, SHARON-ANAMMOX, CANON, OLAND, and DEAMOX have
proved to be very effective in removing ammonia from industrial effluents.
However, most ammonium-rich wastewaters are produced with a certain

concentration range of organics (e.g., old landfill leachates) and the presence
of organics could upset the autotrophic bacteria which are mostly responsi-
ble in all low-cost biological nitrogen-removing technologies except in the
SHARON process. The application of the combined SHARON-ANAMMOX
process has been also limited to wastewater with low total organic carbon
to nitrogen ratio (C/N) ≤0.15. An increase in C/N ratio to more than 0.3 can
lead to competition between heterotrophic bacteria and autotrophic bacteria
in the SHARON process (Wang and Kang, 2005).
Studies have demonstrated that the ANAMMOX and denitrification pro-
cess can coexist in the same environment (Chen et al., 2009). Recently par-
tial nitrification, ANAMMOX and denitrification have been simultaneously
investigated on bench scale in a single-stage nonwoven rotating biological

contractor (NRBC) bioreactor (Chen et al., 2009). The Simultaneous partial
Nitrification, ANAMMOX and Denitrification (SNAD) process was found to
potentially remove ammonium and organics from wastewater in a single
NRBC reactor with total nitrogen removal efficiency of 70% and Chemical
Oxygen Demand (COD) removal efficiency of 94% at nitrogen and COD
loading rates of 0.69 kg N/m3.day and 0.34 kg/m3.day, respectively. Re-
cently, the SNAD process has been demonstrated at full scale for treatment
of landfill leachate (Wang et al., 2010; Xu et al., 2010).
AS/PN-ANAMMOX
The AS/PN-ANAMMOX is a two-stage process comprising the combined acti-
vated sludge process-partial nitrification (AS/PN) followed by the ANAMMOX
process. The concept promotes coexistence of heterotrophic and autotrophic
microorganisms in conventional extended activated sludge process (ASP) for
organic carbon removal and nitrification. Lamsam et al. (2008) demonstrated
that partial nitrification can be achieved in an ASP by controlling three key
parameters, namely, DO (0.3–0.5 mg/L), pH (around 8), and temperature
(35◦ C). It differs from the SHARON process by inclusion of an aeration tank
and a settler with sludge recirculation. For accomplishing partial nitrification
in ASP, sludge retention time (SRT) of 12–15 days was required (Lamsam
et al., 2008). The AS/PN is reported to have several advantages over the con-
ventional extended ASP for organic removal along with nitrification. First,
the process consumes 66% less oxygen, as calculated from the theoretical
equations (8–10) based on the assumption of C18 H19 O9 N as chemical for-
mula for organic matter (Henze et al., 2000). Second, the effluent from AS/PN
contains effluent ammonia and nitrite in the ratio of 1:1, which is desirable
for the downstream ANAMMOX process. In addition, a conventional ASP can
be upgraded into AS/PN by controlling pH, temperature, and DO (Lamsam
et al., 2008).
ASP with nitrification:

C18 H19 O9 N + 19.5O2 → 18CO2 + 9H2 O + H+ + NO−
3 (7)
ASP without nitrification:
C18 H19 O9 N + 17.5O2 + H+ → 18CO2 + 8H2 O + NH+
4 (8)
AS/PN :
C18 H19 O9 N + 18O2 → 18CO2 + 8H2 O + H+ + 0.5NH+ −
4 + 0.5NO2 (9)
Operating conditions of the AS/PN process coupled with the ANAMMOX

process were identified as pH 7.7–8.2 and DO 0.5–0.9 mg/L to achieve over
85% COD removal as well as partial nitrification. Maximum nitrogen removal
rate for the ANAMMOX process was found to be 0.6 kg N/m3.day. The
process was applied for treating sea food processing wastewater at bench
scale.
BABE Process
The Bio-Augmentation Batch Enhanced (BABE) process was developed to
bioaugment nitrification in activated sludge process that operated at subopti-
mal solid retention times (Salem et al., 2003; Salem et al., 2004). The principle
was to implement a nitrification reactor (BABE reactor) in the sludge return
line (Figure 2). The BABE reactor would be fed with an internal N-rich flow
(e.g., effluent from the sludge treatment). Nitrifiers grown in the BABE reactor
would ultimately reach the activated sludge reactor. Hence, the nitrification
capacity of the ASP process can be augmented with nitrifiers grown in the
BABE reactor. Thus the BABE reactor functions basically as a culture tank
supplementing the ASP process with the specially cultivated microorganisms
in the culture tank. The BABE technology has been in full-scale operation at
the WWTP Garmerwolde in Groningen (the Netherlands).
The concept of bioaugmentation may be of special interest in a PN-
ANAMMOX system for bioaugmenting both the cultures in a similar manner
and for seeding into the main reactor.
ENGINEERING CHALLANGES OF AUTOTROPHIC NITROGEN

REMOVAL TECHNOLOGIES
Based on the technologies discussed, it is clear that newly developed am-

monia removal processes are either based on the PN-ANAMMOX concept
or on nitritation-denitritation concept or on the combination of both. Var-

ious nomenclatures were given to the processes based on organisms as-
sumed to be present in the processes (Appendix B). The majority of these
processes are autotrophic and claim to achieve cost reduction through re-
duction in oxygen demand and organic carbon source (Jetten et al., 2005).
Hence, these autotrophic nitrogen removal technologies can achieve sub-
stantial savings in operation and maintenance cost while treating highly rich
ammonia-containing effluents such as reject water, piggery manure, land-
fill leachate, tannery wastewater, and slaughterhouse wastewater (van Hulle
et al., 2010). Some of the engineering challenges faced by such autotrophic
nitrogen-removing technologies are discussed subsequently.
PN or nitritation is an essential prerequisite of the newly developed
autotrophic nitrogen-removing technologies including the ANAMMOX pro-

cess. For nitrite accumulation, the most important environmental parameter
is to inhibit the growth of nitrite oxidizers through free ammonia (FA) or
free nitric acid (FNA) toxicity. FA or FNA toxicity could be achieved through
fine control of pH and temperature. Besides, toxicity, another important pa-
rameter that can cause elimination of nitrite oxidizers is dissolved oxygen
concentration. The difference in the sensitivity of AOB and NOB toward
these influences determines nitrite accumulation in the system. NOB is more
sensitive to detrimental environmental conditions than AOB. Another engi-
neering parameter that is useful for nitritation is difference in the growth
rates of AOB and NOB that can be controlled through maintaining a proper
sludge retention time (SRT). Table 4 lists the factors needed for establishing
nitrite accumulation in a nitritation reactor. However, the potential for using
most of the engineering approaches mentioned in Table 4 for achieving ni-
trite accumulation seems limited because adaptation of the nitrite-oxidizing
bacteria has been reported over long period of operation (Turk and Mavinic,
1989).
The basic engineering challenge of operating a partial nitritation reactor
is to generate an effluent that has ammonia and nitrite in a molar ratio of
1:1.3 for the downstream ANAMMOX reactor. Through years of research by
numerous workers, so far, four basic approaches have evolved to meet this
requirement of ammonia to nitrite ratio (van Hulle et al., 2010): (a) oper-
ation of the reactor at low dissolved oxygen concentration (preferably <
0.5 mg/L); (b) operating at higher pH (7.5–8.0), which increases availability
of substrate for AOB (i.e., free ammonia) and decreases the availability of free
nitrous acid; (c) operation of reactor at high temperature (T > 25◦ C) where
the growth rate of AOB increases over that of NOB; and (day) a shorter
HRT to arrest ammonia oxidation. So far, four types of reactor configurations
have been employed to achieve partial nitritation, namely, fixed film reac-
tors (FFB), continuously stirred tank reactors (CSTR), membrane bioreactors
(MBRs), and SBRs. SRT is the key parameter controlling for nitrite accumula-
tion in the reactors. Unlike in CSTR and SBR, SRT is difficult to manipulate in
TABLE 4. Environmental factors concerningnitritation and the ANAMMOX process
1380
Physiological Values/
state concentrations
Sr. No state of reactor Factors (mg/L) Reference(s) Remarks
1. Partial FA toxicity 0.08–0.82 Anthonisen et al., 1976 NH3 is main inhibitor at pH > 8.0
nitrification FNA toxicity 0.06–0.83 Anthonisen et al., 1976 HNO2 is the main inhibitor at pH < 7.5
pH 7.5–8.0 van Hulle et al., 2007; This pH is favorable for AOB as NH3 is readily
van Hulle et al., 2010 available as substrate at this pH
Temperature 35◦ C Grunditz et al., 2001 Optimum for AOB
35–45◦ C van Hulle et al., 2007 Long term operation at >40◦ C may lead to
deactivation (Hellinga et al., 1999)
>25◦ C Hellinga et al., 1998 Operational temperature at >25◦ C coupled with
SRT of 1–1.5 days was the basis of operating the
SHARON process
DO 0.16 mg O2 /L Hunik et al., 1994 Half saturation (ks ) constant was worked out for
pure culture of Nitrosomonas europaea. But ks in
mixed culture system is dependent on the
biomass density, the floc size, mixing intensity,
and rate of diffusion of oxygen (Munch et al.,
1996)
<0.5 mg O2 /L Hidaka et al., 2002; Nitrite accumulation was achieved in various
Hyungseok et al., reactor configurations through frequent switching
1999; Peng et al., 2004; between oxic and anoxic phases which was
Sin et al., 2006; Jubany controlled through online OUR or DO control
et al., 2009 system
SRT 7–8 hr Bock et al., 1986 Minimum doubling time for AOB
1–2.5 days Van Kempen, 2001 Based on full-scale experience for achieving partial
nitrification
1–1.5 days Hellinga et al., 1998 Basis for developing SHARON process at SRT =
HRT, temperature (>25◦ C) and good aeration
10–40 days Pollice et al., 2002a; Partial nitrification was achieved irrespective of
Pollice et al., 2002b sludge age but under oxygen limitation
30 days Peng and Zhu, 2006 Operation under low temperature (<13◦ C)
Light Light intensity Olson, 1981 Treatment with a low light dose for extended
of 6.64 µmol periods was more damaging to NOB (Guerrero
m−2 s−1 and Jones 1996). Bock (1965) attributed this
greater sensitivity of NOB to the relatively low
cytochrome c content of Nitrobacter compared
to Nitrosomonas.
Others Free hydroxy- Yang et al., 1992; Hu Free hydroxylamine released during oxygen
lamine et al., 1990; Castignetti limitation causes acute and irreversible toxicity to
toxicity and Gunner, 1982; NOB
Stuven et al., 1992
Organics Mosquera-Corral et al., Acetate concentration of 0.2 g C/g N stimulated
(COD) 2005 AOB in the SHARON process. Generally a high
C:N wastewater is not suitable for nitrifiers. But
tolerance to organics depends upon
acclimatization of the sludge and on longer SRT
(2–3 days).
2. ANAMMOX# Nitrite inhibition 350 mg/L Dapena-Mora et al., 2007 Resulted in 50% inhibition of the ANAMMOX
process
>182 mg/L Egli et al., 2001 Experiments with Candidatus Kuenenia
stuttgartiensis
100 mg/L Strous et al., 1999 Pure culture of Candidatus Brocardia
anammoxidans was completely inhibited at
nitrite concentration of 100 mg/L but activity was
restored by adding trace concentrations og
hydroxylamine and hydeazine.
40 mg/L Fux et al., 2003 Long term exposure led to irreversible inhibition of
ANAMMOX process.
(Continued on next page)
1381
1382
TABLE 4. Environmental factors concerningnitritation and the ANAMMOX process (Continued)
Physiological Values/
state concentrations
Sr. No state of reactor Factors (mg/L) Reference(s) Remarks
Phosphate toxicity >155 mg P/L van de Graaf et al., 1996 Studies with pure culture of Candidatus Brocardia
anammoxidans
Up to 620 mg Egli et al., 2001 No inhibition was observed with Candidatus
P/L Kuenenia stuttgartiensis
620 mg P/L Dapena-Mora et al., 2007 Resulted in 50% inhibition of the ANAMMOX
process
55–285 mg P/L Pynaert et al., 2003 Batch studies with highly enriched ANAMMOX
biomass from lab-scale rotating biological
contractor containing Candidatus Kuenenia
stuttgartiensis caused 63% and 80% inhibition of
ANAMMOX process in presence of 55 mg P/L
and 110–285 mg P/L, respectively.
Sulfide toxicity 9.6 mg S/L Dapena-Mora et al., 2007 Resulted in 50% inhibition of the ANAMMOX
process
64 mg S/L van de Graaf et al., 1996 Resistance of ANAMMOX cultures to sulfide
toxicity in presence of nitrate was observed both
in continuous and batch mode experiments
DO Air saturation Egli et al., 2001 Reversible inhibition was observed with
of 0.25–2% Candidatus Kuenenia stuttgartiensis
Air saturation Egli et al., 2001 Irreversible inhibition was observed with
of >18% Candidatus Kuenenia stuttgartiensis
Note. Partial nitrification involved inhibiting NOB and favoring nitrite accumulation. ANAMMOX conditions that are unfavorable for the ANAMMOX process are listed.
biofilm reactors such as FFB and membrane-aerated biofilm reactors (MABR).

Hence, long-term nitrite production without nitrate accumulation is difficult
to achieve in biofilm reactors despite the first three operational strategies
(Biswas et al., 2011; Fux et al., 2004). One advantage of the biofilm sys-
tems is that the fourth strategy of very low HRT can be applied without
sludge washout at higher operational temperature (>25◦ C; Bagchi et al.,
2012). The CSTR used in the SHARON process operates at HRT and SRT of
1–1.5 days and hence leads to loss of biomass. In biofilm systems, HRT and
SRT can be uncoupled and hence can be operated at lower HRT and higher
loading rates, and the size of the unit can be smaller, reducing the foot
print.
In contrast to partial nitritation reactor, the engineering challenge in
the ANAMMOX process is the slow growth rate of ANAMMOX bacteria and
hence extremely long start-up period required for establishing it. Preventing
sludge loss due to washout is essential in ANAMMOX systems as sludge
loss could further aggravate the problem of start-up. Hence, the strategy
of selecting reactor configuration in ANAMMOX systems is to increase SRT
and prevent sludge loss through biofilms or aggregates such as flocs or
granules (Bagchi et al., 2010; van der Star et al., 2008). The ANAMMOX
process is widely studied in a range of reactor configurations, namely, CSRT,
SBR, MBR, fixed bed reactors, fluidized bed reactors, rotating biological bed
contractors, UASB, and gas-lift reactors (Ahn and Choi, 2006; Dapena-Mora
et al., 2004; Fux et al., 2004; Sliekers et al., 2003; Strous et al., 1997; van der
Star et al., 2008, 2002; Third et al., 2005; Third et al., 2005; Wyffels et al.,
2004). Amongst these, SBR was accepted for ANAMMOX enrichment for its
simplicity, homogeneity of mixture in the reactor, and lower footprint area
(Strous et al., 1997; Strous et al., 1998). A next generation reactor, MBR
further proved to be useful in retaining biomass and lowering the start-up
period in the ANAMMOX process (van der Star et al., 2008). Moreover, MBR
enables production of almost pure suspended ANAMMOX cells, avoiding
diffusion limitations in flocs or granules (van der Star et al., 2008). A few
modifications in MBR design such as membrane-SBR (Trigo et al., 2006),
MABR (Gong et al., 2007), and MBR with stirrer (Wang et al., 2009) have also
been tested to achieve more homogeneity, more suspended free cells, and
higher nitrogen removal rate. However, for full-scale applications, biofilm- or
granular-based reactors are preferable over MBR because membranes have
the tendency of fouling and the ANAMMOX bacteria easily form granules and
therefore higher biomass concentrations can be achieved in an economical
way.
Besides the slow growth rate of ANAMMOX bacteria, another ma-
jor challenge of maintaining a high cell number in ANAMMOX reactors
is its susceptibility to inhibition by substrates and products. ANAMMOX
bacteria are severely inhibited by nitrite concentration, though no unifor-
mity has been found among the various reported values in the literature
(Table 4). A remarkable difference in the tolerance of the ANAMMOX bacteria

to nitrite toxicity is probably due to difference in reactor configurations and
other operational conditions. Biofilm- and granular-based systems showed
more tolerance toward nitrite toxicity (Vazquez-Padin et al., 2009b). Dosta
et al. (2008) reported higher nitrite tolerance at higher operating tempera-
ture. Though the ANAMMOX process is not inhibited by high concentrations
of ammonia and nitrite up to concentrations of at least 1 g N per liter (Strous
et al., 1999b). Dapena-Mora et al. (2007) observed a 50% activity loss at am-
monia and nitrate concentration of 770 and 630 mg N per liter, respectively.
An overview of all other inhibitory factors for the ANAMMOX bacteria is
presented in Table 4.
The fourth and the biggest challenge in all the autotrophic processes
is the organic content of wastewater. As the name suggests, autotrophic

nitrogen removal technologies operate in wastewater having low COD:N
ratio. Certain industrial processes generate highly loaded nitrogenous ef-
fluents. Large concentrations of organics (sometimes both recalcitrant and
toxic), sulfides, phosphates, and other inhibitory compounds are also ex-
pected along with the nitrogenous load, making treatment difficult (Biswas
et al., 2010). COD in the effluent is desirable for the denitrification process,
which is often needed as posttreatment of the ANAMMOX and OLAND pro-
cess for nitrate removal (van Hulle at al., 2010). ANAMMOX removes only
90% of the total influent nitrogen as ammonium/nitrite and leave rest 10%
nitrogen as nitrate in the effluent. Hence for complete removal of nitrate,
denitrification might be useful. The desirable C/N ratio for denitrification can
be undesirable for the upstream autotrophic process. The ANAMMOX pro-
cess is not suitable in wastewater having a C:N ratio greater than 1 (Guven
et al., 2005).ANAMMOX is completely and irreversibly inhibited by ethanol
and methanol (Guven et al., 2005). This aspect must be taken into consid-
eration because methanol/ethanol are often employed as an electron donor
for denitrification.
Landfill leachate and reject water originating from sludge dewatering
typically have high ammonia and low COD content and hence are most
suitable for autotrophic technologies (van Hulle et al., 2010). Reject water is
generally recycled to the influent of the WWTP and the combined effluent is
treated in the WWTP. To prevent inhibition of autotrophic microorganisms
in the presence of high COD, the ammonia-rich water from reject water at
WWTP Dokhaven in Rotterdam (the Netherlands), was treated separately
before it was recycled to the COD and ammonia-rich influent of WWTP
(Mulder et al., 2001; van Dongen et al., 2001). The separate treatment of
this stream led to 15% reduction in the total nitrogen load and significantly
reduced the nitrogen concentration in the treated effluent of the main WWTP.
Recently there has been debate on the coexistence of ANAMMOX and
denitrifiers (Chamchoi et al., 2008; Kumar and Lin, 2010; Pathak et al., 2008;
Wang et al., 2010; van Hulle et al., 2010; Wang et al., 2009; Xu et al., 2010).
Several researchers have reported correlation between autotrophic ANAM-

MOX bacteria and heterotrophic denitrifiers in natural ecosystems (Bothe
et al., 2000; Dalsgaard et al., 2003; Dalsgaard et al., 2003; Dalsgaard et al.,
2005; Hulth et al., 2005; Kuypers et al., 2003). Pathak et al. (2007) demon-
strated the symbiotic association of ANAMMOX and denitrifiers when the C/N
ratio of low ammonium-fed immobilized bioreactors is maintained around
0.6. There is a demonstration of the SNAD process under lab and field condi-
tions using synthetic feed and landfill leachate (Chen et al., 2009; Wang et al.,
2010; Xu et al., 2010). Under laboratory conditions, a single, oxygen-limited,
NRBC reactor was used to reduce ammonium and COD from the synthetic
wastewater. An ammonium conversion efficiency of 79%, total nitrogen (TN)
removal efficiency of 70%, and COD removal efficiency of 94% were ob-
tained with the nitrogen and COD loading rate of 0.69 kg N/m3.day and
0.34 kg/m3.day, respectively (Chen et al., 2009). Xu et al. (2010) reported final
maximum activities of aerAOB, anAOB, and denitrification at 2.83 (kg NH4
N/kg/day), 0.65 ((kg NH4 N/kg/day), and 0.11 (kg NH4 N/kg/day), respec-
tively, in the full-scale partially aerated bioreactor treating landfill leachate.
Another full-scale landfill leachate treatment plant is under operation in
Taiwan since 2006 for treating 304 m3/day wastewater at SRT between 12
and 18 (Wang et al., 2010). The COD and ammonia removal of 28% and
80%, respectively, were observed at influent COD, ammonia, and nitrate
concentrations of 554, 634, and 3 mg/L, respectively. A nitrogen mass bal-
ance revealed the TN removal by combined partial nitrification and anaerobic
ammonium oxidation is 68% and the heterotrophic denitrification contributes
to 8% and 23% of TN and COD removals, respectively (Wang et al., 2010).
Besides, landfill leachate and sludge rejects water, there has been very
few reports of ANAMMOX-based processes using COD-bearing industrial
effluents (Reginatto et al., 2005; Sabumon 2007; Toh and Ashbolt, 2002;
Vazquez-Padin et al., 2009b; Waki et al., 2007). Table 5 lists the application
of ANAMMOX-based autotrophic ammonia removal processes in treatment
of COD- and ammonia-bearing industrial effluents.
EXPERIENCES FROM FULL-SCALE APPLICATIONS
The SHARON process was the first process to be scaled up commercially

and installed at six different locations, five in the Netherlands and one in the
United States (Carrio et al., 2003; van Kempen et al., 2005). The process was
first implemented at WWTP Utrecht (the Netherlands) during 1997, while the
most recent application was at WWTP New York Ward Island (USA) in 2006
(Table 2).
The SHARON process needs the addition of an external source of carbon
for the denitritation process, as the organics present in the wastewater are
at times inadequate. Due to the storage problems associated with methanol,
1386
TABLE 5. Application of ANAMMOX-based autotrophic ammonia removal processes in treatment of COD and ammonia bearing industrial effluents
Name of Reactor Type of Wastewater Ammonia removal
the process type wastewater composition efficiencies (%) Remarks Reference
ANNAMOX Sequencing batch Anaerobic sludge NH+4 400–700 mg N/L 69 Bench-scale Vazquez-Padin
reactor digester effluent Inorganic carbon 300–500 mg C/L experiment in 1 L et al., 2009
Total organic carbon 20–50 mg capacity reactor
C/L
PN-ANAMMOX CSTR for PN Anaerobic sludge NH+4 657 ± 56 mg N/L 92 ± 7 Pilot-scale experiment Fux et al.,
SBR for digester effluent NO−2 0.4 ± 0.7 mg N/L
in two 2.5 m3 2002
Anammox NO−3 0.2 ± 0.7 mg N/L
capacity reactors
+
OLAND Sequencing batch Digested black NH4 1065 ± 15 mg N/L 76 Bench-scale Vlaeminck
reactor water NO−2 not detected
experiment et al., 2009b
NO−3 0.2 ± 0.4 mg N/L
CODsol 597 ± 4 mg C/L
+
CANON Sequencing batch Digester NH4 650 ± 50 mg N/L 90 Full-scale application Joss et al.,
reactor supernatant NO−2 < 0.2 mg N/L
at three municipal 2009
NO−3 < 0.2 mg N/L
plant (5 reactor in
CODsol 300 ± 50 mg C/L total, highest
CODtotall 630 ± 50 mg C/L capacity 14000 m3)
SHARON Aeration Tank Sludge rejection NH+4 500–700 mg N/L 75 Full scale (4500 m3) van Kempen
water et al., 2005
SNAD Aeration Tank Landfill leachate NH+
4 634 mg N/L 68 Full scale (192 m3) Wang et al.,
NO−
2 N/L
2010
NO−
3 3 mg N/L
CODsol 554 mg C/L
DEMON SBR Rejection water — 85.8 ± 4.93 500 m3 Full-scale was Wett, 2007
implemented at
WWTP Strass,
Austria.
PN-ANAMMOX Fixed bed Swine wastewater NH+4 2000–4000 mg N/L 70 ± 9 0.95 L Lab-scale reactor Yamamoto
bioreactor NO−2 nondetectable
et al., 2008
NO−3 nondetectable BOD5
3000–5000 mg/L
COD 8000–17000 mg /L
ANAMMOX/ Hybrid anaerobic Slaughter house NH+

4 163.7 ± 31.6 mg N/L 95 0.79 L Lab-scale reactor Reginatto
denitrification reactor wastewater NO−
2 not detected
et al., 2005
connected to an NO−
3 not detected
upflow CODl 614.5 ± 130.4 mg/L
biological filter
with submerged
aeration
ANAMMOX Up-flow anaerobic Pharmaceutical NH+ 4 123−257 mg N/L 85.2 Lab-scale process Tang et al.,
sludge blanket wastewater NO− 2 133−264 mg N/L
2010
(UASB) NO− 3 0–32 mg N/L
CODsol 415−843 mg C/L
ANAMMOX Batch process Animal waste NH+ 4 216 mg N/L 84.5 Batch experiment was Waki et al.,
treatment NO− 2 259 mg N/L
conducted in 723 ml 2007
processes NO− 3 140 mg N/L
glass bottles
wastewater BOD5 32 mg/L
DEAMOX UASB Baker’s yeast total N 993–1,651 mg/L 90 2.53 L lab-scale reactor Kalyuzhnyi
wastewater total P 13–78 mg/L et al., 2006
sulfate 682–3,028 mg/L
CODsol 15000–26600 mg/L
CODtotall 17900–31100 mg/L
ANAMMOX Granular sludge Sludge digester The first full-scale van der Star
reactor effluent (70 m3) ANAMMOX et al., 2007
reactor
1387
various alternative carbon sources have been utilized in various SHARON

plants, such as by-product from biofuel production (Utrecht and Zwolle) and
condensate of sludge drying (Beverwick and Groningen). Besides providing
a cheap and readily available carbon source, the SHARON process needs
precise control of temperature, SRT, and pH for restricting generation of
nitrate, an undesirable intermediate for the process. Avoiding the activity of
NOB forms the key step in the SHARON process (van Dongen et al., 2001).
After the SHARON process, the ANAMMOX process was next to be
scaled up. The first installation took place at Rotterdam (the Netherlands)
in a 70 m3 plant in 2002 after modifying the existing SHARON process into
a two-stage SHARON-ANAMMOX process (van der Star et al., 2007). The
process treated wastewater from an anaerobic sludge digester at a municipal
wastewater treatment plant. Presently this plant is operating at very high am-
monia removal rates of 10 Kg N/m3.day. After the successful demonstration
of ANAMMOX process in combination of SHARON process, it was clear that
the need of addition of an external carbon source for denitrification could be
eliminated and along with this, the problems associated with storing highly
inflammable methanol were also eliminated.
During the same period, another single-stage process based on the PN-
ANAMMOX (CANON) was scaled up in Strass (Austria) in a stepwise manner
from 5l to 300 L, to 2.4 m3, and finally to 500 m3 (Wett, 2006). A full-scale
plant at Glarnerland (Switzerland) was set up in 2007 on a similar technology
(CANON) using the enriched biomass from the plant in Strass, Austria, and is
presently operating at a rate of 0.5 Kg N/m3.day (Wett, 2007). Biomass from
WWTP Strass (Austria) was also used to seed up one of the 1400 m3 SBR at
WWTP, Zurich, operated for single-stage partial nitritation and ANAMMOX
process. Though 50 L acclimatized seed from WWTP Strass was initially
scaled up in a 0.4 m3 pilot plant before seeding into the full-scale SBR in
association with a 200 L of sludge from a conventional nitritation reactor, the
start-up period for reaching the 500 g N m−3d−1 ammonium oxidation rate
took 180 days. During the acclimatization period, sludge age was reduced
from 30 to 4 days, HRT was reduced to 1.2 days, and minimum aeration
was provided to maintain a DO ≤ 1 mg O L−1. The lengths of the aerated
and anaerobic steps were manually adjusted so that the concentration of
nitrite never exceeded 3–8 mg NO2 -N.L−1 (Joss et al., 2009). A comparative
performance of the pilot- and full-scale applications based on two-stage and
single-stage PN-ANAMMOX technology is presented in Table 6.
It is evident from the full-scale experiences that the technical application
of the PN-ANAMMOX process must take into account the inherent problem
of long start-up period. The slow growth rate of ANAMMOX bacteria and the
anaerobicity and toxicity due to the presence of dissolved oxygen, nitrite,
and organic compounds are the few reasons explaining the long start-up
period. The first application at Rotterdam (the Netherlands) took nearly 3
years for stabilization, while that at Strass (Austria) took more than 2.5 years
TABLE 6. Comparative performance of the pilot and full-scale applications based on two-stage and single-stage Partial Nitritation-ANAMMOX
technology
Nitrogen Nitrogen
System Current loading rate removal rate Reactor Efficiency
Process Name Location configuration status (gN/m2·day) (kg N/m3·day) type (%) Reference
Two-reactor
nitritation-ANAMMOX
process
SHARON-ANAMMOX Rotterdam 70 m3 Full scale — 10 Gas lift N.R van der Star
(the Netherlands) et al., 2007
PN-ANAMMOX Stockholm 5 m3 Pilot scale 0.5 — Moving Bed 84 Gut et al.,
(Sweden) Biofilm 2006
Reactor
(MBBR)
PN-ANAMMOX Zurich 1.6 m3 Pilot scale — 2.4 CSTR >90 Fux et al.,
(Switzerland) 2002
BABE Garmerwolde 1250 m3 Full scale N.R N.R Two-stage 75 for nitrification Salem et al.,
(Netherlands) SBR 66 denitrification 2004
One reactor
Nitritation-ANAMMOX
process
DEMON Strass 500 m3 Full scale N.R N.R SBR 83.9 ± 1.8 Wett, 2006
(Austria)
DEMON Glarnerland 400 Full scale 0.68 0.5 SBR 85.8 ± 4.93 Wett, 2007
(Switzerland)
Deammonification Kollikon 33 m3 Full scale 2.6 — RBC 70 of ammonia Siegrist et al.,
(Switzerland) oxidized 1998
Deammonification Mechernich 150 m3 Full scale 2.7 — RBC 50–60 of Seyfried et al.,
(Germany) deammonification 2001
3
One-stage nitritation and Stockholm 2.1 m Pilot scale 1.9 1.5 Moving bed — Cema et al.,
ANAMMOX processes (Sweden) reactor 2006
3
One-reactor partial nitritation Zurich (Switzerland) 1400 m Full scale 0.716 0.3 SBR 90 Joss et al.,
and ANAMMOX processes St. Gallen 300 m3 Full scale N.R 2009
(Switzerland) 160 m3 Full scale 0.4
Niederglatt
1389
to start up (van der Star et al., 2007). Therefore, to avoid such problems, and
reduce the start-up period and save resources, the intermediate pilot-scale
operation was eliminated during the scale-up process in many installations
(Salem et al., 2004; van der Star et al., 2007; van Kempen et al., 2001; Wett,
2006), while the other full-scale applications used enriched biomass from the
existing full-scale processes as inoculums for reducing the start-up period.
For example, at WWTP Niederglatt, 100 m3 acclimatized biomass from WWTP
Zurich was used to seed a 150 m3 single-stage nitritation- ANAMMOX unit.
This unit reached the target ammonia oxidation activity of 400 g N.m−3.day−1
within only a few weeks (Joss et al., 2009)
Besides a long start-up period, achieving a balance between PN and
ANAMMOX is yet another rate-limiting factor for successful operation of
such processes. First, because partial nitritation is itself is a very delicate step
involving partial oxidation of ammonia into nitrite in a controlled manner,
which is not a rule of nature. Second, ANAMMOX is a slow and sensitive
process inhibited by various factors. Thus, to balance between the PN re-
actor and the ANAMMOX reactor is a herculean task, especially during the
stabilization period, and always involves frequent adjustment of the oper-
ating conditions on the basis of the growth and stabilization of AOB and
ANAMMOX bacteria (Joss et al., 2009; van der Star et al., 2007).
Single-stage processes combining PN-ANAMMOX in a single reactor are
mostly operated in an intermittently aerated sequential batch reactor. Again
there are many inherent challenges to obtain a good process performance in
a single-stage reactor. First, the slow growth rate of ANAMMOX bacteria re-
sult is very long start-up periods and hence requires a high biomass retention
period (van der Star et al., 2007; Vlaeminck et al., 2009b; Wett 2006). Sec-
ond, with nitrite being toxic to ANAMMOX bacteria, its accumulation in the
reactor must be restricted. Therefore, growth and activity of aerAOB bacteria
must not exceed the growth rate of anAOB or ANAMMOX bacteria. Third,
high efficiency requires low nitrate production. Nitrate generated by NOB
and ANAMMOX bacteria must be consumed by the heterotrophic denitrifiers
(Vlaeminck et al., 2009b). Maintaining low oxygen concentration of ≤ 1mg
O2 /L and low nitrite concentration of ≤10 mg NO2 -N/L is necessary for the
growth of aerAOB and ANAMMOX bacteria, respectively. Similarly, maintain-
ing a minimum ammonia concentration of 10 mg NH3 -N/L along with low
concentration nitrite and low oxygen are necessary to prevent overgrowth
of NOB. Thus, the most critical parameter in ANAMMOX-based processes
is maintaining an appropriate concentration of dissolved oxygen, which has
to be just sufficient for the growth and activity of AOB, while being insuffi-
cient for the survival of NOB and nontoxic to the ANAMMOX bacteria. The
divergent conditions needed for survival of aerAOB and ANAMMOX bacte-
ria coexisting together in single reactor raises a natural concern about their
ecological relationship in nature.
COEXISTENCE OF aerAOB AND anAOB/ANAMMOX BACTERIA
The discovery of ANAMMOX bacteria in the autotrophic denitrifying plant

raised many speculations about the ecophysiological role of ANAMMOX
bacteria in the nature. The activity of ANAMMOX bacteria depends on the
supply of nitrite. AerAOB are known to oxidize ammonia to nitrite under
aerobic and anoxic conditions (Arp and Stein, 2003; Schmidt and Bock, 1997;
Schmidt et al., 2002). Hence, it is assumed that the aerAOB supply nitrite
for ANAMMOX bacteria and these two groups of microorganisms co-exist
together at oxic-anoxic interphases such as in biofilms and flocs (Schmidt
et al., 2002). Denitrifying bacteria can also supply nitrite for the ANAMMOX
reaction. In fact, there has been lot of debate to quantify the dominant sink
of fixed inorganic nitrogen in marine ecology. Until now, denitrification was

considered as the major source of N2 emission from marine environment.
Oceanic OMZs are responsible for about 35% of the oceanic N2 production
and almost half of it comes from anaerobic activity in the Arabian Sea and
Eastern Tropical South Pacific (ETSP; Lam et al., 2009, Ward et al., 2009).
Only recently it has been argued that ANAMMOX are solely responsible of
N2 loss in the OMZs (Lam et al., 2009). Without detectable denitrification, it
was not clear how substrates are provided for the ANAMMOX reaction.
Ammonia oxidation by aerAOB bacteria, especially by the Nitrosomonas
species, has been shown to occur in both oxygen-dependent and oxygen-
independent–NO2 dependent pathways (Arp and Stein, 2003; Schmidt et al.,
2002). The hypothetical NOx cycle describes the role of NO in ammonia
oxidation by Nitrosomonas species under oxic conditions (Schmidt et al.,
2002). NO was found to stimulate ammonia oxidation even in aerobically
incubated Nitrosomonas species (Schmidt et al., 2001b; Zart et al., 2000).
Hence it is not clear whether NO is the predominant oxidant for the AOB
species rather than O2 as believed always (Arp and Stein, 2003; Schmidt
et al., 2002). The rhetorical question raised by Schmidt et al. (2002) about the
relationship between aerAOB and anAOB as competitors or natural partners
still remains unanswered.
The FISH analysis and activity measurements of biomass from PN-
ANAMMOX reactors mostly detected AOB and ANAMMOX bacteria, but the
aerobic nitrite oxidizers (Nitrobacter or Nitrospira) species were not de-
tected. The plausible explanation for observation was the inability of aerobic
NOB species to withstand competition by aerAOB for oxygen under low oxy-
gen concentration. Another study from quantitative FISH analysis of biomass
from a full-scale OLAND process showed 43% of the population comprised
aerAOB species while the ANAMMOX bacteria constituted merely 8% of the
population (Vlaeminck et al., 2009b). The aerAOB activity determined in this
biomass overestimated the reactor activity. The authors argued that disinte-
gration of biofilms during the batch assay expose the AOB species embedded
within the biofilms to more oxygen concentrations and hence the estimated
rate of ammonia oxidation increased in orders of magnitude as against the
actually observed rates of ammonia oxidation in the full-scale plant. The
ammonia oxidation rate under aerobic conditions is around 100–300 times
greater than that under anaerobic condition (Jetten et al., 2001). Such de-
creased metabolic activity under the anaerobic condition is justified and
hence it has been considered as an adaptation mechanism by aerAOB bac-
teria to survive unfavorable conditions under oxygen stress. However, if the
anaerobic ammonia oxidation pathway by the aerAOB is simply a survival
alternative, then would it be sensible wonder why the anaerobic biomass
from the full-scale OLAND process showed dominance of aerAOB despite
the plant operating under limiting oxygen conditions? Supporting this obser-
vation, some researchers have reported a reduced oxygen consumption rate

during partial nitrification under oxygen-limiting condition, which could not
be explained by the present knowledge of understanding (Ahn and Choi,
2006; Bagchi et al., 2009).
Another source of indirect evidence comes from the application of all the
recent autotrophic ammonia oxidation processes in treating similar kind of
wastewater that is derived from anaerobic digestion. Most of these processes
have been applied for the treatment of digested sludge water, digested black
water, and municipal leachates (Table 5; Cema et al., 2006; van der Star
et al., 2007). The SHARON process has been used for treating high nitrogen-
bearing wastewaters from sludge dewatering, sludge drying or incineration
plants, landfill rejects, and wastewaters from digestion of organic wastes
and manure plants (Mulder et al., 2001; van Kempen et al., 2001). The
BABE process was installed for the treatment of rejection wastewater of
digested sludge dewatering system (Salem et al., 2004). Recently, few more
full-scale applications have been reported for treating rejection wastewater
from a municipal digester in a single-stage SBR through the CANON/OLAND
process (Joss et al., 2009), treatment of digested black water in a single-
stage RBC through the OLAND process (Vlaeminck et al., 2009b) and also
for treatment of landfill leachate through the SNAD process (Wang et al.,
2010).
These applications indicate that the nitrification and ANAMMOX-based
processes are more effective in removing ammonia present in effluents
from anaerobic digestion, which also contain simple organic molecules
such as methanol, ethanol, acetate, propionate, and butyrate that are com-
monly present in anaerobic environments. Both AOB and ANAMMOX
bacteria are known to metabolize some of these organic compounds
(Arp et al., 2007; Hommes et al., 2003; Schmidt, 2009; Strous et al.,
2006).
These evidences strongly suggest natural presence of AOB to anoxic
environment and our incomplete understanding of the ecology in the coex-
istence of AOB and ANAMMOX species under oxic/anoxic environments.
GRAY AREAS IN BIOLOGICAL NITROGEN REMOVAL MECHANISMS
The biological nitrogen removal pathways operating in nature are not well
understood. This is because of the limited understanding of the role of
microorganisms in the global nitrogen cycle. It is also biased by the ex-
perimental evidences on the behavioral pattern of isolated microorganisms
in manmade laboratory conditions. The marine environments are the major
contributors in the global nitrogen cycle. The discoveries on microbial inter-
actions in marine ecological niches are adding to the existing knowledge.
The missing link between the ecology of nitrogen cycle and the redundancy
of microbes and genes in the marine environment is very intriguing. With the
addition of new species of nitrogen-removing microorganisms being added
to the existing list of bacteria, efforts are being taken to decipher their role in
global nitrogen cycle through understanding the mode of energy generation.
Recent findings on the behavioral patterns of AOB have given a de-
tailed insight into their capabilities of surviving under oxygen stress. These
experimental evidences coupled with detection of N2 O and NO fluxes in the
oceanic depths at oxygen-depleted zones have corroborated the knowledge
of existence of AOB under anoxic conditions. These compounds are also
produced in denitrification pathway as intermediates but are not released
into the environment. The source of N2 O and NO, in the marine environ-
ment has been traced to AOB that survive oxygen stress through nitrifiers’
denitrification pathway (Kester et al., 1997). The genes responsible for gen-
eration of N2 O and NO have been discovered (Figure 1) in Nitrosomonas
species (Arp and Stein, 2003). These genes were found to be activated un-
der anaerobic conditions where the microorganisms are forced to survive
through nitrifiers’ denitrification pathway. Because these incompletely oxi-
dized gases or GHG (N2 O and NO) are known to be toxic to living forms,
it is interesting to understand how these microorganisms survive their own
toxic by-products.
The release of N2 O and NO outside the cell could be a probable mech-
anism of surviving the effects of toxic gases (Arp and Stein, 2003). The de-
tection of N2 O and NO fluxes in the marine environment may be related to
such releases. NO is also assumed to play a key role as signal transducer for
transition of one metabolic mechanism to another as proposed in NOx cycle
(Schimdt et al., 2002). It is also suggested that in communities where AOB
and ANAMMOX bacteria work in association in oxic-anoxic interphases, N2 O
and NO are readily used up by the latter as electron acceptor for oxidizing
ammonia to molecular nitrogen (Penton et al., 2006). Hence, it is interest-
ing to understand whether AOB have devised an anaerobic denitrification
pathway to survive under an oxygen stress condition, or if it is an essential
regulatory pathway for supplying signal transducers in anaerobic niches.
As nitrifiers are inhibited by light (Olson, 1981), nitrification can be
assumed to be occurring in the deep sea. This also implies that nitrification is
occurring in the zones having low oxygen fluxes. The deep ocean waters are
mostly found to be rich in nitrate, which is diffused to ocean surfaces through
physical as well as microbiological forces. Nitrification by chemolithotrophic
microorganisms has been understood as an obligatively aerobic process.
Conversely, denitrification by heterotrophic microorganisms has always been
considered as facultatively anaerobic process. Hence, the presence of nitrates
under oxygen-depleted zones in the marine environment is very much in
contrast with the aerobic nature of nitrifying bacteria.
There are many direct evidences that suggest that nitrate generation can
be coupled with Mn reduction, and also that cycles of Mn and N are very
much inherently linked (Hulth et al., 1999; Luther and Popp, 2002). Nitrate
fluxes have also been coincident with iron and sulfur reduction zones in
deep marine sediments (Clement et al., 2005; Zehr and Ward, 2002). The
most likely explanation is that there is recycling of reduced nitrogen com-
pounds in iron and sulfur reduction zones in deep anaerobic layers. It could
suggest that such conversions between reduced nitrogen compounds and
iron and sulfur oxides could be purely chemical processes rather than bio-
logical processes. Also, Luther et al. (1998; 1997) calculated that nitrification
of ammonia with Fe oxides is not thermodynamically favorable at pH <
6.8. However, Mortimer et al. (2002) suggested the role of Fe oxyhydrox-
ides, as against Fe oxides, which are reactive oxides of iron present in such
phases, in oxidation of ammonia to nitrate in deep anaerobic marine sed-
iments. Thus, it is not clear whether it is microbial denitrification followed
by chemical oxidation of N2 to nitrate, or chemical oxidation of ammonia to
nitrate, that is occurring in anaerobic sediments. An illustration depicted in
Figure 4 explains the most probable routes of nitrogen conversions through
biological and chemical pathways in the marine environment.
Incubation of sediments with addition of 15NH3 is reported to generate
15
N2 . This is assumed to take place via aerobic nitrification and anaerobic
denitrification pathways (Jenkins and Kemp, 1984; Seitzinger, 1988). The
flux of N2 generation is measured as denitrification rate in the sediments.
The measured fluxes of N2 in marine sediments have been found to be
more than acetylene inhibited (i.e., O2 dependent nitrification) and labeled
15
NO3 tracer experiments (i.e., anaerobic denitrification). Similar evidences
reported by various researchers have indicated the existence of alternative
N2 removal pathways. Observations at pore water of deep sea sediments
have given evidence of chemo-denitrification (i.e., N2 formation through
reduction of nitrate by dissolved Mn2+; Sørensen et al., 1987). Luther et al.
(1997) proposed two chemical reactions that are thermodynamically feasible
for nitrogen production through the combination of field observations and
laboratory experiments; the first is reduction of nitrate by Mn2+ (chemical
denitrification) and the other is oxidation of inorganic ammonia (NH+ 4 ) and
organic nitrogen to N2 by MnO2 in the presence of oxygen (Figure 5). The
oxidation of NH+ 4 and organic-N to N2 by MnO2 is proposed to occur in
FIGURE 4. Nitrogen conversions in marine environment. Ammonification occurring in the

anaerobic marine sediments makes these zones ammonia rich. Anaerobic ammonia-oxidizing
bacteria (AOB) utilize ammonia under anaerobic condition. AOB oxidize ammonia using
nitrifier denitrification (DN) pathway to generate nitric oxide (NO−) and nitrous oxide (N2 O−)
as end products. N2 O− is released into the atmosphere, while NO− is oxidized into two
molecules of NO− −
2 or one molecule of N2 O4 . NO
−
and N2 O− are used as oxidants for
oxidizing ammonia by AOB and ANAMMOX bacteria, respectively. N2 formed during the
ANAMMOX process is released into the atmosphere. N2 is also generated by DN of nitrate
(NO− −
3 ). NO3 is generated as a result of aerobic nitrification on the oceanic surface and diffuses
to the anaerobic niches at the ocean bed. NO− 3 is also speculated to be generated by chemical
nitrification of inorganic ammonia mediated by metal oxides. On a similar note, metal oxide
mediated chemical DN of NO− 3 to N2 and vice versa is also speculated in ocean beds.
surface sediments. The Mn2+ formed is reoxidized to MnO2 by O2 in a

microbial reaction. Thus, MnO2 serves as the direct oxidant for oxidation of
NH+ 4 and organic-N to N2 , while O2 serves as the ultimate oxidant. It was
concluded that the free energy released during indirect oxidation of NH+ 4 and
organic-N to N2 through MnO2 is much more than during direct oxidation
by O2 . In addition, the role of Fe in many acidic lakes for undergoing similar
reactions as Mn+ −
2 with NO3 was also indicated.
Thus, experiences from full-scale implementations and ecological un-
derstanding in marine environment could be linked together to postulate
a hypothesis that nitrification is basically an anoxic process, if not obliga-
tively anaerobic. In addition, the AOB species oxidize ammonia using indi-
rect oxidizing agents such as nitrite or manganese oxides or iron oxides in
anoxic conditions. The nitrite generated during this process is used up by the
FIGURE 5. Interlinking of Mn and N cycle based on Luther et al. (1997).
anaerobic counterpart (i.e., ANAMMOX bacteria). Hence, AOB and

ANAMMOX species can be considered natural partners under oxygen-free
marine environment, where the presence of ammonia, a common substrate
for both the groups of microorganisms, is naturally abundant (Figure 6).
Anaerobicity is a primitive mechanism of energy generation as per the the-
ories of evolution. An undisturbed ecological environment such as in ocean
beds still harbors such primitive ecosystems. Hence, anoxic AOB species
may be considered aborigines of marine environment that have developed
aerobic pathways in due course of evolution. If this hypothesis is followed,
the need of controlling oxygen concentration for achieving a very unsteady
state of partial nitritation can be avoided (Figure 7). Thus, with this concept
the competition between AOB and NOB populations can also be put to an
end.
FUTURE PERSPECTIVES
The knowledge gained through the long-term studies demonstrate that our
concept of nitrogen cycle as a whole was oversimplified. Many important
clues are still missing, and despite our limited understanding of these mech-
anisms, processes and technologies have been developed on biological ni-
trogen removal based on these complex microbial interactions.
The various nitrogen-removing processes that claim technoeconomi-
cal advantages over the conventional nitrification-denitrification process are
mostly based on the microbial interactions between various communities
FIGURE 6. A hypothetical assumption that ammonia-oxidizing bacteria (AOB) are aborigines

of anaerobic marine environment that are rich source of ammonia (NH3 ). Ammonia is released
in the anaerobic sediments of ocean through ammonification of organic matter. AOB compli-
ments the anaerobic ANAMMOX bacteria with nitrite (NO2 ) as substrate. The proposed NOx
cycle (Schmidt et al., 2002) and the Mn cycle (Hulth et al., 1999; Luther et al., 1997; Sørensen
et al., 1987) are assumed to play the role of oxidants for anaerobic ammonia oxidation by the
AOB.
under oxygen-limiting conditions. Nitrite accumulation, though not a rule

in natural ecosystems, has been the most critical and fragile step in all the
recent biological nitrogen-removing processes. Though conditions for main-
taining the delicate balance of nitrite accumulation in all manmade ecosys-
tems are detailed in many studies, it still remains the most important process
control parameter in all the processes discussed. Apart from nitrite accumu-
lation, understanding the interactions between different nitrogen-removing
microorganisms and the parameters favoring the interactions through pro-
cess optimization are also relevant for achieving effective performances. The
circumstances that lead to development of nitrification, denitrification, het-
erotrophic nitrification, aerobic denitrification, anaerobic ammonia oxidation,
and nitrifier denitrification in natural ecosystem need clearly understanding.
The boundary conditions separating one microbial process from another are
not sharply defined in a natural ecosystem. Hence, it is immature to consider
these processes in isolation.
The underlying microbial principles in all these biological nitrogen re-
moval processes are more or less similar. In spite of that the terminologies
used in describing these processes is confusing and they differ mostly in the
FIGURE 7. The underlying principle in new autotrophic ammonia removal processes is based
on the classical dogma that oxidation of ammonia to nitrite is basically an aerobic process,
while further conversion of nitrite to nitrogen in an anaerobic process. Aerobic oxidation of
ammonia leads to unwanted generation of nitrate due to activity of nitrite-oxidizing bacteria
(NOB). An alternative strategy to avoid nitrate generation is proposed through conversion of
ammonia to nitrite and nitrogen under completely anaerobic condition.
chronological order of their development (Appendix B). Van der Star et al.
(2007) resolved the confusion created in the processes nomenclature by
suggesting some descriptive terminologies that are apparently less ambigu-
ous for the scientific community. Though the recently developed low-cost
technologies for nitrogen removal vaguely describe the underlying microbial
principles, it is not be long before each of these processes will be known
to have similar or more complicated microbial interactions. Hence, until the
fundamental principle in these technologies are unraveled through research
using stable isotopes, inhibition experiments, pure culture experiments, and
modeling it will not be possible to direct them to achieve desirable end
products in a sustainable manner. Thus, long-term process stability in the
application of these technologies is still critical issue, and therefore needs

future research in the areas discussed.
CONCLUSION
Autotrophic ammonia removal processes are presently undergoing through

a phase transformation in order to maintain pace with the stringent environ-
mental protection laws. Many new processes based on the principle of either
PN-ANAMMOX or PN-denitritation are developing that claim to be more tech-
noeconomical as against the conventional nitrification-denitrification process.
PN becomes the key step in all the processes, wherein a very fragile equi-
librium is maintained between aerobic AOB and anaerobic ANAMMOX (or

denitrifying) bacteria through nitrite. The new autotrophic ammonia removal
processes mainly rely on the classical dogma that nitrification is an aerobic
process. Under the aerobic condition, it is very difficult to prevent oxida-
tion of nitrite to nitrate by the activity of aerobic NOB. This is one of the
rate-limiting steps in successful application of such processes.
The recent discovery of the anaerobic ammonia oxidation pathway in
the novel Planctomycetes bacteria and in Nitrosomonas species have brought
in the new concept that can be employed in effectively treating ammonia-
bearing wastewaters through a completely anaerobic process. Under com-
plete anaerobic condition, the unwanted biological conversion of nitrite to
nitrate can be prevented. However, this hypothesis needs to be verified
experimentally before it can be translated into a sustainable process.
ACKNOWLEDGMENTS
The financial support by the Department of Biotechnology, Ministry of Sci-

ence and Technology, Government of India, New Delhi, for this project is
gratefully acknowledged. The authors are thankful to Dr. S.R Wate, Director,
NEERI, Nagpur, India, for his guidance during the project work.
NOMENCLATURE
aerAOB Aerobic ammonia-oxidizing bacteria

anAOB Anaerobic ammonia-oxidizing bacteria
ANAMMOX Anaerobic ammonia oxidation
ART Aerated Retention Time
AS/PN Activated sludge/partial Nitrification
BABE Bio-Augmentation Batch Enhanced
BNR Biological Nitrogen Removal
CANON Completely autotrophic nitrogen removal over nitrite

DEAMOX Denitrifying Ammonium Oxidation
DEMON Deammonification
DN Denitrification
HRT Hydraulic retention time
NOB Nitrite-oxidizing bacteria
OMZ Oxygen Minimum Zone
PN Partial nitrification
RBC Rotating Biological Contractor
SBR Sequential Batch Reactor
SHARON Single reactor High Activity Ammonia Removal Over Nitrite
SNAD Simultaneous nitrification ANAMMOX and denitrification
SND Simultaneous nitrification and denitrification

SRT Sludge Retention Times
WWTP Waste Water Treatment Plant
REFERENCES
Abma, W.R., Schultz, C.E., Mulder, J.W., van der Star, W.R.L., Strous, M., Tokutomi,
T., and van Loosdrecht, M.C.M. (2007). Full scale granular sludge Anammoxs
process. Water Sci. Technol., 55(8–9), 27–33.
Ahn, Y.-H. (2006). Sustainable nitrogen elimination biotechnologies: A review. Pro-
cess Biochem., 41, 1709–1721.
Ahn, Y.-H., and Choi, H.-C. (2006). Autotrophic nitrogen removal from sludge di-
gestor liquids in up-flow sludge bed reactor with external aeration. Process
Biochem., 41, 1945–1950.
Anderson, I.C., and Levine, J.S. (1986). Relative rates of NO and N2 O production by
nitrifiers, denitrifiers and nitrate respirers. Appl. Environ. Microbiol., 51, 938–945.
Anderson, K.K., and Hooper, A.B. (1983). O2 and H2 O are each the source in one O
in NO2 –produced from NH3 by Nitrosomonas; 15N NMR evidence. FEBS Lett.,
164, 236–240.
Anthonisen, A.C., Loehr, R.C., Prakasam, T.B.S., and Srinath, E.G. (1976). Inhibition
of nitrification by ammonia and nitrous acid. J. Water Pollut. Control Fed., 48,
835–852.
Arp, D.J., Chain, P.S.G., and Klotz, M.G. (2007). The impact of genome analyses on
our understanding of ammonia-oxidizing bacteria. Annu. Rev. Microbiol., 61,
503–28
Arp, D.J., Sayavedra-Soto, L.A., and Hommes, N.G. (2002). Molecular biology and
biochemistry of ammonia oxidation by Nitrosomonas europaea. Arch. Micro-
biol., 178, 250–255.
Arp, D.J., and Stein, L. (2003). Metabolism of inorganic N compounds by ammonia-
oxidising bacteria. Crit. Rev. Biochem. Mol. Biol., 38(6), 471–495.
Bagchi, S., Biswas, R., and Nandy, T. (2010a). Start-up and stabilization of Anammox
process from a non-acclimatized sludge in CSTR. J. Ind. Microbiol. Biotechnol.,
37, 943–952.
Bagchi, S., Biswas, R., and Nandy, T. (2010b). Alkalinity and DO as controlling
parameters for single-stage biological nitrogen removal (SBNR) process with
partial nitritation and anammox. J. Ind. Microbiol. Biotechnol., 37, 871–876.
Bagchi, S., Biswas, R., Roychoudhury, K., and Nandy, T. (2009). Stable partial nitri-
fication in an up-flow fixed bed bioreactor under oxygen limiting environment.
Environ. Eng. Sci., 26, 1309–1318.
Bagchi, S., Biswas, R., Vlaeminck, S.E., Roychoudhury K., and Nandy, T. (2012). Sta-
ble performance of non-aerated two-stage partial nitritation/anammox (PANAM)
with minimal process control in Microbial Biotechnology (Available online
doi:10.1111/j.1751-7915.2012.00336.x).
Bano, N., Ruffin, S., Ransom, B., and Hollibaugh, J.T. (2004). Phylogenetic com-
position of Arctic Ocean archaeal assemblages and comparison with Antarctic
assemblages. Appl. Environ. Microbiol., 70, 781–789.
Barraclough, D., and Puri, G. (1995). The use of 15N pool dilution and enrichment to
separate the heterotrophic and autotrophic pathways of nitrification. Soil Biol.
Biochem., 27, 17–22.
Bateman, E.J., and Baggs, E.M. (2005). Contributions of nitrification and denitrifica-
tion to N2O emissions from soils at different water-filled pore space. Biol. Fertil.
Soil, 41, 379–388.
Bazylinski, D.A., and Blakemore, R.P. (1983). Denitrification and assimilatory nitrate
reduction in Aquaspirillum magnetotacticum. Appl. Environ. Microbiol., 46,
1118–1124.
Beaumont, H.J.E., Hommes, N.G., Sayavedra-Soto, L.A., Arp, D.J., Arciero, D.M.,
Hooper, A.B., et al. (2002). Nitrite reductase of Nitrosomonas europaea is not
essential for production of gaseous nitrogen oxides and confers tolerance to
nitrite. J. Bacteriol., 184, 2557–2560.
Beaumont, H.J.E., Lens, S.I., Reijnders, W.N.M., Westerhoff, H.V., and van Span-
ning, R.J.M. (2004a). Expression of nitrite reductase in Nitrosomonas europaea
involves NsrR, a novel nitrite-sensitive transcription activator. Mol. Microbiol.,
54, 148–158.
Beaumont, H.J.E., van Schooten, B., Lens, S.I., Westerhoff, H.V., and van Spanning,
R.J.M. (2004b). Nitrosomonas europaea expresses a nitric oxide reductase during
nitrification. J. Bacteriol., 186, 4417–4421.
Biswas, R., Bagchi, S., Gupta, D., Urewar, C., and Nandy, T. (2010). Treatment
of wastewater from a LTC process industry through biological and chem-
ical oxidation processes for recycle/reuse. Water Sci. Technol., 61, 2563–
2573.
Biswas, R., Bagchi, S., Bihariya, P., Das, A., and Nandy, T. (2011). Stability and
microbial community structure of a partial nitrifying fixed-film bioreactor in
long run. Bioresource Technology, 102, 2487–2494.
Bock, E. (1965). Comparative study of the effect of visible light on Nitrosomonas
europaea and Nitrobacter winogradskyi. Arch. Microbiol., 51, 18–41.
Bock, E. (1995). Nitrogen loss caused by denitrifying Nitrosomonas cells using am-
monium or hydrogen as electron donors and nitrite as electron acceptor. Arch.
Microbiol., 163, 16–20.
Bock, E., Koops, H.P., and Harms, H. (1986). Cell biology of nitrifying bacteria. In
Prosser, J.I. (Ed.), Nitrification (pp. 17–38). Oxford, England: IRL.
Bock, E., and Wagner, M. (2006). Oxidation of inorganic nitrogen compounds as an

energy source. In Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H., and
Stackebrandt, E. (Eds.), The prokaryotes: A handbook on the biology of bacteria –
ecophysiology and biochemistry (3rd ed., Vol. 2, pp. 457–495). Singapore:
Springer.
Bodenstein, M. (1918). Die Geschwindigkeit der Reaktion zwischen Stickoxid und
Sauerstoff. Z. Elektroch., 24, 183–201.
Bothe, H., Jost, G., Schloter, M., Ward, B.B., and Witzel, K.-P. (2000). Molecular
analysis of ammonia oxidation and denitrification in natural environments. FEMS
Microbiol. Rev., 24, 673–690.
Bremner, J.M., and Blackmer, A.M. (1978). Nitrous oxide: emission from soils during
nitrification of fertilizer nitrogen. Science, 199, 295–296.
Broda, E. (1977). Two kinds of lithotrophs missing in nature. Z. allg. Mikrobiol., 17,
491–493.
Cantera, J.J.L., and Stein, L.Y. (2007). Molecular diversity of nitrite reductase genes
(nirK) in nitrifying bacteria. Environ. Microbiol., 9, 765–776.
Carrio, L., Sexton, J., Lopez, A., Gopalakrishnam, K., and Sapienza, V. (2003).
Ammonia-nitrogen removal from centrate, 10 years of testing and operating
experience in New York City. Paper presented at WEFTEC 2003, Session 42:
Municipal Wastewater Treatment Processes, Water Environment Federation.
Castignetti, D., and Gunner, H.B. (1980). Sequential nitrification by Alcaligens sp and
Nitrobacter agilis. Can. J. Microbiol., 26, 1114–1119.
Castignetti, D., and Gunner, H.B. (1982). Differential tolerance of hydroxylamine by
an Alcaligenes sp., a heterotrophic nitrifier, and by Nitrobacter agilis. Can. J.
Microbiol., 28, 148–150.
Castignetti, D., and Hollocher, T.C. (1984). Heterotrophic nitrification among deni-
trifiers. Appl. Environ. Microbiol., 47, 620–623.
Cema, G., Szatkowska, B., Plaza, E., Trela, J., and Surmacz-Gorska, J. (2006). Ni-
trogen removal rates at a technical scale pilot plant with one stage partial
nitritation/ANAMMOX process. Water Sci. Technol., 54, 209–217.
Chamchoi, N., Nitisoravut, S., and Schmidt, J.E. (2008). Inactivation of ANAMMOX
communities under concurrent operation of anaerobic ammonium oxidation
(ANAMMOX) and denitrification. Bioresour. Technol., 99, 3331–3336.
Chen, H.H., Liu, S.T., Yang, F.L., Yuan, X., and Wang, T. (2009). The development
of simultaneous partial nitrification, ANAMMOX and denitrification (SNAD)
process in a single reactor for nitrogen removal. Bioresour. Technol., 100,
1548–1554.
Chopra, I., Storey, C., Falla, T.J., and Pearce, J.H. (1998). Antibiotics, peptidogly-
can synthesis and genomics: the chlamydial anomaly revisited. Microbiol., 144,
2673–2678.
Clement, J.-C., Shrestha, J., Ehrenfeld, J.G., and Jaffe, P.R. (2005). Ammonium oxi-
dation coupled to dissimilatory reduction of iron under anaerobic conditions in
wetland soils. Soil Biol. Biochem., 37, 2323–2328.
Coolen, M.J.L., Abbas, B., van Bleijswijk, J., Hopmans, E.C., Kuypers, M.M.M., Wake-
ham, S.G., and Sinninghe Damsté, S.J. (2007). Putative ammonia-oxidizing Cre-
narchaeota in suboxic waters of the Black Sea: A basin-wide ecological study
using 16S ribosomal and functional genes and membrane lipids. Environ. Mi-
crobiol., 9, 1001–1016.
Cornelius, A., and Rosenwinkel, K.-H. (2002). Aerob/anoxische Deam-

monifikation stickstoffhaltiger Abwässer im KALDNESR -Biofilmverfahren, KA-
Wasserwirtschaft, Abwasser, Abfall 49, 1398–1403 (in German).
Dalsgaard, T., Canfield, D.E., Petersen, J., Thamdrup, B., and Acuna-Gonzalez, J.
(2003). N2 production by the anammox reaction in the anoxic water column of
Golfo Dulce, Costa Rica. Nature, 422, 606–608.
Dalsgaard, T., and Thamdrup, B. (2002). Factors controlling anaerobic ammo-
nium oxidation with nitrite in marine sediments. Appl. Environ. Microbiol., 68,
3802–3808.
Dalsgaard, T., Thamdrup, B., and Canfield, D.E. (2005). Mini-review: Anaerobic
ammonium oxidation (anammox) in the marine environment. Res. Microbiol.,
56, 457–464.
Dapena-Mora, A., Arrojo, B., Campos, J.L., Mosquera-Corral, A., and Mendez, R.
(2004a). Improvement of the settling properties of ANAMMOX sludge in an

SBR. J. Chem. Technol. Biotechnol., 79, 1417–1420.
Dapena-Mora, A., Campos, J.L., Mosquera-Corral, A., Jetten, M.S.M., and Mendez, R.
(2004b). Stability of the ANAMMOX process in a gas-lift reactor and a SBR. J.
Biotechnol., 110(2), 159–170.
Dapena-Mora, A., Fernandez, I., Campos, J.L., Mosquera-Corral, A., Mendez, R., and
Jetten, M.S.M. (2007). Evaluation of activity and inhibition effects on Annamox
process by batch tests based on the nitrogen gas production. Enzyme Microb.
Technol., 40, 859–865.
Dosta, J., Fernández, I., Vázquez-Padı́n, J.R., Mosquera-Corral, A., Campos, J.L., Mata-
Álvarez, J., and Méndez, R. (2008). Short- and long-term effects of temperature
on the Anammox process. J. Hazard. Mater., 154, 688–693.
Egli, K., Fanger, U., Alvarezz, P.J.J., Siegrist, H., van der Meer, J.R., and Zehnder,
A.J.B. (2001). Enrichment and characterization of an ANAMMOX bacterium from
a rotating biological contactor treating ammonium rich leachate. Arch. Micro-
biol., 175, 198–207.
Fiencke, C., Spieck, S., and Bock, E. (2005). Nitrifying bacteria. In Werner, D.,
and Newton, W.E. (Eds.), Nitrogen fixation: Origins, applications, and research
progress (Vol. 4, pp. 255–276). Amsterdam, the Netherlands: Springer.
Francis, C.A., Beman, J.M., and Kuypers, M.M.M. (2007). New processes and players
in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia
oxidation. ISME J., 1, 19–27.
Francis, C.A., Roberts, K.J., Beman, J.M., Santoro, A.E., and Oakley, B.B. (2005).
Ubiquity and diversity of ammonia-oxidizing archaea in water columns and
sediments of the ocean. Proc. Nat. Acad. Sci. USA, 102, 14683–14688.
Fuerst, J.A. (2005). Intracellular compartmentation in planctomycetes. Annu. Rev.
Microbiol., 59, 299–328.
Fux, C. (2003). Biological nitrogen elimination of ammonium-rich sludge digester
liquids. Doctoral dissertation, ETH-Zürich, Switzerland.
Fux, C., Böhler, M., Huber, P., Bruner, I., and Siegrist, H. (2002). Biological treatment
of ammonium-rich wastewater by partial nitritation and subsequent anaerobic
ammonium oxidation (ANAMMOX) in a pilot plant. J. Biotechnol., 99, 295–306.
Fux, C., Marchesi, V., Brunner, I., and Siegrist, H. (2004). Anaerobic ammonium
oxidation of ammonium-rich waste streams in fixed-bed reactors. Water Sci.
Technol., 49(11–12), 77–82.
Fux, C., and Siegrist, H. (2004). Nitrogen removal from sludge digester liquids by
nitrification/denitrification or partial nitritation/ANAMMOX: Environmental and
economical considerations. Water Sci. Technol., 50(10), 19–26.
Gong, Z., Yang, F., Liu, S., Bao, H., Hu, S., and Furukawa, K. (2007). Feasibility of a
membrane-aerated biofilm reactor to achieve single-stage autotrophic nitrogen
removal based on ANAMMOX. Chemosphere, 69, 776–784.
Grunditz, C., and Dalhammar, G. (2001). Development of nitrification inhibition
assays using pure cultures of Nitrosomonas and Nitrobacter. Water Res., 35,
433–440.
Guerrero, M.A., and Jones, R.D. (1996). Photoinhibition of marine nitrifying bacteria.
1. Wavelength-dependent response. Mar. Ecol-Prog. Ser., 141(1–3), 183–192.
Gupta, A.B. (1997). Thiosphaera pantotropha: A sulphur bacterium capable of simul-
taneous heterotrophic nitrification and aerobic denitrification. Enzyme Microb.
Technol., 21, 589–595.

Gupta, A.B., and Gupta, S.K. (2001). Simultaneous carbon and nitrogen removal
from high strength domestic wastewater in an aerobic RBC biofilm. Water Res.,
35, 1714–1722.
Gut, L., Płaza, E., Trela, J., Hultman, B., and Bosander, J. (2006). Combined partial
nitritation/anammox system for treatment of digester supernatant. Water Sci.
Technol., 53, 149–159.
Guven, D., Dapena-Mora, A., Kartal, B., Schmid, M.C., Maas, B., van de Pas-
Schoonen, K.T., Sozen, S., Mendez, R., Op den Camp, H.J.M., Jetten, M.S.M.,
Strous, M., and Schmidt, I. (2005). Propionate oxidation and methanol inhibition
of the anaerobic ammonium oxidizing bacteria. Appl. Environ. Microbiol., 71,
1066–1071.
Guven, D., van de Pas-Schoonen, K., Schmid, M.C., Strous, M., Jetten, M.S.M., Sozen,
S., Orhon, D., and Schmidt, I. (2004). Implementation of the ANAMMOX process
for improved nitrogen removal. J. Environ. Sci. Health Part A Tox. Haz. Subst.
Environ. Eng., 39, 1729–1738.
Hallam, S.J., Mincer, T.J., Schleper, C., Preston, C.M., Roberts, K., and Richardson,
P.M., et al. (2006). Pathways of carbon assimilation and ammonia oxidation
suggested by environmental genomic analyses of marine crenarchaeota. PLoS
Biol., 4, e95.
Hao, X., Heijnen, J.J., and van Loosdrecht, M.C.M. (2001). Sensitivity analysis of a
biofilm model describing a one-stage completely autotrophic nitrogen removal
(CANON) process. Biotechnol. Bioeng., 77, 266–277.
Hayatsu, M., Tago, K., and Saito, M. (2008). Various players in the nitrogen cy-
cle: Diversity and functions of the microorganisms involved in nitrification and
denitrification. Soil Sci. Plant Nutr., 54, 33–45.
Hellinga, C., Schellen, A.A.J.C., Mulder, J.W., van Loosdrecht, M.C.M., and Heijnen,
J.J. (1998). The SHARON process: An innovative method for nitrogen removal
from ammonium rich wastewater. Water Sci. Technol., 37, 135–142.
Hellinga, C., van Loosdrecht, M.C.M., and Heijnen, J.J. (1997). The Sharon process
for nitrogen removal in ammonium rich waste water. Mededelingen Faculteit
Landbouwwetenschappen, Universiteit Gent, 62 (4b), pp-1743–1750.
Hellinga, C., van Loosdrecht, M.C.M., and Heijnen, J.J. (1999). Model based design of
a novel process for nitrogen removal from concentrated flows. Math. Comput.
Modell. Dyn. Syst., 5, 351–371.
Henze, M., Gujer, W., Matsuo, T., and van Loosdrecht, M.C.M. (2000). Activated
sludge models ASM1, ASM2, ASM2d and ASM3. Scientific and Technical Reports.
London, England: IWA.
Herndl, G.J., Reinthaler, T., van Teira, E.A.H., Veth, C., Pernthaler, A., and Pernthaler,
J. (2005). Contribution of Archaea to total prokaryotic production in the deep
Atlantic Ocean. Appl. Environ. Microbiol., 71, 2303–2309.
Hershberger, K.L., Barns, S.M., Reysenbach, A.-L., Dawson, S.C., and Pace, N.R.
(1996). Wide diversity of Crenarchaeota. Nature, 384, 420–420.
Hidaka, T., Yamada, H., Kawamura, M., and Tsuno, H. (2002). Effect of dissolved
oxygen conditions on nitrogen removal in continuously fed intermittent-aeration
process with two tanks. Water Sci. Technol., 45, 181–188.
Hippen, A., Helmer, C., Kunst, S., Rosenwinkel, K.-H., and Seyfried, C.F. (2001). Six
years’ practical experience with aerobic/anoxic deammonification in biofilm
systems. Water Sci. Technol., 44, 39–48.

Hippen, A., Rosenwinkel, K.-H., Baumgarten, G., and Seyfried, C.F. (1997). Aerobic
deammonification: A new experience in the treatment of wastewaters. Water
Sci. Technol., 35, 111–120.
Hommes, N.G., S-Soto, L.A., and Arp, D.J. (2003). Chemolithoorganotrophic growth
of Nitrosomonas europaea on fructose. J. Bacteriol., 185, 6809–6814.
Hooper, A.B., di Spirito, A.A., Olson, T.C., Anderson, K.A., Cunningham, W., and
Taaffe, L.R. (1984). Generation of the proton gradient by a periplasmic dehy-
drogenase. In Crawford, R.L., and Hanson, R.S. (Eds.), Microbial growth on C1
compounds (pp. 53–58). Washington DC: American Society for Microbiology.
Hooper, A., and Terry, K. (1979). Hydroxylamine oxidoreductase of Nitrosomonas:
Production of nitric oxide from hydroxylamine. Biochim. Biophys. Acta, 571,
12–20.
Hooper, A.B., Vannelli, T., Bergmann, D.J., and Arciero, D.M. (1997). Enzymology
of the oxidation of ammonia to nitrite by bacteria. Antonie van Leeuwenhoek,
71, 59–67.
Hu, S.S. (1990). Acute substrate-intermediate-product related inhibition of nitrifiers.
Master’s thesis, School of Civil Engineering, Purdue University, West Lafayette,
Indiana, USA.
Hulth, S., Aller, R.C., Canfield, D.E., Dalsgaard, T., Engstrfm, P., Gilbert, F., Sundback,
K., and Thamdrup, B. (2005). Nitrogen removal in marine sediments: Recent
findings and future research challenges. Mar. Chem., 94, 125–145.
Hulth, S., Aller, R.C., and Gilbert, F. (1999). Coupled anoxic nitrification/manganese
reduction in marine sediments. Geochim. Cosmochim. Acta, 63, 49–66.
Hunik, J.H., Tramper, J., and Wijffels, R.H. (1994). A strategy to scale-up nitrification
processes with immobilized cells of Nitrosomonas europaea and Nitrobacter
agilis. Bioprocess Eng., 11, 73–82.
Hwang, I.S., Min, K.S., Choi, E., and Yun, Z. (2005). Nitrogen removal from pig-
gery waste using the combined SHARON and ANAMMOX process. Water Sci.
Technol., 52, 487–494.
Hyman, M.R., Page, C.L., and Arp, D.J. (1994). Oxidation of methyl fluoride and
dimethyl ether by ammonia monooxygenase in Nitrosomonas europaea. Appl.
Environ. Microbiol., 60, 3033–3035.
Hyungseok, Y., Kyu-Hong, A., Kwang-Hwan, L., Youn-Ung, K., and Kyung-
Guen, S. (1999). Nitrogen removal from synthetic wastewater by simultaneous
nitrification and denitrification (SND) via nitrite in a intermittently-aerated reac-

tor. Water Res., 33, 145–154.
Ingalls, A.E., Shah, S.R., Hansman, R.L., Aluwihare, L.I., Santos, G.M., Druffel, E.R.M.,
and Pearson, A. (2006). Quantifying archaeal community autotrophy in the
mesopelagic ocean using natural radiocarbon. Proc. Nat. Acad. Sci. USA, 103,
4242–4247.
Jardin, N., Hippen, A., Seyfried, C.F., Rosenwinkel, K.-H., and Greulich, F. (2001).
Deammonification of sludge liquor at the wastewater treatment plant Hattingen
with a moving bed system. GWF, 142, 479–484 (in German).
Jenkins, M.C., and Kemp, W.M. (1984). The coupling of nitrification and denitrifica-
tion in two estuarine sediments. Limnol. Oceanog., 29, 609–619.
Jetten, M.S.M., Cirpus, I., Kartal, B., van Niftrik, L., van De Pas-Schoonen, K.T.,
Sliekers, O., Haaijer, S., van der Star, W., Schmid, M., van de Vossenberg,
J., Schmidt, I., Harhangi, H., van Loosdrecht, M., Kuenen, J.G., den Camp,
H.O., and Strous, M. (2005). 1994–2004: 10 years of research on the anaerobic
oxidation of ammonium. Biochem. Soc. Trans., 33, 119–123.
Jetten, M.S.M., Horn, S.J., and van Loosdrecht, M.C.M. (1997a). Towards a more
sustainable municipal wastewater treatment system. Water Sci. Technol., 35,
171–180.
Jetten, M.S.M., Logemann, S., Meyzer, G., Robertson, L.A., de Vries, S., van Loos-
drecht, M.C.M., and Kuenen, J.G. (1997b). Novel principles in the microbial
conversions of nitrogen compounds. Antonie van Leeuenhoek, 71, 75–93.
Jetten, M.S.M., Sliekers, A.O., Kuypers, M.M.M., Dalsgaard, T., van Niftrik, L.,
Cirpus, I., van de Pas-Schoonen, K.T., Lavik, G., Thamdrup, B., Le Paslier,
D., Op den Camp, H.J.M., Hulth, S., Nielsen, L.P., Abma, W., Third, K.,
Engstrom, P., Kuenen, J.G., Jorgensen, B.B., Canfield, D.E., Sinninghe, D.J.S.,
Revsbech, N.P., Fuerst, J., Weissenbach, J., Wagner, M., Schmidt, I., Schmid,
M., and Strous, M. (2003). Anaerobic ammonium oxidation by marine
and freshwater Planctomycete-like bacteria. Appl. Microbiol. Biotechnol., 63,
107–114.
Jetten, M.S.M., Strous, M., van de Pas-Schoonen, K.T., Schalk, J., van Dongen,
U.G.J.M., and van de Graaf, A.A. (1999). The anaerobic oxidation of ammo-
nium. FEMS Microbiol. Rev., 22, 421–437.
Jetten, M.S.M., Wagner, M., Fuerst, J., van Loosdrecht, M.C.M., Kuenen, G., and
Strous, M. (2001). Microbiology and application of the anaerobic ammonium
oxidation (‘ANAMMOX’) process. Curr. Opin. Biotechnol., 12, 283–288.
Jordan, K., Kondrashov, F.A., Adzhubei, I.A., Wolf, Y.I., Koonin, E.V., Kondrashov,
A.S., and Sunyaev, S.A. (2005). Universal trend of amino acid gain and loss in
protein evolution. Nature, 433, 633–638.
Joss, A., Salzgeber, D., Eugster, J., Konig, R., Rottermann, K., Burger, S., Fabijan, P.,
Leumann, S., Mohn, J., and Siegrist, H. (2009). Full-scale nitrogen removal from
digester liquid with Partial Nitritation and ANAMMOX in one SBR. Environ. Sci.
Technol., 43, 5301–5306.
Jubany, I., Lafuente, J., Baeza, J.A., and Carrer, J. (2009). Total and stable washout
of nitrite oxidizing bacteria from a nitrifying continuous activated sludge system
using automatic control based on oxygen uptake rate measurements. Water
Res., 43, 2761–2772.
Kalyuzhnyi, S.V., and Gladchenko, M. (2009). DEAMOX: New microbiological pro-

cess of nitrogen removal from strong nitrogenous wastewater. Desalination,
248, 783–793.
Kalyuzhnyi, S.V., Gladchenko, M.A., Kang, H., Mulder, A., and Versprille, A. (2008).
Development and optimisation of VFA driven DEAMOX process for treatment
of strong nitrogenous anaerobic effluents. Water Sci. Technol., 57, 323–328.
Kalyuzhnyi, S.V., Gladchenko, M., Mulder, A., and Versprille, B. (2006).
DEAMOX—New biological nitrogen removal process based on anaerobic am-
monia oxidation coupled to sulphide-driven conversion of nitrate into nitrite.
Water Res., 40, 3637–3645.
Kalyuzhnyi, S.V., Gladchenko, M., Mulder, A., and Versprille, B. (2007). Comparison
of quasisteady-state performance of the DEAMOX process under intermittent
and continuous feeding and different nitrogen loading rates. Biotechnol. J., 2,
894–900.
Karner, M.B., DeLong, E.F., and Karl, D.M. (2001). Archaeal dominance in the
mesopelagic zone of the Pacific Ocean. Nature, 409, 507–510.
Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb,
R.I., Schouten, S., Fuerst, J.A., Damsté, J.S., Jetten, M.S.M., and Strous, M.
(2007). Candidatus “Anammoxoglobus propionicus” a new propionate oxidiz-
ing species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol.,
30, 39–49.
Kartal, B., van Niftrik, L., Sliekers, O., Schmid, M.C., Schmidt, I., van de Pas-
Schoonen, K., Cirpus, I., van der Star, W., van Loosdrecht, M.C.M., and Abma, W.
(2004). Application, eco-physiology and biodiversity of anaerobic ammonium-
oxidizing bacteria. Rev. Environ. Sci. Biol./Technol., 3, 255–264.
Kester, R.A., de Boer, W., and Laanbroek, H.J. (1997). Production of NO and N2 O by
pure cultures of nitrifying and denitrifying bacteria during changes in aeration.
Appl. Environ. Microbiol., 63, 3872–3877.
Khin, T., and Annachhatre, A.P. (2004). Novel microbial nitrogen removal processes.
Biotechnol. Adv., 22, 519–532.
Killham, K. (1986). Heterotrophic nitrification. In Prosser, J.I. (Ed.), Nitrification
(pp. 117–126). Oxford, England: IRL Press.
Klotz, M.G., and Stein, L.Y. (2008). Nitrifier genomics and evolution of the nitrogen
cycle. FEMS Microbiol. Lett., 278, 146–156.
Konneke, M., Bernhard, A.E., de la Torre, J.R., Walker, C.B., Waterbury, J.B., and
Stahl, D.A. (2005). Isolation of an autotrophic ammonia-oxidizing marine ar-
chaeon. Nature, 437, 543–546.
Kuai, L.P., and Verstraete, W. (1998). Ammonium removal by the oxygenlimited
autotrophic nitrification–denitrification system. Appl. Environ. Microbiol., 64,
4500–4506.
Kuenen, J.G. (2008). ANAMMOX bacteria: From discovery to application. Nat. Rev.
Microbiol., 6, 320–326.
Kuenen, J.G., and Roberston, L.A. (1994). Combined nitrification-denitrification pro-
cesses. FEMS Microbiol. Rev., 15, 109–117.
Kumar, M., and Lin, J.-G. (2010). Co-existence of anammox and denitrification for
simultaneous nitrogen and carbon removal—strategies and issues. J. Hazard.
Mater., 178, 1–9.
Kuypers, M.M.M., Sliekers, A.O., Lavik, G., Schmid, M., Jorgensen, B.B., Kuenen,
J.G., Damste, J.S.S., Strous, M., and Jetten, M.S.M. (2003). Anaerobic ammonium
oxidation by anammox bacteria in the Black Sea. Nature, 422, 608–611.
Laanbroek, H.J., Bar-Gilissen, M.J., and Hoogveld, H.L. (2002). Nitrite as a stimulus
for ammonia-starved Nitrosomonas europaea. Appl. Environ. Microbiol., 68,
1454–1457.
Ladiges, G., Thierbach, R.D., Beier, M., and Focken. (2006). Versuche zur zweistu-
figen Deammonifikation im Hamburger Klärwerksverbund. [Attempts to two-
stage deammonification in the wastewatertreatment union of Hamburg]. 6. Aach-
ener Tagungmit Informationsforum: Stickstoffrückbelastung-Stand der Technik
2006-, Aachen (Ger), ATEMIS GmbH.p. Fachbeitrag 13, 13.
Lam, P., Lavik, G., Jensen, M.M., van de Vossenberg, J., Schmid, M., Woebken, D.,
Gutierrez, D., Amann, R., Jetten, M.S.M., and Kuypers, M.M.M. (2009). Revising
the nitrogen cycle in the Peruvian oxygen minimum zone. Proc. Natl Acad. Sci.
USA, 106, 4752–4757.
Lamsam, A., Laohaprapanon, S., and Annachhatre, A.P. (2008). Combined activated
sludge with partial nitrification (AS/PN) and ANAMMOX processes for treatment
of seafood processing wastewater. J. Environ. Sci. Health. Part-A Tox. Hazard.
Subst. Environ. Eng., 43, 1198–1208.
Leininger, S., Urich, T., Schloter, M., Schwark, L., Nicol, J., Qi, G.W., Prosser, J.I.,
Schuster, S.C., and Schleper, C. (2006). Archaea predominate among ammonia-
oxidizing prokaryotes in soils. Nature, 442, 806–809.
Lieu, P.K., Hatozaki, R., Homan, H., and Furukawa, K. (2005). Single stage nitro-
gen removal using ANAMMOX and partial nitritation (SNAP) for treatment of
synthetic landfill leachate. Jpn. J. Water Treat. Biol., 41(2), 103.
Lieu, P.K., Homan, H., Kurogl, A., Kawagoshi, Y., Fujii, T., and Furukawa, K. (2006).
Characterization of sludge from single-stage nitrogen removal using ANAMMOX
and partial nitritation (SNAP). Jpn. J. Water Treat. Biol., 42, 53–64.
Lipschultz, F., Zafiriou, O.C., Wofsy, S.C., McElroy, M.B., Valois, F.W., and Watson,
S.W. (1981). Production of NO and N2 0 by soil nitrifying bacteria. Nature, 294,
641–643.
Lopez, H., Puig, S., Ganigue, R., Ruscalleda, M., Balaguer, M.D., and Colprim, J.
(2008). Start-up and enrichment of a granular ANAMMOX SBR to treat high
nitrogen load wastewaters. J. Chem. Technol. Biotechnol., 83, 233–241.
Luther, G.W., Brendel, P.J., Lewis, B.L., Sundby, B., Lefrancois, L., Silverberg, N.,
and Nuzzio, D.B. (1998). Simultaneous measurement of O2 , Mn, Fe, I−, and S2−
in marine pore waters with a solid state voltametric microoelectrode. Limnol.
Oceanogr., 43, 325–333.
Luther, G.W., and Popp, J.I. (2002). Kinetics of the abiotic reduction of poly-
meric manganese dioxide by nitrite: An anaerobic nitrification reaction. Aquatic
Geochem., 8, 15–36.
Luther, G.W., Sundby, B., Lewis, B.L., Brendel, P.J., and Silverberg, N. (1997). Inter-
actions of manganese with the nitrogen cycle: Alternative pathway to dinitrogen.
Geochimi. Cosmochim. Acta, 61, 4043–4052.
Massana, R., DeLong, E.F., and Pedrós-Alió, C. (2000). A few cosmopolitan phylo-
types dominate planktonnic archaeal assemblages in widely different oceanic
provinces. Appl. Environ. Microbiol., 66, 1777–1787.
Meiberg, J.B.M., Bruinenberg, P.M., and Harder, W. (1980). Effect of dissolved oxy-
gen tension on the metabolism of methylated amines in hyphomicrobium X
in the absence and presence of nitrate: Evidence for ‘aerobic’ denitrification. J.
Gen. Microbiol., 120, 453–463.
Meyer, R.L., Risgaard-Petersen, N., and Aller, D.E. (2005). Correlation between anam-
mox activity and the microscale distribution of nitrite in a subtropical mangrove
sediment. Appl. Environ. Microbiol., 71, 6142–6149.
Mortimer, R.J.G., Krom, M.D., Harris, S.J., Hayes, P.J., Davies, I.M., Davison, W., and
Zhang, H. (2002). Evidence for complex recycling processes within sedimentary
biogeochemical zones. Marine Ecol. Prog. Series, 236, 31–35.
Mosquera-Corral, A., González, F., Campos, J.L., and Méndez, R. (2005). Partial
nitrification in a SHARON reactor in the presence of salts and organic carbon
compounds. Process Biochem., 40, 3109–3118.
Mulder, A. (1992). U.S. Patent No. 5,078,884. Washington, DC: U.S. Patent and
Trademark Office.
Mulder, J.W., van Loosdrecht, M.C.M., Hellinga, C., and van Kempen, R. (2001). Full
scale application of the SHARON process for treatment of rejection water of
digested sludge dewatering. Water Sci. Technol., 43(11), 127–134.
Münch, E.V., Lant, P., and Keller, J. (1996). Simultaneous nitrification and denitrifi-
cation in bench-scale sequencing batch reactors. Water Res., 30, 277–284.
Nicol, G.W., and Schleper, C. (2006). Ammonia-oxidising renarchaeota: Important
players in the nitrogen cycle? Trends Microbiol., 14, 207–212.
Olson, R.J. (1981). Differential photoinhibition of marine nitrifying bacteria: A pos-
sible mechanism for the formation of the primary nitrite maximum. J. Mar. Res.,
39, 227–238.
Paredes, D., Kuschk, P., Mbwette, T.S.A., Stange, F., Muller, R.A., and Koser, H.
(2007). New aspects of microbial nitrogen transforms in the context of wastew-
ater treatment: A review. Eng. Life Sci., 7(1), 13–25.
Park, H.D., and Noguera, D.R. (2007). Characterization of two ammonia-oxidizing
bacteria isolated from reactors operated with low dissolved oxygen concentra-
tions. J. Appl. Microbiol., 102, 1401–1417.
Pathak, B.K., Kazama, F., Saiki, Y., and Sumino, T. (2007). Presence and activity
of anammox and denitrification process in low ammonium-fed bioreactors.
Bioresour. Technol., 98, 2201–2206.
Peng, Y., Chen, Y., Wang, S., Peng, C., Liu, M., Song, X., and Cui, Y. (2004). Ni-
trite accumulation by aeration controlled in sequencing batch reactors treating
domestic wastewater. Water Sci. Technol., 50(10), 35–43.
Peng, Y., and Zhu, G. (2006). Biological nitrogen removal with nitrification and
denitrification via nitrite pathway. Appl. Environ. Microbiol., 73, 15–26.
Penton, C.R., Devol, A.H., and Tiedje, J.M. (2006). Molecular evidence for the broad
distribution of anaerobic ammonium-oxidizing bacteria in freshwater and ma-
rine sediments. Appl. Environ. Microbiol., 72, 6829–6832.
Pochana, K., and Keller, J. (1999). Study of factors affecting simultaneous nitrification
and denitrification (SND). Water. Sci. Technol., 39(6), 61–68.
Pollice, A., Tandoi, V., and Lestingi, C. (2002a). Influence of aeration and sludge
retention time on ammonium oxidation to nitrite and nitrate. Water Res., 36,
2541–2546.
Pollice, A., Valter, T., and Carmela, L. (2002b). Influence of aeration and sludge
retention time on ammonium oxidation to nitrite and nitrate. Water Res., 36,
2541–2546.
Poth, M. (1986). Dinitrogen production from nitrite by a Nitrosomonas isolate. Appl.
Poth, M., and Focht, D.D. (1985). N-15 kinetic-analysis of N2 O production by Ni-
trosomonas europaea: An examination of nitrifier denitrification. Appl. Environ.
Microbiol., 49, 1134–1141.
Pynaert, K., Smets, B.F., Wyffels, S., Beheydt, D., Siciliano, S.D., and Verstraete,
W. (2003). Characterization of an autotrophic nitrogen-removing biofilm from a
highly loaded lab-scale rotating biological contactor. Appl. Environ. Microbiol.,
69, 3626–3635.
Reginatto, V., Teixeira, R.M., Pereira, F., Schmidell, W., Furigo, A. Jr., Menes, R.,
Etchebehere, C., and Soares, H.M. (2005). Anaerobic ammonium oxidation

in a bioreactor treating slaughterhouse wastewater. Braz. J. Chem. Eng., 22,
593–600.
Robertson, L.A. (1988). Aerobic denitrification and heterotrophic nitrification in This-
phaera pantatropha and other bacteria. Doctoral dissertation, Delft University
of Technology, Delft, the Netherlands.
Robertson, L.A., Cornelisse, R., Vos, D.E., Hadioetomo, R., and Kuenen, J.G. (1989a).
Aerobic denitrification in various heterotrophic nitrifiers. Antonie van Leeuwen-
hoek, 56, 289–299.
Robertson, L.A., Cornelisse, R., Zeng, R., and Kuenen, J.G. (1989b). The effect of
thisulphate and other inhibitors of autrophic nitrification on heterotrophic nitri-
fiers. Antonie van Leeuwenhoek, 56, 301–309.
Robertson, L.A., and Kuenen, J.G. (1983). Thisphaera pantotropha gen. nov. sp.
nov., a facultatively anaerobic, facultatively autotrophic sulphur bacterium. J.
Gen Microbiol., 129, 2847–2855.
Robertson, L.A., and Kuenen, J.G. (1988). Hetrotrophic nitrification in Thiosphaera
pantotropha: Oxygen uptake and enzyme studies. J. Gen. Microbiol., 134,
857–863.
Robertson, L.A., and Kuenen, J.G. (1990). Combined heterotrophic nitrification and
aerobic denitrification in Thiosphaera pantotropha and other bacteria. Antonie
van Leeuwenboek, 57, 139–152.
Robertson, L.A., and Kuenen, J.G. (1992). Aerobic sulphur oxidizing bacteria. In
Balows, A., Truper, H.G., Dworkin, M., Harder, W., and Schleifer, K.H. (Eds.),
The prokaryotes (pp. 385–413). Berlin: Springer-Verlag.
Sabumon, P.C. (2007). Anaerobic ammonia removal in presence of organic matter:
A novel route. J. Hazard. Mater., 149, 49–59.
Salem, S., Berends, D., van Loosdrecht, M.C.M., and Heijnen, J.J. (2003). Bioaug-
mentation by nitrification with return sludge. Water Res., 37, 1794–1804.
Salem, S., Berends, D.H.J.G., van der Roest, H.F., van der Kuij, R.J., and van Loos-
drecht, M.C.M. (2004). Full scale application of BABER technology. Water Sci.
Technol., 50(7), 87–96.
Schmid, M.C., Risgaard-Petersen, N., van de Vossenberg, J., Kuypers, M.M., Lavik,
G., Petersen, J., Hulth, S., Thamdrup, B., Canfield, D., Dalsgaard, T., Rysgaard,
S., Sejr, M.K., Strous, M., den Camp, H.J., and Jetten, M.S. (2007). Anaerobic
ammonium-oxidizing bacteria in marine environments: widespread occurrence

but low diversity. Environ. Microbiol., 9, 1476–1484.
Schmid, M., Twachtmann, U., Klein, M., Strous, M., Juretschko, S., Jetten, M., Metzger,
J., Schleifer, K.H., and Wagner, M. (2000). Molecular evidence for genus level
diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst.
Appl. Microbiol., 23, 93–106.
Schmidt, I. (2009). Chemoorganoheterotrophic growth of Nitrosomonas europaea
and Nitrosomonas eutropha. Curr. Microbiol., 59, 130–138.
Schmidt, I., and Bock, E. (1997). Anaerobic ammonia oxidation with nitrogen dioxide
by Nitrosomonas eutropha. Arch. Microbiol., 167, 106–111.
Schmidt, I., and Bock, E. (1998). Anaerobic ammonia oxidation by cell-free extracts
of Nitrosomonas eutropha. Antonie van Leeuwenhoek, 73, 271–278.
Schmidt, I., Bock, E., and Jetten, M.S.M. (2001a). Ammonia oxidation by Nitro-
somonas eutropha with NO2 as oxidant is not inhibited by acetylene. Microbiol.,

147, 2247–2253.
Schmidt, I., Look, C., Bock, E., and Jetten, M.S.M. (2004a). Ammonium and hy-
droxylamine uptake and accumulation in Nitrosomonas. Microbiology, 150,
1405–1412.
Schmidt, I., Sliekers, O., Schmid, M., Bock, E., Fuerst, J., Kuenen, J.G., Jetten, M.S.M.,
and Strous, M. (2003). New concepts of microbial treatment processes for the
nitrogen removal in wastewater. FEMS Microbiol. Rev., 27, 481–492.
Schmidt, I., Sliekers, O., Schmid, M., Cirpus, I., Strous, M., Bock, E., Kuenen,
J.G., and Jetten, M.S.M. (2002). Aerobic and anaerobic ammonia oxidizing
bacteria—competitors or natural partners? FEMS Microbiol. Ecol., 39, 175–181.
Schmidt, I., van Spanning, R.J.M., and Jetten, M.S.M. (2004b). Denitrification and
ammonia oxidation by Nitrosomonas europaea wild-type, and NirK- and NorB-
deficient mutants. Microbiology, 150, 4107–4114.
Schmidt, I., Zart, D., and Bock, E. (2001b). Effects of gaseous NO2 on cells of
Nitrosomonas eutropha previously incapable of using ammonia as an energy
source. Antonie Van Leeuwenhoek, 79, 39–47.
Schubert, C.J., Durisch-Kaiser, E., Wehrli, B., Thamdrup, B., Lam, P., and Kuypers,
M.M.M. (2006). Anaerobic ammonium oxidation in a tropical freshwater system
(Lake Tanganyika). Environ Microbiol., 8, 1857–1863.
Seitzinger, S.P. (1988). Denitrification in freshwater and marine eco-system: Ecolog-
ical and geochemical significance. Limnol. Oceanogr., 33, 702–724.
Seyfried, C.F., Hippen, A., Helmer, C., Kunst, S., and Rosenwinkel, K.-H. (2001).
One-stage deammonification: nitrogen elimination at low costs. Water Sci. Tech;
Water Supply, 1(1), 71–80.
Shaw, L.J., Nicol, G.W., Smith, Z., Fear, J., Prosser, J.I., and Baggs, E.M. (2006).
Nitrosospira spp. can produce nitrous oxide via a nitrifier denitrification pathway.
Shrestha, N.K., Hadano, S., Kamachi, T., and Okura, I. (2001). Conversion of am-
monia to dinitrogen in wastewater by Nitrosomonas europaea. Appl. Biochem.
Biotechnol., 90, 221–232.
Siegrist, H., Reithaar, S., and Lais, P. (1998). Nitrogen loss in a nitrifying rotating
contactor treating ammonium rich leachate without organic carbon. Water Sci.
Technol., 37(4–5), 589–591.
Sin, G., Villez, K., and Vanrolleghem, P.A. (2006). Application of amodel-based
optimization methodology for nutrient removing SBRs leads to falsification of
the model. Water Sci. Technol., 53(4–5), 95–103.
Sinha, B., and Annachhatre, A.P. (2007). Partial nitrification-operational parameters
and microorganisms involved. Rev. Environ. Sci. Biotechnol., 6, 285–313.
Sinninghe Damsté, J.S., Rijpstra, W.I.C., Hopmans, E.C., Prahl, F.G., Wakeham, S.G.,
and Schouten, S. (2002a). Distribution of membrane lipids of planktonic Cre-
narchaeota in the Arabian Sea. Appl. Environ. Microbiol., 68, 2997–3002.
Sinninghe Damsté, J.S., Schouten, S., Hopmans, E.C., van Duin, A.C.T., and
Geenevasen, J.A.J. (2002b). Crenarchaeol: The characteristic core glycerol dibi-
phytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic crenar-
chaeota. J. Lipid Res., 43, 1641–1651.
Sliekers, A.O., Derwort, N., Gomez, J.L., Strous, M., Kuenen, J.G., and Jetten, M.S.M.
(2002). Completely autotrophic nitrogen removal over nitrite in one single re-
actor. Water Res., 36, 2475–2482.
Sliekers, A.O., Haaijer, S., Schmid, M., Harhangi, H., Verwegen, K., Kuenen, J.G.,
and Jetten, M.S.M. (2004). Nitrification and ANAMMOX with urea as the energy
source. Syst. Appl. Microbiol., 27, 271–278.
Sliekers, A.O., Haaijer, S.C.M., Stafsnes, M.H., Kuenen, J.G., and Jetten, M.S.M.
(2005). Competition and coexistence of aerobic ammonium and nitrite-oxidizing
bacteria at low oxygen concentrations. Appl. Microbiol. Biotechnol., 68,
808–817.
Sliekers, A.O., Third, K.A., Abma, W., Kuenen, J.G., and Jetten, M.S.M. (2003).
CANON and anammox in a gas-lift reactor. FEMS Microbiol. Lett., 218, 339–344.
Sørensen, J., Sørensen, K.S., Colley, S., Hydes, D.J., Thomson, J., and Wilson, T.R.S.
(1987). Depth localization of denitrification in deep-sea sediment from the
Maderia Abyssal Plain. Limnol. Oceanogr., 32, 758–762.
Stein, L.Y., Arp, D.J., Berube, P.M., Chain, P.S., Hauser, L., Jetten, M.S., Klotz, M.G.,
Larimer, F.W., Norton, J.M., Op den Camp, H.J., Shin, M., and Wei, X. (2007).
Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas
eutropha C91: Implications for niche adaptation. Environ. Microbiol., 9,
2993–3007.
Strous, M. (2000). Microbiology of anaerobic ammonium oxidation. Doctoral disser-
tation, TU Delft, the Netherlands.
Strous, M., Fuerst, J.A., Kramer, E.H.M., Logemann, S., Muyzer, G., and van de Pas
Schoonen, K.T. (1999a). Missing lithotroph identified as new PlanctomyIcete.
Nature, 400, 446–449.
Strous, M., van Gueven, E., Kuenen, J.G., and Jetten, M.S.M. (1997). Ammonia re-
moval from concentrated waste streams with the anaerobic ammonium oxida-
tion (ANAMMOX) process in different reactor configurations. Water Res., 31,
1955–1962.
Strous, M., Heijnen, J.J., Kuenen, J.G., and Jetten, M.S.M. (1998). The sequenc-
ing batch reactor as a powerful tool for the study of slowly growing anaer-
obic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol., 50,
589–596.
Strous, M., and Jetten, M.S.M. (2004). Anaerobic oxidation of methane and ammo-
nium. Annu. Rev. Microbiol., 58, 99–117.
Strous, M., Kuenen, J.G., Fuerst, J.A., Wagner, M., and Jetten, M.S.M. (2002).
The ANAMMOX case-a new experimental manifesto for microbiological eco-
physiology. Antonie van Leeuwenhoek, 81, 693–702.
Strous, M., Kuenen, J.G., and Jetten, M.S.M. (1999b). Key physiology of anaerobic
ammonium oxidation. Appl. Environ. Microbiol., 65, 3248–3250.
Strous, M., Pelletier, E., Mangenot, S., Rattei, T., Lehner, A., Horn, M., Daims, H., et al.
(2006). Deciphering the evolution and metabolism of an anammox bacterium
from a community genome. Nature, 440, 790–794.
Stüven, R., Vollmer, M., and Bock, E. (1992). The impact of organic matter
on nitric oxide formation by Nitrosomonas europaea. Arch. Microbiol., 158,
439–443.
Sun, S.-P., Nàcher, C.P., Merkey, B., Zhou, Q., Xia, S.-Q., Yang, D.-H., Sun, J.-H., and
Smets, B.F. (2010). Effective biological nitrogen removal treatment processes for
domestic wastewaters with low C N ratios: A review. Environ. Eng. Sci., 27,
111–126.
Sutka, R.L., Ostrom, N.E., Ostrom, P.H., Breznak, J.A., Gandhi, H., Pitt, A.J., and
Li, F. (2006). Distinguishing nitrous oxide production from nitrification and
denitrification on the basis of isotopomer abundances. Appl. Environ. Microbiol.,
72, 638–644.
Tal, Y., Watts, J.E., and Schreier, H.J. (2005). Anaerobic ammoniaoxidizing bacteria
and related activity in Baltimore inner harbor sediment. Appl. Environ. Micro-
biol., 71, 1816–1821.
Tang, C.-J., Zheng, P., Chen, P.P., Zhang, J.-Q., Mahmood, Q., Chen, X.-G., Chen, J.-
W., Wu, D.-T. (2010). Enhanced nitrogen removal from pharmaceutical wastew-
ater using SBA-ANAMMOX process. Water Res., 45, 201–210.
Third, K.A., Paxman, J., Schmid, M., Strous, M., Jetten, M.S.M., and Cord-Ruwisch,
R. (2005). Enrichment of anammox from activated sludge and its application in
the CANON process. Microbial. Ecol., 49, 236–244.
Third, K.A., Sliekers, A.O., Kuenen, J.G., and Jetten, M.S.M. (2001). The CANON
system (completely autotrophic nitrogen-removal over nitrite) under ammonium
limitation: Interaction and competition between three groups of bacteria. Syst.
Appl. Microbiol., 24, 588–596.
Toh, S.K., and Ashbolt, N.J. (2002). Adaptation of anaerobic ammonium-oxidizing
consortium to synthetic coke-ovens wastewater. Appl. Microbiol. Biotechnol.,
59, 344–352.
Toh, S.K., Webb, R.I., and Ashbolt, N.J. (2002). Enrichment of autotrophic anaerobic
ammonium-oxidizing consortia from various wastewaters. Microbiol. Ecol., 43,
154–167.
Treusch, A.H., Leininger, S., Kletzin, A., Schuster, S.C., Klenk, H.P., and Schleper,
C. (2005). Novel genes for nitrite reductase and Amo-related proteins indicate
a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environ.
Microbiol., 7, 1985–1995.
Trigo, C., Campos, J.M., Garrido, J.M., and Mendez, R. (2006). Start-up of the anam-
mox process in a membrane bioreactor. J. Biotechnol., 126, 475–487.
Tsushima, I., Kindaichi, T., and Okabe, S. (2007). Quantification of anaerobic
ammonium-oxidizing bacteria in enrichment cultures by real-time PCR. Water
Res., 41, 785–794.
Turk, O., and Mavinic, D.S. (1989). Maintaining nitrite buildup in a system acclimated
to free ammonia. Water Res., 23, 1383–1388.
van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S.M., and Kuenen, J.G.
(1996). Autotrophic growth of anaerobic ammonium-oxidizing microorganisms
in a fluidized bed reactor. Microbiology., 142, 2187–2196.
van der Star, W.R.L., Miclea, A.I., van Dongen, U.G.J.M., Muyzer, G., Picioreanu, C.,
and van Loosdrecht, M.C.M. (2008). The membrane bioreactor: A novel tool to
grow anammox. Biotechnol. Bioeng., 101, 286–294.
van der Star, W.R.L., Wiebe, R., Abma, W.R., Blommers, D., Mulder, J.W., Tokutomi,
T., Strous, M., Picioreanu, C., and van Loosdrech, M.C.M. (2007). Startup of
reactors for anoxic ammonium oxidation: Experiences from the first full-scale
ANAMMOX reactor in Rotterdam. Water Res., 41, 4149–4163.
van Dongen, U., Jetten, M.S.M., and van Loosdrecht, M.C.M. (2001). The SHARON-
ANAMMOX process for treatment of ammonium rich wastewater. Water Sci.

Technol., 44, 153–160.
van Hulle, S.W.H., Vandeweyer, H.J.P., Meesschaert, B.D., Vanrolleghem, P.A., De-
jans, P., and Dumoulin, A. (2010). Engineering aspects and practical application
of autotrophic nitrogen removal from nitrogen rich streams. Chem. Eng. J., 162,
1–20.
van Hulle, S.W.H., Volcke, E.I.P., López Teruel, J., Donckels, B., van Loosdrecht,
M.C.M., and Vanrolleghem, P.A. (2007). Influence of temperature and pH on
the kinetics of the SHARON nitritation process. J. Chem. Technol. Biotechnol.,
82, 471–480.
van Kempen, R., Mulder, J.W., Uijterlinde, C.A., and van Loosdrecht, M.C.M. (2001).
Overview: Full scale experience of the SHARONR process for treatment of
rejection water of digested sludge dewatering. Water Sci. Technol., 44(1),
145–152.
van Kempen, R., ten Have, C.C.R., Meijer, S.C.F., Mulder, J.W., Duin, J.O.J., Uijter-
linde, C.A., and van Loosdrecht, M.C.M. (2005). SHARON process evaluated
for improved wastewater treatment plant effluent quality. Water Sci. Technol.,
52(4), 55–62.
van Loosdrecht, M.C.M., Ha, O.X., Jetten, M.S.M., and Abma, W. (2004). Use of
ANAMMOX in urban wastewater treatment. Water Sci. Technol.; Water Supply.,
4(1), 87–94.
van Niel, E.W.J., Robertson, L.A., and Kuenen, J.G. (1987). Heterotrophic nitrification
in denitrifying bacteria. Proc. 4th Eur. Cong. Biotechnol., 3, 363.
van Niftrik, L.A., Fuerst, J.A., Sinninghe Damst, J.S., Kuenen, J.G., Jetten, M.S.M.,
and Strous, M. (2004). The anammoxosome: an intracytoplasmic compartment
in anammox bacteria. FEMS Microbiol. Lett., 233, 7–13.
Vazquez-Padin, J.R., Fernádez, I., Figueroa, M., Mosquera-Corral, A., Campos, J.L.,
and Mendez, R. (2009a). Applications of anammox based processes to treat
anaerobic digester supernatant at room temperature. Bioresour. Technol., 100,
2988–2994.
Vazquez-Padin, J.R., Pozo, M.J., Jarpa, M.. Figueroa, M., Franco, A., Mosquera-Corral,
A., Campos, J.L., and Mendez, R. (2009b). Treatment of anaerobic sludge di-
gester effluents by the CANON process in an air pulsing SBR. J. Hazard. Mater.,
166, 336–341.
Venter, J.C., Remington, K., Heidelberg, J.F., Halpern, A.L., Rusch, D., Eisen, J.A.,
et al. (2004). Environmental genome shotgun sequencing of the Sargasso Sea.
Science, 304, 66–74.
Verstraete, W. (1975). Heterotrophic nitrification in soils and aqueous media. Izvestija
Akademi NAuk SSSR Ser. Bio., 4, 541–558.
Vlaeminck, S.E., Cloetens, L.F.F., de Clippeleir, H., Carballa, M., and Verstraete, W.
(2009a). Grannular biomass capable of partial nitritation and ANAMMOX. Water
Sci. Technol., 59, 610–617.
Vlaeminck, S.E., Terada, A., Smets, B.F., van der Linden, D., Boon, N., Verstraete, W.,
and Carballa, M. (2009b). Nitrogen removal from digested black water by one-
stage partial nitritation and ANAMMOX. Environ. Sci. Technol., 43, 5035–5041.
Voysey, P.A., and Wood, P.M. (1987). Methanol and formaldehyde oxidation by an
autotrophic nitrifying bacterium. J. Gen. Microbiol., 133, 283–290.
Waki, M., Tokutomi, T., Yokoyama, H., and Tanaka, Y. (2007). Nitrogen removal
from animal waste treatment water by ANAMMOX enrichment. Bioresour. Tech-
nol., 98, 2775–2780.
Wang, C.C., Lee, P.H., Kumar, M., Huang, Y.T., Sung, S., and Lin, J.G. (2010). Simul-
taneous partial nitrification, anaerobic ammonium oxidation and denitrification
(SNAD) in a full-scale landfill-leachate treatment plant. J. Hazard. Mater., 175,
622–628.
Wang, J., and Kang, J. (2005). The characteristics of anaerobic ammonia oxidation
(ANAEROBIC AMMONIA REMOVAL) by granular sludge from an ESGB reactor.
Process Biochem., 40, 1973–1978.
Wang, T., Zhang, H., Yang, F., Liu, S., Fu, Z., and Chen, H. (2009). Start-up of
the anammox process from the conventional activated sludge in a membrane
bioreactor. Bioresour. Technol., 100, 2501–2506.
Ward, B.B., Devol, A.H., Rich, J.J., Chang, B.X., Bulow, S.E., Naik, H., Pratihary, A.,
and Jayakumar, A. (2009). Denitrification as the dominant nitrogen loss process
in the Arabian Sea. Nature, 461, 78–81.
Wett, B. (2006). Solved up-scaling problems for implementing deammonification of
rejection water. Water Sci. Technol., 53(12), 121–128.
Wett, B. (2007). Development and implementation of a robust deammonification
process. Water Sci. Technol., 56(7), 81–88.
Woebken, D., Lam, P., Kuypers, M.M.M., Naqvi, S.W.A., Kartal, B., Strous, M., Jetten,
M.S., Fuchs, B.M., and Amann, R. (2008). A microdiversity study of marine
anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine
oxygen minimum zones. Environ. Microbiol., 10, 3106–3119.
Wrage, N., Velthof, G.L., Oenema, O., and Laanbroek, H.J. (2004). Acetylene and
oxygen as inhibitors of nitrous oxide production in Nitrosomonas europaea and
Nitrosospira briensis: A cautionary tale. FEMS Microbiol. Ecol., 47, 13–18.
Wrage, N., Velthof, G.L., van Beusichem, M.L., and Oenema, O. (2001). Role of
nitrifier denitrification in the production of nitrous oxide. Soil. Biol. Biochem.,
33, 1723–1732.
Wyffels, S., Boeckx, P., Pynaert, K., Zhang, D., Van Cleemput, O., Chen, G., and
Verstraete, W. (2004). Nitrogen removal from sludge reject water by a two-
stage oxygen limited autotrophic nitrification denitrification process. Water Sci.
Technol., 49(5–6), 57–64.
Xu, Z.Y., Zeng, G.M., Yang, Z.H., Xiao, Y., Cao, M., Sun, H.S., Ji, L.L., and Chen, Y.
(2010). Biological treatment of landfill leachate with the integration of partial
nitrification, anaerobic ammonium oxidation and heterotrophic denitrification.
Bioresour. Technol., 101, 79–86.
Yang, L., and Alleman, J.E. (1992). Investigation of batchwise nitrite build-up by an
enriched nitrification culture. Water Sci. Technol., 26, 997–1005.
Zart, D., and Bock, E. (1998). High rate of aerobic nitrification and denitrification by
Nitrosomonas eutropha grown in a fermenter with complete biomass retention
in the presence of gaseous NO2 or NO. Arch. Microbiol., 169, 282–286.
Zart, D., Schmidt, I., and Bock, E. (2000). Significance of gaseous NO for ammonia
oxidation by Nitrosomonas eutropha. Antonie van Leeuwenhoek, 77, 49–55.
Zehr, J.P., and Ward, B.B. (2002). Nitrogen cycling in the ocean: New perspectives
on processes and paradigms. Appl. Environ. Microbiol., 68, 1015–1024.
Zhang, L., Zheng, P., Tang, C-J., and Jin, R-C. (2008). Anaerobic ammonium oxida-
tion for treatment of ammonium-rich wastewaters. J. Zhejiang Univ. Sci B., 9,
416–426.
Zhang, M., Tay, J.H., Qian, Y., and Gu, X.S. (1998). Coke plant wastewater treatment
by fixed biofilm system for COD and NH3 removal. Water Res., 32, 519–527.
Zumft, W.G. (1997). Cell biology and molecular basis of denitrification. Microbiol.
Mol. Biol. Rev., 61, 533–616.
APPENDIX A
Discovery of ANAMMOX
Generally unusual observations made during biological processes lead

to the investigation for understanding the underlying microbial phe-
nomenon. Conversely, sometimes ideas on certain biological processes
come first followed by actual hunt on their existence in nature. This hap-
pened when an Austrian chemist, Boarda (1977) wondered about the exis-
tence of certain nitrifying microorganisms which could catalyze a hitherto
unknown reaction based on thermodynamic calculations.
NH+ −
4 + NO2 → N2 + 2H2 O
Unaware of such predictions, Mulder detected unusual disappearance of

nitrogen in his denitrifying pilot plant in 1999. The reactor received two
effluent streams, one from the methanogenic pilot plant containing am-
monium, sulfide and organic compounds and another from a nitrifying
plant. Unexpectedly after 100 days of operations, ammonia was decreas-
ing with no built up of NO− −
2 or NO3 . He coined the term ANAMMOX for
anaerobic ammonium oxidation in presence of NO− 3 . However, Mulder
was not sure of the biological nature of the reaction. Research carried
out in collaboration with J. G. Kuenen and his group, comprising Astrid
van de Graaf, Mike Jetten and Marc Strous, not only established the bi-
ological nature of the reaction and its mechanism, but also established
the identity of the responsible microorganism that belonged to the group
Planctomycetes. The first ANAMMOX bacteria identified was Candita-
tus Brocardia, ANAMMOXidans (Strous et al., 1999a). Further research
through physiological and genomic analysis revealed many unexpected
facts. CO2 can be used as carbon source to produce biomass and NO− 2
functions not only as electron acceptor for ammonium oxidation, but also
an electron donor for reduction of CO2 .
−
NH+ +
4 + 1.32NO2 + 0.066HCO3 + 0.13H →
1.02N2 + 0.26NO−
3 + 0.066CH2 O0.5 N0.15 + 2.03H2 O
Besides NO− −
2 , NO3 can also act as electron acceptor. The autotrophic
ANAMMOX bacteria can oxidize formate, acetate and propionate to CO2
through dissimilatory pathway. Assimilatory pathway for utilization of or-
ganic carbon source does not exist. The organism can generate NO− 2 and
NH3 form NO− 3 using formate, acetate and propionate as electron donor;
and thereby capable of producing its electron donors and acceptors.
APPENDIX B
The Ambiguous Process Nomenclatures
Chronologically, SHARON was the first process to be developed in the

year 1997 based on the concept of partial nitrification and denitrifica-
tion in a single unit. As this process was developed before the Planc-
tomycetes group of bacteria were discovered, the role of ANAMMOX
bacteria was eventually not considered. This was followed by OLAND
process, in 1998. Although the ANAMMOX reaction was first detected by
Mulder in the year 1989, it was not before 1999, the biological nature
of the reaction was established (Strous et al., 1999a) and a technology
was developed on partial nitrification and ANAMMOX. For partial nitri-

fication, the concept of SHARON was adopted and named the process
as SHARON-ANAMMOXR . The similar process in single stage systems
was termed differently by different workers, such as OLAND (Kuai and
Verstraete, 1998), CANON (Third et al., 2001), aerobic/anoxic deammonifi-
cation (Hippen et al., 2001), DEMON for deammonification (Seyfried et al.,
2001), SND for simultaneous nitrification and denitrification (Pochana and
Keller, 1999), SNAP for Single stage Nitrogen removal using ANAMMOX
and Partial nitratation (Lieu et al., 2005; 2006), SNAD for Simultaneous par-
tial Nitrification, ANAMMOX, and Denitrification (Chen et al, 2009), DE-
MON (Wett, 2006; 2007) and DIB for Deammonification in interval aerated
Biofilm systems (Ladiges et al., 2006). After the discovery of ANAMMOX
process in 1999, many technologies developed having some component
of ANAMMOX reaction in it. Different research groups adopted differ-
ent terms often based on the reactor configurations or on the assumed
underlying microbial principles. The three processes, namely, SHARON
(Hellinga et al., 1997; 1998; Jetten et al., 1997; Mulder et al., 2001; van
Kempen et al., 2001), SNAD (Chen et al., 2009) and AS/PN-ANAMMOX
(Lamsam et al., 2008) were applicable for simultaneous removal of or-
ganics and ammonia from industrial effluents. The SHARON process and
SNAD process differ by the inclusion of ANAMMOX process in the later
(Chen et al., 2009). In contrast to SNAD process, the AS/PN-ANAMMOX
process excludes the role of denitrifying bacteria. The AS/PN-ANAMMOX
process also differs from the SHARON process merely by the requirement
of aeration as in conventional activated sludge process. Thus, the under-
lying microbial principles of these processes are more or less similar, and
they differ mostly in the chronological order of their development. van
der Star et al. (2007) has resolved the confusion created in the processes
nomenclature by suggesting some descriptive terminologies which are
apparently less ambiguous for the scientific community.
View publication stats

10CriticalReviews Anammox 2012

Uploaded by

Copyright:

Available Formats

You might also like

10CriticalReviews Anammox 2012

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

10CriticalReviews Anammox 2012

Uploaded by

Copyright:

Available Formats

See discussions, stats, and author profiles for this publication at: https://www.researchgate.

Critical Reviews in Environmental Science and Technology

Article in Critical Reviews in Environmental Science and Technology · August 2012

Samik Bagchi Rima Biswas

SEE PROFILE SEE PROFILE

Chemo-biochemical desulfurization of industrial emissions containing oxides of sulfur View project

REWATER View project

The user has requested enhancement of the downloaded file.

Critical Reviews in Environmental

Autotrophic Ammonia Removal

Available online: 30 Aug 2011

To link to this article: http://dx.doi.org/10.1080/10643389.2011.556885

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

Autotrophic Ammonia Removal Processes:

SAMIK BAGCHI, RIMA BISWAS, and TAPAS NANDY

The last decade has witnessed rapid spur in technoeconomic au-

KEY WORDS: ammonia, ammonia oxidation, anaerobic ecology,

Ammonia removal through biological oxidation has always been a matter

Address correspondence to Rima Biswas, National Environmental Engineering Research

denitrification processes are mutually opposite. On one hand, nitrification is

somonas species and the discovery of novel pathway of anaerobic ammonia

Ammonia Oxidation by Nitrosomonas Species

Autotrophism was considered to be the only mode of growth for such

FIGURE 1. The three energy-evolving pathways in Nitrosomonas species. Figure modified,

In nitrification, N2 O is produced as a by-product during ammonia oxida-

centration (Cantera and Stein, 2007).

ANAEROBIC AMMONIA OXIDATION

oxidation in cell-free extracts. Nitrite was found to be essential for stimu-

Oxidation of ammonia to hydroxylamine is catalyzed by ammonia

reaction was not inhibited in presence of acetylene (Schmidt et al., 2001a).

ANAEROBIC AMMONIA OXIDATION BY ANAMMOX BACTERIA

bacteria as given in equation 5 (Jetten et al., 2001; Strous et al., 1998).

After the discovery of ANAMMOX bacteria by Strous et al. (1998; Ap-

reviews are reported describing the physiological, metabolic, and genomic

TABLE 1. Microbial diversity of ANAMMOX bacteria discovered so far

Species Source Reference

Candidatus “Brocadia anammoxidans” Denitrifying reactor Strous et al., 1999a

by the discovery of aerobic ammonium oxidation within the domain of

have identified an amo homologue of A, B, and C genes on an archaeal asso-

Heterotrophic Nitrification and Aerobic Denitrification

Similar to other heterotrophic nitrifiers, Paracoccus denitrificans does not

(Robertson and Kuenen, 1990). Through several electron acceptor utiliza-

ner, 2006). Nevertheless, the unique ability of heterotrophic nitrification and

TECHNOLOGICAL ADVANCEMENTS IN AUTOTROPHIC AMMONIA

Based on the knowledge available on nitrogen-removing microorganisms, a

temperature, pH, hydraulic retention time, substrate concentration, and dis-

bon source has to be additionally supplied. Besides, temperature (between

Since in System Wastewater Nitrogen loading Reactor Efficiency

of ANAMMOX bacteria in CANON biomass with absence of aerobic NOB

OLAND and DEMON

by autotrophic organisms (Kuai and Verstraete, 1998). Initially, the process

Wastewater Reactor NLRmax NRRmax

CANON Synthetic SBR 0.131 0.075 Sliekers et al., 2002

of partial nitritation by introducing denitritation of nitrate using sulfide as an

The process is applicable for sulfur-bearing nitrogenous wastewater

However, most ammonium-rich wastewaters are produced with a certain

investigated on bench scale in a single-stage nonwoven rotating biological