Rev Environ Sci Biotechnol (2018) 17:285–321
https://doi.org/10.1007/s11157-018-9463-4
REVIEW PAPER
Ammonia-Oxidizing Bacteria (AOB): opportunities
and applications—a review
Moomen Soliman . Ahmed Eldyasti
Published online: 23 March 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract Recently, partial nitrification has been
adopted widely as a first step of both nitrite shunt
and deammonification processes towards efficient and
economical nitrogen removal from wastewater. Effec-
tive partial nitrification relies on stimulating the first
step of nitrification while inhibiting the second step
and by consequence accumulating ammonia-oxidizing
bacteria (AOB). Successful AOB accumulation
depends upon the knowledge of their microbial
characteristics and kinetics parameters as well as the
main parameters that can selectively inhibits NOBs’
growth or allow AOBs to outcompete them. Several
bioreactors configurations either in suspended or
attached growth have been used towards achieving
partial nitrification using different inhibition condi-
tions. This review aims to illustrate an up to date
version of the metabolism and factors affecting AOB
growth and summarize the current bioreactors config-
urations in all lab-scale and full-scale applications for
AOB. Moreover, successful partial nitrification
attempts in the literature in suspended and attached
growth systems have been complied. Additionally, the
possibility of improving the current applications of
AOB and the integration into the operation of existing
M. Soliman  A. Eldyasti (&)
Department of Civil Engineering, Lassonde School of
Engineering, York University, Toronto, ON M3J 1P3,
Canada
e-mail: Ahmed.eldyasti@lassonde.yorku.ca
WWTPs in order to transform into water resources
recovery facility has been presented.
Keywords Ammonia oxidizing bacteria (AOB) 
Partial nitrification  Anammox  Full scale  Attached
growth  Kinetics
1 Introduction
Nitrogen is abundant naturally in the environment and
is provided for plants and animals nutrition through
the nitrogen cycle, however human manipulation of
the environment has altered significantly the nitrogen
cycle affecting water, soil and atmosphere. In term of
water, excess nitrogenous compounds discharged in
water bodies from sewage results in overstimulation of
the growth of algae consequently decreasing dissolved
oxygen concentrations and causing eutrophication
which may results in the death of all aquatic organ-
isms. Hence, in order to protect the aquatic ecosystems
reducing nutrient levels in the effluent of wastewater
treatment plants becomes an urgent need. The most
common method of removing nitrogen from wastew-
ater is the biological nitrogen removal (BNR) com-
prising nitrification and denitrification as it is
considered economical and efficient. Nitrification is
a two-steps biological oxidation process performed by
two different groups of aerobic autotrophic bacteria.
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The first step of nitrification is the oxidation of
ammonium (NH4) to nitrite (NO2) carried out by the
ammonia-oxidizing bacteria (AOB), while the second
step is the oxidation of nitrite (NO2) to nitrate (NO3)
carried out by the nitrite-oxidizing bacteria (NOB). On
the other hand, denitrification is the reduction of
nitrate to nitrite followed by its further reduction to
form nitrogen gas carried out by a group of anaerobic
heterotrophic bacteria. Nevertheless, BNR process
encounters some problems including (1) slow growth
of the microorganisms involved in these biological
processes, (2) high oxygen and carbon requirements,
(3) controlling the simultaneous nitrification/denitrifi-
cation conditions. To overcome the aforementioned
challenges, Shortcut Biological Nitrogen Removal
(SBNR) has been intensively studied in the past
several years. SBNR process is based on the fact that
nitrite is an intermediary compound in both nitrifica-
tion and denitrification, hence ammonium oxidation
step is halted at the nitrite stage which can be directly
reduced to nitrogen gas either by heterotrophic
denitrifies (conventional denitritation) or by anaerobic
ammonia oxidizing (Anammox) bacteria using the
nitrite produced as electron acceptor. Therefore,
SBNR reduces the oxygen consumption in the aerobic
phase by 25% resulting from suppressing the nitrite
oxidation step and consequently reduces the total
energy for aeration by 60%. Additionally, SBNR
eradicates the electron donor requirement due to
avoiding nitrate reduction to nitrite by 40% in the
anoxic zone, in which the carbon dioxide emissions
are reduced by 20% and decrease the sludge produc-
tion in nitrification and denitrification processes by 35
and 55%, respectively (Peng and Zhu 2006).
As previously stated, the first step of SBNR process
relies on suppressing the second step of nitrification
which is the nitrite oxidation to nitrate without having
an inhibitory effect on the former ammonia oxidation
rate known as partial nitrification process. Hence,
Successful partial nitrification could be achieved by
accumulating AOB and inhibiting NOB. AOB accu-
mulation depends upon the knowledge of their micro-
bial characteristics and kinetics parameters as well as
the main parameters that can selectively inhibit NOBs’
growth or allow AOBs to outcompete them. These
parameters may include appropriate regulation of the
reactor’s dissolved oxygen concentration (DO), tem-
perature, pH, hydraulic retention time (HRT), solids
retention time (SRT), alkalinity as well as the presence
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Rev Environ Sci Biotechnol (2018) 17:285–321
of free ammonia. Moreover, numerous bioreactors
configurations have been used towards achieving
partial nitrification in wastewater treatment plants
each comprising several advantages as well as some
drawbacks. These biological systems are either based
on suspended growth technologies where bacteria are
grown in flocs which refer to an assemblage of
individual cells or micro colonies that take place in a
reactor under particular conditions or after the addition
of an agent to the medium (Boonaert et al. 2002) or
attached growth technologies where bacteria are
grown in biofilm which is a complex coherent
structure of cells and cellular products which can
grow as large, dense granules or attached on a static
solid surface or attached on to suspended carriers
(Nicolella et al. 2010).
Thus, this review paper aims to explore the
microbial characteristics and kinetics parameters of
AOB as well as investigating the optimum values of
the main parameters that can selectively inhibits NOB
growth or allow AOB to outcompete NOB. Moreover,
successful partial nitrification attempts in the literature
in suspended and attached growth systems have been
complied. Additionally, the possibility of improving
the current applications of AOB and the integration
into the operation of existing WWTPs in order to
transform into water resources recovery facility
(WRRF) has been presented.
2 Aerobic AOBs microbial characteristics
2.1 AOB’s phylogeny
Since the first AOB isolation in 1890, numerous
studies have been performed to identify their phylo-
genetic diversity (Koops and Pommerening-Röser
2001; Purkhold et al. 2000). As a result for these
efforts, five AOB genera have been recognized and
classified in the Proteobacteria class. Four of which
lies in the b-Proteobacteria subclass including Nitro-
somonas (including Nitrosococcus mobilis), Nitrosos-
pira, Nitrosovibrio and Nitrosolobus, while one
cluster of Nitrosococcus belongs within the c-Pro-
teobacteria subclass. However, the investigation of
AOB enumeration, diversity and abundance in engi-
neering systems or their natural environments
remained a complex process using the conventional
culture-dependent technique due to the complexity
Rev Environ Sci Biotechnol (2018) 17:285–321 287

and long period accompanied with the cultivation of ranging from straight rods cells with polar to subpolar
these microorganisms—till the evolution of culture motile cells having Peripheral flattened vesicles with
independent molecular techniques (Geets et al. 2006). cell size of (0.7–1.5 9 1.0–2.4 lm) for Nitrosomonas
Furthermore, the culture dependent methods are clusters to (1.5–1.8 9 1.7–2.5) lm spherical ellip-
thought to underrate the actual cell numbers due to soidal cells with Tuft of flagella for motility having
potential defective cell suspension or disturbance in Peripheral or central stacks of vesicles observed in
flasks and microcolonies or some cell damage Nitrosococcus. On the other hand, Nitrosospira
(Kowalchuk and Stephen 2001). On the other hand, species are reported to be tightly coiled spirals with
the development of culture independent molecular Peritrichous motile cells having Invaginations in the
methods like fluorescence in situ hybridization intracytoplasmic membranes with cell size of
(FISH), polymerase chain reaction (PCR), quantitative (0.3–0.8 9 1.0–8.0) lm, while, Nitrosolobus species
PCR (qPCR), denaturing gradient gel electrophoresis are reported to be Pleomorphic lobate cells with
(DGGE) and Quinone profile techniques has allowed Peritrichous flagella as well but having a compart-
an accurate detection and identification of AOB mentalized intracytoplasmic membrane with cell size
communities. of (1.0–1.5 9 1.0–2.5) lm. Moreover, Nitrosovibrio
species are polar to subpolar slender curved rods
2.2 AOB’s morphology measuring (0.3–0.4 9 1.1–3.0) lm having as well
Invaginations in the intracytoplasmic membrane
The morphological diversity of AOB can be classified (Koops and Pommerening-Röser 2001; Sinha and
into four main categories: cell shape, cell size, Annachhatre 2007).
flagellation of motile cells, and arrangement of
intracytoplasmic membranes as shown in Fig. 1.
These categories vary from one cluster to another

AOB

Beta Gamma
Proteobacteria Proteobacteria

Nitrosomonos Nitrosospira Nitrosovibrio Nitrosolobus Nitrosococcus

Nitrosomonas Nitrosomonas Nitrosomonas Nitrosococcus Nitrosomonas Nitrosomonas Nitrosomonas Nitrosomonas Nitrosomonas Nitrosomonas Nitrosomonas Nitrosospira Nitrosovibrio Nitrosolobus Nitrosococcus Nitrosococcus
europaea eutropha halophila mobilis communis nitrosa oligotropha ureae marina aestuarii cryotolerans briensis tenuis multiformis oceani halophilus
Rod to pear
Short Rods Large rods Spheres or Spheres or Spheres or Slender rods Tightly closed
shaped with Coccoid or Slender Pleomorphic Spherical or Spherical or
Cell Shape with pointed Coccoid with rounded rods with rods with rods with with rounded Rod shaped Rod shaped spirals and
one or both rod shaped curved rods lobate ellipsoidal ellipsoidal
ends ends rounded ends rounded ends rounded ends ends vibrio forms
ends pointed

(0.8-1.1 x 1.0- (1.0-1.3 x 1.6- (1.1-1.5 x 1.5- (1.5-1.7 x 1.5- (1.0-1.4 x 1.7- (1.3-1.5 x 1.4- (0.8-1.2 x 1.1- (0.8-1.2 x 1.1- (0.7-0.9 x 1.7- (1.0-1.3 x 1.4- (2.0-4.0 x 1.2- (0.3-0.8 x 1.0- (0.3-0.4 x 1.1- (1.0-1.5 x 1.0- (1.5-1.8 x 1.7- (1.5-1.8 x 1.7-
Cell size
1.7) µm 2.3) µm 2.2) µm 2.1) µm 2.2) µm 2.2) µm 2.4) µm 2.4) µm 2.2) µm 2.0) µm 2.2) µm 8.0) µm 3.0) µm 2.5) µm 2.5) µm 2.5) µm

Tuft of Tuft of Peritrichous Subpolar Peritrichous Tuft of Tuft of

Motility Not observed Motile Not observed Not observed Not observed Not observed Not observed Not observed Not observed
flagella flagella flagella flagella flagella flagella flagella

• No obligate • No obligate • Obligate • Obligate • No obligate • No obligate • No obligate • No obligate • Obligate • Obligate • Obligate • No obligate • No obligate • No obligate • Obligate • Obligate
Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt • Max. Salt
requirement conc. 400 conc. 400 conc. 900 conc. 500 conc. 250 conc. 300 conc. 150 conc. 200 conc. 800 conc. 600 conc. 550 conc. 250 conc. 100 conc. 200 conc. 1100 conc. 1800
mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl

• 30-61 µM • 14-43 µM • 19-46 µM • 1.9-4.2 µM • 50-52 µM • 42-59 µM

Max. amm. Max. amm. Max. amm. Max. amm. Max. amm. Max. amm. Max. amm. Max. amm. Max. amm. Max. amm.
Substrate • Max. amm. • Max. amm. • Max. amm. • Max. amm. • Max. amm. • Max. amm.
conc. 600 mN conc. 400 mN conc. 250 mN conc. 50 conc. 400 mN conc. 200 mN conc. 100 mN conc. 50 mN conc. 1000 conc. 500 mN
affinity conc. 400 conc. 250 conc. 100 conc. 200 conc. 200 conc. 400
NH4Cl NH4Cl NH4Cl mN NH4Cl NH4Cl NH4Cl NH4Cl NH4Cl mN NH4Cl NH4Cl
mN NH4Cl mN NH4Cl mN NH4Cl mN NH4Cl mN NH4Cl mN NH4Cl

• Sewage • Eutrophic
• Marine
disposal • Sewage • Moderate freshwater • Natural soils
• Eutrophic environment • Soils
plants disposal eutrophic • Marine • Oligotrophic • Oligotrophic • Freshwater • Marine
Preferred • Brackish environment • Marine • Marine • Low • Sewage • Marine
• Eutrophic plants pH neutral environment freshwater freshwater environment • Natural soils environment
habitat water • Aquatic environment environment temperature disposal environment
freshwater • Eutrophic soils • Wastewater • Natural soils • Natural soils • Marine • Salt lakes
environment as low as plants
• Fertilized environment • Freshwater treatment environment
5 °C
soils plants

Fig. 1 Morphological and eco-physiological characteristics of reported ammonia oxidizing bacteria’s species

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2.3 AOB’s ecophysiology
The microbial ecology, phylogeny and morphology
are precursors of the organism’s biological activities
within a specific environment. Therefore, bacterial
clusters characterization by eco-physiological meth-
ods would indicate the presence of specific symbiotic
associations between groups of bacteria that utilize
nitrogen derivatives as their substrate; such as ammo-
nia oxidizing bacteria (AOB) and nitrite oxidizing
bacteria (NOB). Based on the fact that the former two
bacteria differ phylogenetically, each of them will
have its unique metabolic processes in terms of type
and efficiency depending on the bacterial cluster and
lineage. As shown in Fig. 1, AOBs can be divided into
three groups, each has its unique eco-physiological
parameters and preferred habitats. The first group is
Nitrosomonas genus which is divided into six lin-
eages. The first lineage which comprises four
species—Nitrosomonas europaea, Nitrosomonas
eutropha, Nitrosomonas halophila, and N. mobilis—
is characterized by its moderate salt requirement,
negative urease activity, substrate affinity of (30–61)
lM, and can be isolated from sewage disposal plants,
eutrophic fresh water and brackish water as shown in
Fig. 1. Whereas, the second Nitrosomonas lineage
which comprises three species—Nitrosomonas com-
munis, Nitrosomonas sp. I, Nitrosomonas sp. II—has
no salt requirement, as well no urease activity with less
substrate affinity of (14–43) lM and is present in non-
acidic soils. Furthermore, the third lineage which
comprises Nitrosomonas nitrosa species has no salt
requirement as well but with positive urease activity
and moderate substrate affinity (19–46) lM and is
commonly found in eutrophic fresh water. Moreover,
the fourth Nitrosomonas lineage which comprises two
species—Nitrosomonas ureae and Nitrosomonas olig-
otropha has no salt requirement, positive urease
activity with the least substrate affinity (1.9–4.2)
lM, and its preferred habitats are oligotrophic fresh
water and natural salts. Lastly, the fifth and sixth
lineage comprising Nitrosomonas marina, Nitro-
somonas sp. III, and Nitrosomonas aestuarii and
Nitrosomonas cryotolerans, respectively both are
obligate halophilic, have positive urease activity and
usually found in marine environment, and have
highest substrate affinity (50–52) and (42–59) lM
for the fifth and sixth lineage, respectively. On the
other hand, the second group of AOBs comprises
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Nitrosolobus multiformis, Nitrosovibrio tenuis and
Nitrosospira sp. I having all similar eco-physiological
parameters of no salt requirement, positive or negative
urease activity and are found in soils, rocks and fresh
water. Whereas, the last group comprises Nitrosococ-
cus oceani and Nitrosococcus halophilus which are
obligate halophilic and found in marine environment
with positive urease activity for the former and
negative urease activity for the latter (Koops and
Pommerening-Röser 2001).
Moreover, AOB characteristics and predominant
species vary in engineering systems according to the
reactor type (i.e. Continuous stirred tank reactors
(CSTR), Sequential batch reactors (SBR), Single
reactor for high activity ammonia removal over nitrite
(SHARON), Moving bed biofilm reactors (MBBR),
etc.), influent characteristics (i.e. freshwater, raw
wastewater, etc.) and operational conditions (i.e.
temperature, pH, DO, etc.) which strongly affect the
microbial ecology (Aoi et al. 2000). It may be
speculated that there are two types of AOB according
to the affinity of ammonia: (1) k-strategists which has
lower growth rate and higher ammonia affinity, thus
dominate in ammonia limited conditions and (2)
r-strategists which has higher growth rate and lower
ammonia affinity and by consequence dominate in
abundant ammonia conditions (Zhang et al. 2004). In
terms of different influent characteristics, Burrell et al.
(2001) reported during studies on freshwater based on
FISH analysis that N. marina outcompeted N. euro-
paea in low ammonia concentrations while in high
concentrations N. europaea was dominant (Burrell
et al. 2001). Therefore, AOB communities were
investigated in 12 wastewater treatment plants (8
SBRs and 4 activated sludge) using DGGE and it was
reported that in 11 wastewater treatment plants
(WWTPs) where the influent was characterized with
high ammonia concentrations the dominant species
were N. europaea and N. eutropha whereas in WWTP
with low ammonia concentrations N. ureae, N.
oligotropha and N. marina were dominant (Otawa
et al. 2006). Additionally, in salty wastewaters N.
mobilis (falling within the Nitrosomonas species) was
reported to be dominant (Schmidt et al. 2003).
Additionally, in terms of reactor configuration it
can be divided to two main systems: suspended growth
reactors and attached growth reactors. In the case of
suspended growth systems, in a partial nitrification
SBR treating landfill leachate with extremely high
Rev Environ Sci Biotechnol (2018) 17:285–321 289

nitrogen concentration N. europaea and N. eutropha consists of the conversion of nitrite to nitrate carried
were detected using PCR amplification and DGGE out by nitrite oxidizing bacteria (NOB) (Eq. 2).
fingerprinting (Gabarró et al. 2012). Moreover, it was AOB
found that the addition of landfill leachates into NHþ  þ
3 þ 1:5O2 ! NO2 þ H þ H2 O ð1Þ
municipal wastewater in a partial nitrification SBR
NOB
had an effect on the morphologic structure of AOB NO 
2 þ 0:5O2 ! NO3 ð2Þ
communities decreasing their diameter and increasing
The first step of nitrification carried out by ammonia
the microcolonies distribution in the flocks (Fudala-
oxidizing bacteria is called nitritation and comprises
Ksiazek et al. 2014). On the other hand in a partial
two steps with hydroxylamine (NH2OH) as an inter-
nitrification CSTR treating synthetic wastewater with
mediate product. The first step of nitritation is the
high ammonia concentrations and devoid of any
oxidation of ammonia to hydroxylamine catalyzed by
organic carbon, qPCR analysis revealed that the
the membrane bound ammonia monooxygenase
dominant AOB cluster was N. europaea suggesting
(AMO). This step requires a molecular oxygen and a
it could be part of r-strategists (Ahn et al. 2008).
pair of electrons (Eq. 3). In the second step, hydrox-
Further, analysis of a 16S rRNA gene revealed that in
ylamine is further oxidized to nitrite catalyzed by the
the single reactor system for high activity ammonia
hydroxylamine oxidoreductase (HAO) using oxygen
removal over nitrite (SHARON) N. eutropha was
from water and an additional molecular oxygen as a
dominant clone with 69% of the clones (Logemann
terminal electron acceptor (Eq. 4). This step generates
et al. 1998). In the case of attached growth systems, the
two pairs of electrons, one pair of which is compen-
diversity of AOB communities was compared in a
sated for the support of the first step of ammonia
biological aerated filter (BAF) and a trickling filter
oxidation, whereas the other pair is passed to the
treating identical wastewater using PCR analysis
terminal oxidase via an electron transport chain,
targeting 16S rRNA gene sequence combined with
generating a proton motive force (Sinha and Annach-
DGGE. A difference in the community structure was
hatre 2007).
noted between the two systems with a higher diversity
of AOB in the trickling filter than in the BAF although NHþ þ 
3 þ O2 þ 2H þ 2e ! NH2 OH þ H2 O ð3Þ
N. mobilis dominated all the samples analyzed in both
reactors (Rowan et al. 2003). NH2 OH þ H2 O ! NO þ
2 þ 5H þ 4e


2.4 AOB’s physiology 2Hþ þ 0:5O2 þ 2e ! H2 O ð4Þ

The previous reaction (Eq. 1) serves as energy-yield-
Nitrogen is usually present in wastewater in three
ing reaction for AOB which utilize ammonia as their
forms: (1) organic nitrogen compounds, (2) ammo-
sole source of energy. Besides being AOB energy
nium (NH4?), and (3) trace amounts of nitrite (NO2-)
source, part of the ammonia is used for their cell
and nitrate (NO3-). However, the organic fractions
growth as nitrogen source while carbon dioxide serves
such as proteins, amino acids, and amino sugars are
as their chief carbon source. If the empirical formu-
degraded immediately to ammonium (NH4?) either in
lation C5H7NO2 for the gross composition of the
the sewer systems or in the bioreactor (Law et al.
biomass is considered acceptable for AOB, the growth
2012). In conventional BNR plants, ammonia which
can be expressed by the following reaction (Eq. 5):
was suggested to be the true substrate for the oxidation
process and not ammonium (Suzuki et al. 1974) is 15CO2 þ 13NHþ 
3 ! 10NO2 þ 3C5 H7 NO2 þ 23H
þ

oxidized to nitrate via autotrophic nitrification fol- þ 4H2 O

lowed by its reduction to nitrogen gas via hetero- ð5Þ
trophic denitrification.
Nitrification is a two-step aerobic biological oxi- The energy released from the ammonia oxidation
dation process. The first velocity-limiting process step (Eq. 1) is utilized in the synthesis reaction (Eq. 5).
consists of the conversion of ammonia to nitrite Moreover, the free acid (H?) produced from the
carried out by ammonia oxidizing bacteria (AOB)
(Eq. 1). While the second step which is a rapid step

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oxidation and synthesis reactions reacts to produce
carbonic acid (H2CO3) according to the following
reactions (Eq. 6):
CO2 þ H2 O $ H2 CO3 $ Hþ þ HCO
3 ð6Þ
Therefore, the energy and synthesis reactions can be
reformulated to Eqs. (7) and (8), respectively after
taking in consideration the effect of the previous
reaction.
NHþ  
3 þ 1:5O2 þ 2HCO3 ! 2H2 CO3 þ H2 O þ NO2
ð7Þ
13NHþ  
3 þ 23HCO3 ! 8H2 CO3 þ 19H2 O þ 10NO2
þ 3C5 H7 NO2
ð8Þ
It can be deduced from the past two reactions that the
nitritation process results in destruction of alkalinity
through the production of hydrogen ions.
2.5 AOB’s enzymology
As discussed in the previous section, the ammonia
oxidation to nitrite is a two steps process, ammonia
oxidation to hydroxylamine followed by its further
oxidation to nitrite. The second step—hydroxylamine
oxidation to nitrite—is carried out by a periplasm-
associated enzyme hydroxylamine oxidoreductase
(HAO) generating four electrons that serves as the
sole source of reducing equivalents for the first step of
ammonia oxidation as well as cell synthesis. These
electrons are transported from HAO through cyto-
chrome c554—an electron acceptor to HAO—to
membrane cytochrome c552 then distributed by the
ubiquinone pool (Arp et al. 2002). Two of the four
generated electrons are returned to a membrane-bound
multisubunit enzyme ammonia monooxygenase
(AMO) to perform ammonia conversion to hydroxy-
lamine (Hooper et al. 1997). Furthermore, 1.65
electrons of the remaining pair of electrons are passed
through cytochrome c552 to cytochrome aa3 to the
terminal electron acceptor O2 and reduced to form
H2O while the remaining 0.35 electrons are used for
the reduction of NAD? to NADH through ‘reverse
electron flow’ performed by the embedded electron
carrier NADH dehydrogenase as illustrated in Fig. 2.
It was also suggested that in case of low dissolved
oxygen concentrations a portion of the 1.65 electrons
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Rev Environ Sci Biotechnol (2018) 17:285–321
passed to the terminal oxidase might pass to nitrite
reductase (NiR) and nitric oxide reductase (NoR)
enzymes producing nitric oxide (NO), nitrous oxide
(N2O) and trace amounts of nitrogen gas (N2) (Whit-
taker et al. 2000).
2.6 AOB’s kinetics
The AOB and NOB kinetics have been thoroughly
studied due to their crucial importance in controlling
and optimizing nitrogen removal processes. However,
an evident variation has been observed in the reported
kinetics values as shown in Tables 1 and 2. This wide
range in the values of kinetics parameters is due to the
different conditions that vary from a study to another
such as: wastewater characteristics (low or high
strength), operational conditions (temperature, pH,
DO), reactor configuration (suspended or attached
growth), identification technique (experimental or
model based) (Sin et al. 2008).
As a matter of fact, temperature is a critical
parameter that governs nitrifies kinetics as at higher
temperature AOB was reported to outcompete NOB
whereas at lower temperature NOB are able to oxidize
nitrite much faster (Wiesmann 1994). It was reported
in several studies that temperature significantly affects
the maximum specific growth rate for both AOB and
NOB with higher values for AOB at higher temper-
ature which is the condition the SHARON process
relied on to achieve partial nitrification (Hellinga et al.
1998; Kim et al. 2008; Melcer 2004). Another
parameter that might alter the kinetics value is the
DO concentrations. Canziani et al. (2006) studied the
effect of oxygen concentration on nitrifiers kinetics in
a MBR treating landfill leachate (Canziani et al. 2006).
It was reported that low DO concentrations increases
the difference between the maximum specific growth
rate (lmax) of AOB and NOB. At high DO ([ 1.5 mg/
L), lmax of AOB and NOB were 0.625 and 0.555 d-1
respectively, decreasing the DO to the range of
0.0–0.5 mg/L resulted in a decrease in both AOB
and NOB growth rates but at a higher rate for that of
NOB which lost around 75% of its lmax against only
28% for that of AOB. Moreover after slightly
increasing the DO to the range of 0.5–1.5 mg/L,
AOB completely recovered its maximum specific
growth rate whereas NOB lmax was still 30% lower
than the value calculated at high DO. The previous
results implying the inhibition of NOB at low DO
Rev Environ Sci Biotechnol (2018) 17:285–321
4e-
HAO
HNO2+4H++4e-
Periplasm NH2OH+H2O
NH3+O2+2e-
NADH 2e-
AMO
DH
+
2H
Cytoplasm NAD+ NADH
0.35e-
Fig. 2 Electron transport pathways in AOB. HAO hydroxy-
lamine oxidoreductase, Cyt c 554 cytochrome c554, Cyt cm 552
membrane cytochrome c552, QH2 ubiquinol; AMO ammonia
monooxygenase, NADH DH NADH dehydrogenase, Cyt bc1
concentrations may be attributed to the higher affinity
for oxygen of AOB than NOB (Wiesmann 1994).
3 Parameters affecting AOB
Effective partial nitrification could be achieved by
accumulating AOB and inhibiting NOB. Successful
AOB accumulation depends upon the knowledge of
the parameters affecting their growth. These param-
eters may include appropriate regulation of the
reactor’s dissolved oxygen concentration (DO), tem-
perature, pH, hydraulic retention time (HRT), solids
retention time (SRT), alkalinity as well as the presence
of free ammonia.
3.1 Dissolved oxygen concentration
Controlling the dissolved oxygen concentration in the
reactor is a possible way for enhancing nitrite accu-
mulation and by consequence accumulating AOB. It is
based on the differences between the Monod satura-
tion constant of oxygen for AOB and NOB that are
known to be 0.3 and 1.1 mg/L, respectively (Wies-
mann 1994). Thus, low DO concentration might be
more restrictive for the growth of NOB than AOB due
to the higher affinity for oxygen of AOB as shown in
Fig. 3. This was clearly illustrated in the report of
Hanaki et al. (1990) where low DO concentration
(0.5 mg/L) produced no effect on ammonia oxidation
291
NO N2O
HNO2
NiR NoR
Cyt c 554
4e- Cyt c 552 CCP
2H+
Cyt cm H 2O 2 2H2O
1.
552
65
1.65e-
-
e
4e-
1.65e-
QH2 Cyt bc1 Cyt aa3
2H+
½ O2 H2O
Complex 3, Cyt c 552 cytochrome c552, Cyt aa3 cytochrome c
oxidase, NiR nitrite reductase, NoR nitric oxide reductase, CCP
cytochrome c peroxidase
while nitrite oxidation was strongly inhibited. More-
over, low levels of DO elevated the growth yield of
AOB by double while the growth rate of NOB was
unchanged (Hanaki et al. 1990). However, limited DO
conditions might cause sludge filamentous bulking
problems and result in lower nitrification rate (Guo
et al. 2010). On the other hand, Stenstrom and Poduska
(1980) reported that there is no clear DO concentration
for optimum nitrification. According to this study, one
possible reason for the wide variation reported for DO
concentration is the effect of the oxygen diffusion with
the flocs (Stenstrom and Poduska 1980). This might
explain the variation in the critical DO concentration
values recorded in the literatures for controlling partial
nitrification. Suspended growth and attached growth
reactor systems have been conducted widely in
different DO concentrations to obtain higher nitrite
accumulation.
In the case of suspended growth systems, a DO
concentration of 0.4–0.5 mg/L in an activated sludge
reactor resulted in a nitrite accumulation rate of 96%
and an AOB population of 5.33 9 108 cell/mL.
Moreover, complete nitrification was obtained at a
DO concentration of 1.5–2.5 mg/L with almost unob-
vious nitrite accumulation rate while at a very low DO
condition (0.2 mg/L) nitrite build up did not occur and
nitrite accumulation rate was about 1.7% on average
(Mohammed et al. 2014). Wei et al. (2014) reported
that high DO concentration could damage nitrite
accumulation directly. According to their tests, in a
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Table 1 Summary of reported AOB kinetic parameters

Symbol Name Unit Reactor Temperature Range References
type

Suspended growth
lmax Maximum d-1 CSTR Ambient 0.24–1.21 Ahn et al. (2008), Blackburne et al. (2007),
growth rate Carrera et al. (2004), Chiellini et al. (2013),
Jubany et al. (2008), Liu and Wang (2013),
Munz et al. (2010), Wiesmann (1994)
High 0.9–2.1 Galı́ et al. (2007a, b), Hellinga et al. (1999),
Jones et al. (2007), Keen and Prosser (1987),
Magrı́ et al. (2007), Van Hulle et al. (2007)
SBR Ambient 0.46–1.06 Gao et al. (2010), Kampschreur et al. (2007),
Rongsayamanont et al. (2010)
High 0.94–1.96 Galı́ et al. (2007a, b), Liu et al. (2016),
Pambrun et al. (2006), Soliman and Eldyasti
(2017), Vadivelu et al. (2006a, b)
baerobic Aerobic d-1 CSTR Ambient 0.048–0.32 Ahn et al. (2008), Jubany et al. (2008), Liu and
decay rate Wang (2013), Munz et al. (2010), Wiesmann
(1994)
High 0.17–0.23 Jones et al. (2007), Magrı́ et al. (2007)
SBR Ambient 0.071 Kampschreur et al. (2007)
High 0.245–0.26 Liu et al. (2016), Vadivelu et al. (2006a, b)
KNH4 Substrate mg N/L CSTR Ambient 0.14–0.7 Jubany et al. (2008), Manser et al. (2005),
(NH4) half Wiesmann (1994)
saturation High 0.44–0.75 Hellinga et al. (1999), Jones et al. (2007),
constant Magrı́ et al. (2007), Van Hulle et al. (2007)
SBR Ambient 1.0–1.35 Gao et al. (2010), Kampschreur et al. (2007),
Rongsayamanont et al. (2010)
High 0.5 Pambrun et al. (2006), Soliman and Eldyasti
(2017)
Ko Oxygen half mg O2/L CSTR Ambient 0.3–0.79 Daebel et al. (2007), Guisasola et al. (2005),
saturation Jubany et al. (2008), Manser et al. (2005),
constant Wiesmann (1994)
High 0.25–0.94 Galı́ et al. (2007a, b), Jones et al. (2007),
Magrı́ et al. (2007), Van Hulle et al. (2007)
SBR Ambient 0.307–0.5 Gao et al. (2010), Kampschreur et al. (2007),
Rongsayamanont et al. (2010)
High 0.34–0.36 Galı́ et al. (2007a, b), Liu et al. (2016)
Y Yield mg COD/mg N CSTR Ambient 0.11–0.18 Ahn et al. (2008), Blackburne et al. (2007),
Jubany et al. (2008), Wiesmann (1994)
High 0.15–0.2 Galı́ et al. (2007a, b), Jones et al. (2007)
SBR Ambient 0.15–0.39 Gao et al. (2010), Kampschreur et al. (2007)
High 0.18–0.39 Dan (2014), Galı́ et al. (2007a, b), Soliman and
Eldyasti (2017), Vadivelu et al. (2006a, b)
Attached growth
lmax Maximum d-1 MBR– Ambient 0.18–1.08 Carrera et al. (2004), Chiellini et al. (2013),
growth rate FBBR– Munz et al. (2010), Pérez et al. (2009)
BAS High 0.33–3.40 Canziani et al. (2006), Lackner et al. (2010),
Vannecke and Volcke (2015)
b Aerobic d-1 MBR Ambient 0.08 Munz et al. (2010)
decay rate High 0.017–0.17 Vannecke and Volcke (2015)

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Table 1 continued
Symbol Name Unit Reactor Temperature
type
KNH4 Substrate mg N/L MBR Ambient
(NH4) half FBBR High
saturation
constant
KO2 Oxygen half mg O2/L MBR- Ambient
saturation FBBR
constant MBR High
Y Yield mg COD/mg N FBBR Ambient
MBR High
sequencing batch reactor (SBR) the initial nitrite
accumulation rate was decreased from 95.43 to 3.09%
under a DO concentration of 2.0–4.0 mg/L while
decreasing the DO concentration to 0.8 mg/L resulted
in the nitrite accumulation being 93.7% (Wei et al.
2014). Stenstrom and Poduska (1980) suggested that
at higher SRT, nitrification can be achieved at DO
concentrations at the level of 0.5–1.0 mg/L and at
lower SRT higher concentrations may be needed
(Stenstrom and Poduska 1980). This was illustrated in
the test performed in a CSTR operated at a SRT of
3 days where the DO concentration needed to main-
tain partial nitrification was 1.54 ± 0.87 mg O2/L
(Ahn et al. 2008).
In the case of biofilm systems the conversion rate is
usually limited by the oxygen transfer from liquid to
biofilm (Van Loosdrecht and Heijnen 1993). Hence, in
a biofilm airlift suspension reactor a dissolved oxygen
concentration of around 1.5 mg/L was required to
achieve an effluent with a high nitrite concentration
while maintaining a good ammonia conversion.
Whereas at dissolved oxygen concentrations below
1 mg/L ammonia oxidation decreased and resulted in
lower nitrite concentrations in the effluent. Moreover,
ammonia was completely converted to nitrate when
the dissolved oxygen in the reactor was over 2.5 mg/L
(Heijnen 1997). Additionally, in a static sequencing
batch worm reactor a DO concentration of 1.5 mg/L
was reported to inhibit NOB (Liu et al. 2014). It was
suggested that the high ammonia oxidation rate
compared to the nitrite oxidation rate at lower oxygen
concentrations may be due to a localization of the
NOB in the inner biofilm regions as well as the lower
293
Range References
0.13 Manser et al. (2005)
0.72 Schramm et al. (1999)
0.176–0.57 Daebel et al. (2007), Manser et al. (2005),
Pérez et al. (2009)
0.07–0.68 Lackner et al. (2010), Vannecke and Volcke
(2015)
0.17 Pérez et al. (2009)
0.09–0.41 Vannecke and Volcke (2015)
oxygen half-saturation constant for AOB (Tanaka and
Dunn 1982).
3.2 Temperature
Temperature has a clear effect on the ammonia
oxidation in partial nitrification systems. Nitrification
usually proceeds better in warmer seasons. The effect
of temperature on nitrogen removal was investigated
in batch biofilm reactors. The ammonia removal
efficiency was higher as the reactors temperatures
increased to reach more than 80% at 25 °C while it
went below 30% at 15 °C and lower (Kim and Lee
2011). Moreover, it was noted that nitrite accumulated
in an activated sludge plant especially over the
summer period (Tonkovic 1998). It has been sug-
gested that NOB has slower growth rates than AOB
when temperature goes up from 24 °C, while low
temperatures lead to NOB domination over AOB in
partial nitrification (Rodriguez-Sanchez et al. 2014).
In the same manner, Nitrobacter (usually the NOB
dominant species in nitrification) was reported to be
active at a range of temperature between 10 and 20 °C
while nitrite build-up remained low despite the FA
concentration that normally inhibits the Nitrobacter
activity which shows that the temperature effect of the
Nitrobacter activation prevailed over its inhibition by
FA. On the other hand, raising temperature to a range
between 20 and 25 °C resulted in an activation of the
ammonia oxidation activity together with a slowing of
the nitrite oxidation activity (Balmelle et al. 1992).
Moreover, in an inverse turbulent bed reactor raising
the temperature from 30 to 35 °C resulted in a
decrease in nitrate concentration in the outlet of the
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Table 2 Summary of reported NOB kinetic parameters

Symbol Name Unit Reactor Temperature Range References
type

Suspended growth
lmax Maximum d-1 CSTR Ambient 0.16–2.6 Ahn et al. (2008), Blackburne et al. (2007),
growth rate Carrera et al. (2004), Chiellini et al. (2013),
Jubany et al. (2008), Liu and Wang (2013),
Munz et al. (2010), Wiesmann (1994)
High 0.56–1.05 Hellinga et al. (1999), Jones et al. (2007),
Keen and Prosser (1987), Magrı́ et al. (2007)
SBR Ambient 0.48–0.56 Kampschreur et al. (2007), Vadivelu et al.
(2006a, b)
High 0.674–2.51 Liu et al. (2016), Pambrun et al. (2006),
Soliman and Eldyasti (2017)
baerobic Aerobic d-1 CSTR Ambient 0.045–1.7 Ahn et al. (2008), Jubany et al. (2008), Liu and
decay rate Wang (2013), Munz et al. (2010), Wiesmann
(1994)
High 0.04–0.17 Jones et al. (2007), Magrı́ et al. (2007)
SBR Ambient 0.069–0.08 Kampschreur et al. (2007), Vadivelu et al.
(2006a, b)
High 0.245 Liu et al. (2016), Soliman and Eldyasti (2017)
KNO2 Substrate mg N/L CSTR Ambient 0.28–1.6 Carrera et al. (2004), Manser et al. (2005)
(NO2) half SBR High 0.02–0.05 Jones et al. (2007), Magrı́ et al. (2007)
saturation
Ambient 1.49–3 Kampschreur et al. (2007), Vadivelu et al.
constant
(2006a, b)
High 1.62 Pambrun et al. (2006)
Ko Oxygen half mg O2/L CSTR Ambient 0.43–1.75 Blackburne et al. (2007), Daebel et al. (2007),
saturation Guisasola et al. (2005), Jubany et al. (2008),
constant Manser et al. (2005), Wiesmann (1994)
High 0.5–0.73 Jones et al. (2007), Magrı́ et al. (2007)
SBR Ambient 0.357–1.0 Kampschreur et al. (2007), Rongsayamanont
et al. (2010)
High 0.54 Liu et al. (2016), Soliman and Eldyasti (2017)
Y Yield mg COD/mg N CSTR Ambient 0.04–0.2 Ahn et al. (2008), Blackburne et al. (2007),
Jubany et al. (2008), Wiesmann (1994)
High 0.09 Jones et al. (2007)
SBR Ambient 0.041 Kampschreur et al. (2007)
High 0.1 Liu et al. (2016)
Attached growth
lmax Maximum d-1 MBR– Ambient 0.162–1.53 Carrera et al. (2004), Chiellini et al. (2013),
growth rate FBBR– Downing and Nerenberg (2008), Munz et al.
BAS (2010), Pérez et al. (2009)
High 0.129–3.54 Canziani et al. (2006), Lackner et al. (2010),
Vannecke and Volcke (2015)
b Aerobic d-1 MBR Ambient 0.11 Munz et al. (2010)
decay rate High 0.012–0.18 Vannecke and Volcke (2015)
KNH4 Substrate mg N/L MBR– Ambient 0.17–5.04 Carrera et al. (2004), Carvallo et al. (2002),
(NH4) half BAS Downing and Nerenberg (2008), Manser
saturation et al. (2005), Pérez et al. (2009)
constant FBBR High 0.46 Schramm et al. (1999)

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Table 2 continued
Symbol Name Unit Reactor Temperature Range References
type

KO2 Oxygen half mg O2/L MBR- Ambient 0.176–0.57 Daebel et al. (2007), Downing and Nerenberg
saturation FBBR (2008), Manser et al. (2005), Pérez et al.
constant (2009)
High 1.78–1.98 Lackner et al. (2010), Schramm et al. (1999)
Y Yield mg COD/mg N FBBR Ambient 0.06 Pérez et al. (2009)
MBR High 0.02–0.2 Vannecke and Volcke (2015)

Furthermore, temperature affects the chemical equi-

libriums of free ammonia (FA) and free nitrous acid
(FNA) which influences the performance of partial
nitrification. In a sequencing batch reactor, FA concen-
trations increased from 20.76 ± 4.23 mg N–NH3/L on
average at a temperature of 25 °C to
122.92 ± 27.23 mg N–NH3/L on average at a temper-
ature of 35 °C, whereas FNA levels showed the opposite
behavior decreasing from 0.47 ± 0.09 mg N–HNO2/L
to 0.12 ± 0.02 mg N–HNO2/L (Gabarró et al. 2012).
In practice, partial nitrification reactors are commonly
operated at a temperature of range between 30 and
35 °C.

3.3 pH, free ammonia (FA) and free nitrous acid

(FNA)

The value of pH influences the equilibrium of free

Fig. 3 Performance of Partial nitrification suspended systems
during different DO concentrations in terms of: a nitrite
accumulation rate (NAR); b ammonia removal efficiency
(ARE)
reactor combined with a simultaneous increase in the
nitrite concentration (Bougard et al. 2006). However,
partial nitrification could be achieved at low temper-
atures if the conditions for AOB to outcompete NOB
were created (Ge et al. 2015).
ammonia (FA) and free nitrous acid which have an
inhibitory effect on both AOB and NOB, thus
regulating pH is commonly used to achieve partial
nitrification. The nitrite oxidizers appears to be more
sensitive to free ammonia as a value of 0.1–1.0 mg
FA/L was reported to inhibit NOB while 10–150 mg
FA/L is necessary for the AOB inhibition (Anthonisen
et al. 1976). In that manner, almost similar ranges were
reported in other studies where NOB was inhibited at a
range of 0.1–4.0 mg FA/L in batch reactors (Bae et al.
2001) and 1–5 mg FA/L in Anaerobic–Aerobic
Treatment of High-Strength Ammonium Wastewater
whereas AOB was inhibited at FA concentrations
higher than 7 mg/L and stopped at 20 mg/L (Abeling
and Seyfried 1992). However, free ammonia was
reported to only inhibit NOB not to kill them and after
a long period of cultivation nitrite oxidizers can be
adapted to high free ammonia concentration and
recover activity (Beccari et al. 1983). The relationship

123
296
between free ammonia concentration and pH is as
follows (Anthonisen et al. 1976):
mg 17
NH3  N  10pH
FA ¼    ð9Þ
L 14 10pH þ exp 6344
273þT
Along with free ammonia concentration, nitrite oxi-
dizers are more sensitive to free nitrous acid than
ammonia oxidizers. FNA concentration of
0.42–1.72 mg-N/L resulted in a 50% reduction in
AOB activity while lower concentration of
0.011–0.07 mg-N/L started to inhibit NOB and
0.026–0.22 mg-N/L could completely inhibit NOB
(Zhou et al. 2011). Moreover in an activated sludge,
FNA acted as an uncoupler by donating a proton inside
the cell which directly interferes with the transmem-
brane pH gradient required for ATP synthesis causing
the inhibition (Glass et al. 1997). The relationship
between free nitrous acid and pH is as follows:
mg 46 NO2  N
FNA ¼    ð10Þ
L 14 10pH  exp  2300
273þT
Therefore, most of the literature suggests that pH in
the range of 7.5–8.5 is most suited to inhibit NOB. It
has been reported that the optimal pH for Nitro-
somonas species ranges between 7.9 and 8.2, while for
Nitrobacter species it ranges between 7.2 and 7.6
(Alleman 1985). Likewise, a pH between 7.5 and 7.8
was suggested to favor partial nitrification (He et al.
2012). Moreover, in a nitrifying biofilm activity nitrite
accumulation started above pH 7.5 and increased to
85% at pH of 8.5 (Villaverde et al. 1997). These results
were similar with those of Abeling and Seyfried
(1992) who reported that pH should be maintained
over 7.5 to inhibit NOB and accumulate nitrite
(Abeling and Seyfried 1992).
3.4 HRT and SRT
HRT and SRT adjustments can lead to change in
microbial community in wastewater treatment plants
resulting in washing out NOB populations knowing
that the minimum doubling time of AOB is 7–8 h
shorter than that of NOB 10–13 h (Bock et al. 1986).
Conventionally, HRT and SRT are set up to be the
same time in partial nitrification reactors. However,
the development of decoupled HRT and SRT partial
nitrification reactors has been attempted by the
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Rev Environ Sci Biotechnol (2018) 17:285–321
biomass recycling or the attached growth systems
which increases the SRT with respect to HRT.
The effect of HRT on partial nitrification reactors
has been studied in several studies. In the case of
suspended growth systems, in an activated sludge at
HRT of 9.1 h a nitrite accumulation rate of 96% was
achieved (Mohammed et al. 2014). Moreover, the
effect of HRT on partial nitrification was investigated
in a SBR using precultured aerobic granules in
continuous flow reactor. At HRT of 12 and 7.2 h,
both the removal efficiency of ammonia and the nitrite
accumulation rate exceeded 90%. Whereas the
removal efficiency of ammonia was decreased and
fluctuated (20–56%) at HRT of 2.4 h (Wan et al.
2014). On the other hand, in a sequencing batch
reactor treating acrylic fiber wastewater the optimal
HRT was 20 h. At this HRT, the ammonium removal
rate reached 97% with a nitrite accumulation of 87%
(Li et al. 2013a, b).
In the case of attached growth systems, on average
82% of ammonium was converted to nitrite in a MBR
when HRT was controlled at 10 h (Shen et al. 2014).
Moreover, the influence of HRT on biofilms was
investigated in a plastic SHARON bioreactor con-
structed as a submerged biofilter with PVC carriers. It
was found that at HRT of 12 h 100% of the ammonia
was converted to nitrite, while decreasing the HRT to
9.6 h resulted in the reduction of the quantity on
ammonia converted to nitrite to 60%. Furthermore, the
HRT of 12 h resulted in the formation of highly
specialized biofilms mainly by Nitrosomonas species
which are effective in the ammonia to nitrite biotrans-
formation. In contrast, controlling the reactor at an
HRT of 9.6 h facilitated the formation heterogeneous
biofilms that allow a closer ammonium to nitrite ratio
(González-Martı́nez et al. 2013). However, in a hybrid
moving bed biofilm reactor, an HRT of 9.5 h led to the
maximum population of AOB (Leyva-Dı́az et al.
2015),
The SRT also has a crucial influence on bacterial
communities in partial nitrification reactors. In sus-
pended growth systems, based on experiences in a
CSTR, controlling the reactor at a SRT of 3 days led to
NOB washout (Ahn et al. 2008). However, successful
partial nitrification were reported under longer sludge
age in other studies. In an activated sludge process a
SRT of 10–13 days was reported to be more appro-
priate for AOB accumulation with a nitrite accumu-
lation rate of 95%. Furthermore, under shorter SRT
Rev Environ Sci Biotechnol (2018) 17:285–321
stable partial nitrification deteriorated, whereas
increasing the SRT to 16 days resulted as well in a
decrease in nitrite accumulation rate from 93 to 37%
(Mohammed et al. 2014).
4 Suspended partial nitrification technologies
4.1 Sequential batch reactor (SBR)
In recent years, sequencing batch reactors (SBR) have
been adopted widely as an efficient technology for
wastewater treatment due to its simple configuration.
These systems have been successfully used to treat
both municipal and industrial wastewater. SBRs are
considered as fill and draw version of the activated
sludge, basically a batch reactor operating under a
series of operations that constitute the SBR cycle. This
cycle typically includes the following operations: fill,
react, settle, decant and idle. Figure 4 illustrates
examples of the SBRs cycle used in partial nitrification
systems. The difference between SBR and activated
sludge technologies is that the former performs
biological treatment and sedimentation within the
same reactor using a time control sequence while the
latter uses separate reactors for treatment and
sedimentation.
SBRs have shown great success in achieving nitrite
accumulation at high nitrogen loading rates. This type
of reactors has discontinuous feeding which allows the
reactor to maintain high ammonia concentration.
Thus, SBR are suitable to treat ammonia-rich organic
wastewater with variable nitrogen loads such as
landfill leachate and slaughterhouse wastewater. Dis-
turbance effects resulting from receiving high ammo-
nium loading shocks contained in leachates would be
less important due to the high biomass concentration
inside the reactor as well as the sequencing of the
feeding phase would help to control possible FA and
FNA accumulations inside the reactor and by conse-
quence inhibiting NOB. Provided that partial nitrifi-
cation in SBRs relies on NOB inhibition by FA & FNA
concentrations, NOB washout will be extremely
influenced by ammonia concentration and nitrogen
loading rate. Moreover, AOB could also be inhibited
by higher FA & FNA concentrations, thus controlling
them is a key factor to maintain partial nitrification in
SBRs.
297
Stable partial nitrification in a SBR treating landfill
leachate with extremely high nitrogen concentration
was achieved at both 25 and 35 °C (Gabarró et al.
2012). The SBR cycle had a total duration of 1440 min
divided into 14 sub-cycles of 100 min each, a 20 min
settling phase and a 20 min draw phase. Each sub-
cycle consisted of 10 min of aerobic feeding and
90 min of aerobic reaction. DO and pH were main-
tained at over 2 mg O2/L and below 8.0 respectively.
Both reactors were started at an specific nitrogen rate
of 0.2 kg N/kg VSS. d and was progressively raised to
0.81 ± 0.11 kg N/kg VSS. d and 0.84 ± 0.24 kg N/
kg VSS. d at 25 and 35 °C respectively by increasing
the daily influent flow. At stable state, HRT was about
4.5 days and 12.0 days at 25 and 35 °C respectively,
additionally the reactor suspended solids concentra-
tion was 1306 ± 620 mg VSS/L. The molar alkalinity
to ammonium ratio was 1.16 ± 0.06 and
1.12 ± 0.06 mol HCO3/mol NH4 at 20 and 35 °C
respectively. It was noticed that nitrate reached its
maximum concentration during the first 10 days and
then steadily decreased till the end of the experiments
while nitrite started accumulating from the beginning
of the experiments reaching stable concentration val-
ues of around 3500 mg N/L from day 22nd and 18th at
25 and 35 °C respectively. Effluent characteristics
were similar for both experiments NH4, NO2, NO3
were 2725.9 ± 153.2, 3719.2 ± 174.5, 41.9 ± 25.0
and 2629.9 ± 123.4, 3245.5 ± 115.7, and
25.8 ± 3.0 mg N/L at 25 and 35 °C respectively. It
is remarkable that the nitrate concentration was lower
than 1% of the total influent nitrogen for both
experiments. It was suggested that FA and FNA
concentration were the main factors for NOB inhibi-
tion given the extremely high ammonium content of
the landfill leachate. Furthermore, a high ammonia
removal efficiency (with an average of 98.6 ± 2.8%)
accompanied with a stable nitrite accumulation (with
an average of 93.0 ± 0.7%) was reached at different
nitrogen loading rate ranging from 0.375 to 1.2 kg/m3/
d. in a SBR treating synthetic wastewater devoid of
any organic substrate with a stepwise increase of
ammonium concentration through a novel DO control
strategy depending on using variable mixing regime
(Soliman and Eldyasti 2016). The SBR had a total
cycle duration of 4 h consisting of 5 min of feeding
200 min of aerobic reaction. 30 min of settling and
5 min of decanting. Temperature and pH were main-
tained at 31 °C and 7.9–8.2 respectively. The reactor
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298 Rev Environ Sci Biotechnol (2018) 17:285–321

SBRs cycle duration (min)

Zhang et al., 2017
Yan et al., 2013
Wei et al., 2014
Wang et al., 2017
Wang et al., 2015
Wang et al., 2014
Soliman and Eldyasti, 2016
Liu et al., 2016
Li et al., 2008

0 60 120 180 240 300 360 420 480 540 600 660 720
Soliman
Li et al., Liu et al., and Wang et Wang et Wang et Wei et Yan et Zhang et
2008 2016 Eldyasti, al., 2014 al., 2015 al., 2017 al., 2014 al., 2013 al., 2017
2016
fill 7 20 5 10 10 10 5 5 5
reaction 393 300 200 300 335 285 385 300 480
settle 30 20 30 45 60 60 20 40 30
decant and iddle 50 20 5 5 75 5 70 15 205

Fig. 4 SBR cycle duration for different partial nitrification studies

operation comprised three stages with different oper-
ation conditions. Stage I aimed to reach complete
nitrification thus DO was controlled at the conven-
tional range of 1.5–3.5 mg/L. During this stage, 2
NLRs of 0.3 and 0.4 kg/m3/d were introduced to the
reactor as well HRT was controlled at 12 h. After
13 days of operation, ammonia removal efficiency
(ARE) increased from 90 to 100% with 99.9% of the
ammonia oxidized to nitrate indicating the occurrence
of complete nitrification. Whereas, Stage II aimed to
start partial nitrification through introducing NOB
inhibition conditions by decreasing the DO range to
0.5–1.8 mg/L which resulted in an instant drop in the
ARE to 65% due to the new oxygen limitation
conditions. However, the reactor was able to recover
its performance afterwards reaching an ARE of 100%.
In addition, nitrite started to build up during this stage
and nitrate was suppressed implying the success of
NOB inhibition through low DO concentration. The
average ammonia removal efficiency (ARE) and
nitrite accumulation rate (NAR) during this stage
were 96.2 ± 2.9 and 89.4 ± 3.2% respectively. After-
wards, stage III aimed to reach higher partial nitrifi-
cation at higher NLRs reaching a NLR of 1.2 kg/m3/d,
hence the DO was slightly increased to the range of
0.6–1.2 mg/L to match the higher oxygen requirement
of higher ammonia concentrations. The SBR success-
fully maintained a high ARE and NAR during all the
NLRs phases with an average of 98.6 ± 2.1 and
94.3 ± 0.7% respectively. The successful nitrite
accumulation and nitrate suppression while maintain-
ing high ammonia removal was referred to the
combination of NOB inhibition conditions such as
low DO concentrations, high temperature, high pH and
sufficient alkalinity in addition to maintaining high FA
and FNA concentrations.
Moreover, nitrogen removal via nitrite of a mixture
of real municipal wastewater (RWW) and an increas-
ing quantity of real landfill leachates (RLL) in a
sequencing batch reactor was investigated (Fudala-
Ksiazek et al. 2014). SBR was inoculated with
nitrifying activated sludge and was fed with RWW
during a 9 weeks start-up period then with a mixture of
RWW and increasing quantities (1, 2, 5 and 10% of
volume) of RLL over the following 17 weeks. The
SBR cycle had a total duration of 12 h for 1 and 2%
addition of landfill leachate and 24 h for 5 and 10%.
Each cycle consisted of six phases: anaerobic filling,
aerobic reaction, anoxic reaction, aerobic reaction,
settling and decantation. Temperature, DO in the
aerobic phase and SRT were controlled at 20 ± 1 °C,
1.0 ± 0.5 mg O2/L and 70–92 days respectively. In
the stable portion of stable period, high ammonia
removal efficiency (up to 96%) was achieved with
nitrate domination in the effluent (up to 100% of the
TN). During the co-treatment of RWW with the

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addition of RLL, the removal efficiency of ammonia
was higher with values of 99% for RM1 and RM2,
91% for RM5 and 72% for RM10 with an average
amount of nitrite of 62, 66, 61 and 22% for RM1,
RM2, RM5 and RM10 respectively. Whereas, nitrate
concentration in the effluent decreased with the
addition of landfill leachates to reach an average of
31, 9, 8 and 4% for RM1, RM2, RM5 and RM10
respectively. It was suggested that the decrease of the
nitrate concentration was due to the inhibition of NOB
resulting from the increase in FA concentration with
continuous increase of N–NH4 (pH above 8) from
0.49 mg N–NH3/L for RWW to 2.06, 3.78, 8.05 and
8.91 mg N–NH3/L for RM1, RM2, RM5 and RM10
respectively.
Furthermore, SBR can be used as a first step in the
anammox process where anaerobic ammonia oxidiz-
ing bacteria oxidize ammonium to nitrogen gas using
nitrite as the electron acceptor. One of the key features
of an anammox reactor is the availability of suit-
able influent composed of 1:1.32 ammonium: nitrite
molar ratio. Thus, more than half of the ammonium in
the wastewater influent must previously be partially
oxidized to nitrite, avoiding further oxidation of nitrite
to nitrate which can be achieved in a partial nitrifica-
tion SBR. This was illustrated by the experiments
performed in a SBR treating urbane landfill leachates
to attain a suitable influent for an anammox reactor
(Ganigué et al. 2007). Raw leachate was collected
from a landfill with NH4, COD and BOD concentra-
tions on average of 1623 ± 424 mg N–NH4/L,
4512 ± 649 and 558 ± 257 mg/L respectively. The
SBR cycle had a duration of 8 h consisted of 360 min
of aerobic feeding, 80 min of aerobic reaction, 15 min
of settling and 25 min of decanting. Temperature, DO
in the aerobic reaction phase, pH and HRT were
controlled at 36 ± 1 °C, 2 mg/L, 6.8–7.1 and
1.5 days. SRT was not controlled but calculated
considering reactor MLSS and effluent suspended
solids concentration and found to be 5 days on
average. The SBR was inoculated with nitrifying
sludge and was fed with a mixture of synthetic
wastewater and urban landfill leachate with a leachate
proportion in the feed increasing until reaching a
100% raw leachate on day 167. The molar ammonium
to alkalinity ratio was always adjusted to 1:1 with
sodium bicarbonate additions. Nitrite percentages
were between 40 and 60% during the majority of the
experiment, obtaining a composition close to a molar
299
ammonium to nitrite ratio of 1:1. Despite this perfor-
mance, a lower percentage of ammonium oxidation
was observed each time the ammonium loading rate
was increased. Nevertheless, the system recovered its
performance after a period of stable influent ammonia
concentration. It was suggested that this effect could
be due to the possible slow response of AOB to the
increasing loading rate. The stable phase was reached
after 130 days of operation when the reactor was
operated with 75% of raw leachate and an influent
ammonium concentration of 1440 mg N/L. Under
such conditions, NH4, NO2 and NO3 concentrations in
the effluent were 725, 672 and 0.4 mg N/L respec-
tively. Additionally, the FA and FNA concentrations
were 5.58 and 0.18 mg N/L respectively. Thus, the
low formation of nitrate could be explained by the FA
concentration which was enough to inhibit NOB
without inhibiting AOB. On the other hand, the
consumption of alkalinity was very close to the
ammonium oxidation by the combined effect of
biological ammonium oxidation (autotrophic growth
and pH regulation) and the stripping effect caused by
aeration.
4.2 Continuous stirred tank reactor (CSTR)
Another type of reactors where partial nitrification can
be performed is continuous stirred tank reactor
(CSTR). This type of reactors run at steady state and
has a continuous flow for reactants and products. The
effect of ammonia loading rate on partial nitrification
in a CSTR without biomass recycle operated over a
wide range of HRT was investigated (Daalkhaijav and
Nemati 2014). The bioreactor was inoculated by a
nitrifying culture mainly comprising Nitrosomonas
and Nitrobacter species and was fed in a continuous
mode by pumping a medium of 17.6 ± 0.2 mM
ammonia at a low flow rate of 1 mL/h which was
increased incrementally to a reach a highest value of
115 mL/h. Mixing was provided by a magnetic stirrer
and aeration was carried out through a porous air
diffuser at a rate of 100 mL/min. pH was adjusted to
7.5–8.5 using sodium bicarbonate solution. The CSTR
was operated at room temperature (25 °C) for
243 days. The reactor was operated under a number
of applied flow rates representing low, medium and
high ammonia loading rates. It was noted that at low
ammonia loading rates (up to 1.0 mM/h) ammonia
was almost totally oxidized with a nitrate domination
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(95–99% of the effluent). The average DO concentra-
tion measured was 4.8 ± 0.1 mg O2/L. Increasing
ammonia loading rates to the range of 1.0–3.1 mM/h
resulted in an increase in nitrite concentrations and a
decrease in nitrate concentration. Furthermore,
increasing loading rate from 3.1 to 5.4 mM/h
enhanced the production rate of nitrite to a maximum
value of 2.5 mM/h (48.8 ± 8.9% of the effluent) and a
decrease in ammonia removal rate (44.7 ± 6.1%
ammonia in the effluent) at a corresponding HRT of
3.7 h. The average DO concentration measured was
4.8 ± 0.2 mg O2/L. While, increasing loading rates
over 5.4 mM/h led to decreasing trends of nitrite as
well as a decrease in ammonia removal rate. The
average DO concentration measured was
5.2 ± 0.1 mg O2/L. It was suggested that the sharp
decrease in ammonia removal rate following the
increases in loading rate was due to cell washout that
occurred in short HRTs. Results of this experiment
revealed that ammonia loading rate can be used as an
alternative operating variable to control partial nitri-
fication and may also be used to generate a suitable in-
fluent for the Anammox process.
Moreover, a stable accumulation of 50% ammonia
and nitrite to feed an anammox reactor was achieved
in a CSTR followed by a settling tank operated at room
temperature by pH controlled partial nitrification
(Durán et al. 2014). Regulating the ammonium to
inorganic carbon ratio (NH4/IC) was selected as the
strategy to control pH and the inhibitory effects of NA
and FNA, thus inhibiting NOB activity and achieving
partial nitrification. The feeding media consisted of
pig slurry pretreated in an aerobic granular plant with
NH4 and COD concentration of 399 ± 25 mg NH4–
N/L and 103 ± 43 mg COD/L respectively. Aeration
was carried out through air spargers. HRT, tempera-
ture and DO were controlled at 3 days, 22–25 °C and
over 2.0 mg O2/L respectively. The CSTR was
inoculated with an amount of 0.45 g VSS/L of
nitrifying activated sludge occupying 25% of the
liquid volume. The applied ammonia nitrogen loading
rate (NLR) in the reactor was 0.13 ± 0.01 kg NH4–N/
(m3 d). The reactor was operated in four different
stages with different NH4/IC ratio in the feeding
ranging from 1.19 g N/g C in stage I to 0.82 g N/g C
in stage IV. The NH4/IC ratio was controlled by the
addition bicarbonate without the control of pH in the
influent. It was noted that in stage I 24% of the fed
ammonium was oxidized to nitrite and 7% to nitrate
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with a nitrite to ammonium (NO2/NH4) ratio of
0.35 ± 0.05 in the effluent. Similar results were
obtained in stage II and III where the NH4/IC ratio
was decreased with a NO2/NH4 ratio of 0.49 ± 0.07 in
the effluent. In both stages, nitrate was present in the
effluent but in small amounts. Whereas, decreasing the
NH4/IC ratio to 0.82 g N/g C resulted in the oxidation
of 50% of ammonium to nitrite without significant
production of nitrate at a corresponding pH of 6.0. It
was suggested that the nitrite accumulation that
occurred in the reactor was due to the inhibitory effect
of the FA and FNA. In stage I, both AOB and NOB
were inhibited by FA concentration, while once the pH
was decreased due to the decrease in NH4/IC ratio in
the following stages only NOB were inhibited.
Whereas, FNA concentration was high enough to
inhibit NOB only during stage IV.
4.3 Single reactor for high activity ammonia
removal over nitrite (SHARON)
A single reactor for high activity ammonia removal
over nitrite (SHARON) process is operated in a
continuous stirred tank reactor (CSTR) at a relatively
high temperature (30–40 °C) and without sludge
retention which means that SRT equals HRT. Several
studies have reported problems in maintaining partial
nitrification in long-term operation due to NOB
acclimation to the non-favoring conditions during
long periods. Thus, the total washout of NOB is crucial
for maintaining stable partial nitrification and to
achieve this SRT is a key control parameter. Given
that the specific growth rate of NOB is lower than the
specific growth rate of AOB, the lower SRT the easier
NOB are washout. Thus Sharon process is operated at
an equal HRT and SRT which is the lowest value of
SRT. Furthermore, temperature is controlled over
30 °C to favor AOB growth and washout NOB from
the system. However, in most of wastewater treatment
plants room temperature is under 30 °C, thus temper-
atures around this value could not be maintained
without a significant operational cost. Therefore,
Sharon process could be costly.
An effluent ready for anammox process was
obtained in a Sharon process treating real reject water
(Galı́ et al. 2007a). Reject water was obtained from a
mesophilic anaerobic digester with NH4 and COD
concentrations of 800–900 mg NH4–N/L and
1500–2000 mg COD/L respectively. The reactor was
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inoculated with 250 ± 25 mg VSS/L of autotrophic
biomass. HRT, pH and temperature were
1.2–1.4 days, 8 and 35 °C. The inorganic to ammo-
nium ratio in the feeding was 0.98 mol HCO3/mol N.
The ammonium and nitrite concentrations in the
effluent had an average of 350 ± 25 mg NH4–N/L
and 400 ± 25 mg NO2–N/L respectively, giving a
removal efficiency of 0.3 kg NH4–N/(m3 d) which
was transformed to nitrite. It was suggested that partial
nitrification in Sharon would be the most economical
treatment when combined with an anammox reactor
due to the savings costs in terms of oxygen, methanol
and reactors volume.
Moreover, the treatment of nitrogen rich refinery
wastewater by partial nitrification was evaluated in a
Sharon process and investigated the high potential
toxic effect of the real wastewater on the biomass
(Milia et al. 2012). Real wastewater was taken from
the outlet of a stripping unit of a refinery plant with
high alkalinity (up to 1380 mg CaCO3/L) and a
dissolved organic carbon (DOC) of 240 ± 92 mg/L
and toxic compounds such as sulfides, cyanides and
phenols. The reactor was water-jacketed allowing the
temperature to be controlled by a thermostatic water
bath. Complete mixing was applied and aeration was
supplied by a membrane pump and introduced through
a fine bubble aerator at the bottom of reactor. The
reactor was inoculated with conventional activated
sludge from a municipal wastewater treatment plant.
Temperature, DO concentration and pH were con-
trolled at 35 ± 0.5 °C, 2.0 mg/L and 6.5–7.5. HRT
and SRT were maintained at 1–1.25 days. The reactor
was operated in 3 phases. During the first phase, it was
fed with a synthetic influent containing only NH4 as a
substrate with a concentration increasing from 100 to
1000 mg/L with an applied volumetric nitrogen load-
ing rate of 0.1–1.0 kg N/(m3 d). During phase II, real
wastewater was gradually added to the synthetic
medium with increasing ratios and the ammonium
concentration was also increased up to 2000 mg/L.
4.4 Novel systems
In a novel pilot-scale six tanks activated sludge
process treating real domestic wastewater a 94%
nitrite accumulation was achieved through a combi-
nation of short HRT and low DO level (Mohammed
et al. 2014). Raw wastewater was obtained from the
main manhole of a campus with NH4 and COD
301
concentrations of 40.6 mg NH4-N/L and 125.3 mg
COD/L on average respectively. The system was
composed of a rectangular compartment divided by
baffles to form six-compartment reactor with the last
one operated as a clarifier. Mixing was provided by
mechanical mixers and aeration was supplied by an air
compressor through an air diffuser inside the reactor.
The system was inoculated with seed sludge taken
from a municipal wastewater treatment plant with SS
concentration of 2820–3100 mg/L and VSS to SS ratio
of 83%. Temperature was maintained at 24 °C and pH
varied from 7.12 to 7.43. The system was divided to
six phases alternating aerobic, anaerobic and anoxic
zones. It was run for six successive runs. The first two
runs were implemented to investigate nitrite accumu-
lation throughout a combination of short HRTs of
9.1 h with normal DO of 1.5–2 mg/L concentration to
compare it with runs number IV and V. Run number III
was to investigate nitrite accumulation rate under a
very low DO concentration of 0.2 mg/L. Run number
IV and V was performed to investigate the influence of
a combination of low DO of 0.4–0.5 mg/L with low
HRT of 9.1 h control on partial nitrification perfor-
mance. The SRT was extended from 13 to 16 days in
Run number VI to investigate SRT effect on stable per-
formance of partial nitrification. It was noted that in
runs number I and II at normal DO levels, the system
showed very good complete nitrification whereas the
nitrite accumulation was almost unobvious during
these runs. Subsequently, nitrification deteriorated in
run number III at DO concentration of 0.2 mg/L and
also nitrite build up did not occur. Partial nitrification
via nitrite was successfully achieved during run
number IV with nitrite accumulation rate of 95%,
however short HRT of 9.1 h and SRT of 10 days
resulted in poor NH4 removal of 50%. Increasing HRT
to 13 h and SRT to 13 days enhanced NH4 removal to
over 97% with nitrite accumulation rate stabilized at
over 94%. However, the extension of SRT to 16 days
resulted in a decline in the nitrite accumulation rate to
less than 44%.
Moreover, in an activated sludge pilot plant treating
a high strength synthetic wastewater mimicking reject
water with a configuration of three continuous reactors
in series plus a settler partial nitrification was achieved
through a combination of free ammonia inhibition and
DO limitation linked to a properly selected SRT for the
selective washout of NOB (Torà et al. 2012). The
synthetic influent mimicked the reject water from the
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dewatering process of anaerobic digested sludge with
high ammonium concentration and low COD concen-
tration of 1150 ± 150 mg NH4-N/L and 30–35 mg
COD/L respectively. The system was inoculated with
activated sludge of a municipal wastewater treatment
plant with total biomass concentration of 2100 mg
VSS/L composed of 97 ± 2% heterotrophs,
2 ± 0.5% AOBs and \ 1% NOB. The three reactors
were connected in series and worked under complete
mixing conditions. A fraction of reactor 3 effluent was
recycled to reactor 1 (internal recycle) as well as an
external recycle from the settling tank to reactor 1 to
maintain the biomass concentration in the reactors.
Temperature, SRT and DO concentration were main-
tained at 30 °C, 8 ± 3 days and 2.0 mg/L respec-
tively. The pH was controlled at 8.3 ± 0.1 in reactor 1
and 2 to increase the fraction of free ammonia and
maintained at 8.2 in reactor 3. The start-up of partial
nitrification from the activated sludge with the low
percentage of nitrifying bacteria was achieved in
30 days resulting in a nitrifying system with a biomass
concentration of 1200 mg VSS/L and an AOB fraction
of 72 ± 10%. It was suggested that the decrease in
biomass concentration from 2100 to 1200 mg VSS/L
was due to the decay of heterotrophic bacteria because
of the low COD concentration in the effluent. After the
start-up period, the system was successfully operated
for 800 days and it was noted that the inlet ammonium
was fully oxidized to nitrite during the whole opera-
tion period except for some short periods when nitrite
accumulation rate decreased to 70%. The nitrate
accumulation during these periods was suggested to
be caused by an SRT increase in the system due to the
unexpected improvement in settling properties which
increased the biomass concentrations in the reactors
and by consequence allowed NOB growth. Other than
that the AOB fraction was maintained around
80 ± 7% while the NOB population was around \ 1
± 0.4% during the long term operation. A high
volumetric ammonium nitrogen oxidation rate of
2.0 ± 0.4 g N/L/d was achieved in this system.
5 Attached partial nitrification technologies
In conventional treatment systems bacteria are grown
in flocs which refer to an assemblage of individual
cells or micro colonies that take place in a reactor
under particular conditions or after the addition of an
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agent to the medium (Boonaert et al. 2002). These
flocs are usually prone to be washed-out easily which
could be a restraint for slow-growing bacteria like
AOB. An alternate way for bacteria growth are
biofilms systems. A biofilm is a complex coherent
structure of cells and cellular products which can grow
as large, dense granules or attached on a static solid
surface or attached on suspended carriers (Nicolella
et al. 2010). In biofilm systems, substrate (e.g. oxygen
and nitrogen sources) have to cross the biofilm–liquid
interface by diffusion to reach the microbial cells and
be consumed. The depth of layer diffused by the
substrate depends on the biofilm porosity, substrate
concentration, mass transfer in the aggregate–liquid
interface and the biofilm reaction rate. As a result,
conditions in biofilm reactors are not homogeneous
and by consequence organisms in the biofilm experi-
ence different conditions depending on the distance
from the biofilm surface according to the diffusion
gradients. In multi-species biofilm systems, this will
lead to a biofilm with a layered structure, giving
species with different ecophysiological characteristics
the opportunity to survive. In this layered structure,
organisms with higher growth rate like NOB in
nitrification process will be found at the outside layer
of the biofilm, whereas organisms with slower growth
rate such as AOB will be found in the inside layer
(Heijnen et al. 1989). As a result slower growing
organisms will be more protected from external shear
forces and are less likely to be washed out through
detachment, hence AOBs are often grown in biofilm
systems.
Compared to suspended growth systems, attached
growth systems have extra advantages such as (1)
higher settling velocity of solids (around 50 m/h
compared to 5 m/h for suspended growth systems)
which may lead to the elimination of clarification
stage, (2) smaller area requirements (3) higher
biomass concentration can be retained in the bioreac-
tor without the need of biomass and effluent separation
(around 30 kg/m3 compared to 3 kg/m3 for suspended
growth systems), and (4) higher sludge age with lower
sludge production (several weeks). However, attached
growth technologies might encounter some challenges
such as (1) clogging of media pores due to biofilm
growth, (2) long start-up period due to biofilm
formation, (3) controlling biofilm thickness is difficult,
and (4) controlling substrate concentration and
biomass distribution gradients due to larger size of
Rev Environ Sci Biotechnol (2018) 17:285–321
biofilms (usually 0.5–3.0 mm compared to flocs
usually 10–150 lm) and lower porosity which make
the diffusional transport slower (Nicolella et al. 2010).
Attached growth systems are commonly used in
biological treatment in the following cases (Van
Loosdrecht and Heijnen 1993):
1. Diluted wastewater with high flowrates are to be
treated due to high biomass retention in these
systems. Otherwise in high substrate concentra-
tion ([ 10 g COD/L) and rapidly growing organ-
isms biofilm formation is unnecessary as
sufficient biomass will be formed to metabolize
the substrate within short residence time.
2. Microorganisms which readily form biofilms are
used
3. Processes that need to be operated with high
biomass concentration without using settlers and
biomass recirculation.
5.1 Biofilm processes
Biofilm reactors could be either fixed film reactors
(static biofilms) like trickling filter—the oldest form of
biofilm reactors—, membrane bioreactors (MBR) or
suspended carriers reactors (particulate biofilms) like
moving bed biofilm ractor (MBBR), biofilm airlift
suspension (BAS), biofilm up flow sludge blanket
(USB) (Fig. 5).
5.1.1 Fixed film reactors (static biofilm)
In fixed film reactors, the biomass is attached to the
carrier that is retained fixed in the reactor. These
systems are characterized by their simple configura-
tion and low maintenance cost. On the other hand, the
biofilm surface area of these reactors does not exceed
200 m2/m3.
5.1.1.1 Membrane bioreactor (MBR) Membrane
bioreactor (MBR) technology (Fig. 5a) is a reliable
process for wastewater treatment that has become
increasingly used in the past decade to overcome many
of the limitations of conventional systems. These
systems allows a higher biomass concentration to be
maintained allowing smaller reactors to be used
compared to conventional systems. Moreover, MBRs
have been often operated with long SRTs which
results in less sludge production. However, these
303
systems have some drawbacks mainly for their high
operating costs requirements. These costs include
membrane cleaning to mitigate the fouling concerns,
the energy costs for air scouring to control bacterial
growth on membrane surface and the possible usage of
chemicals to produce biosolids acceptable for disposal
to overcome the settleability issues.
MBRs systems have been adopted widely for
nitrogen removal process. These systems avoid cell
washout by maintaining complete biomass in the
reactor which favors the growth of nitrifying bacteria
and by consequence increase the nitrification effi-
ciency. Shen et al. (2014) investigated in their
experiments the performance of a nitritation mem-
brane bioreactor treating synthetic wastewater devoid
of organic carbon at 30 °C (Shen et al. 2014). An
ammonium conversation rate of about 0.8 kg N/
(m3 d) was achieved through controlling DO concen-
trations in the range of 0.5–0.8 mg/L and pH in the
range of 8.0–8.5. A hollow fiber ultrafiltration mem-
brane module of 0.01 lm pore size and 0.1 m2
effective area was submerged inside the reactor. It
was physically flushed with highly pressurized water
when the fouling rates were low and chemically
cleaned the later days. The reactor was operated at
HRT of 10 h and at a prolonged SRT. Different
nitrogen loading rate were applied through different
ammonium concentrations in the effluent. It was noted
that nitrate dominated rather than nitrite during lower
loading rate in the startup period which was
attributable to the presence of NOB which decreased
gradually with the increase of the loading rates most
likely due to the low and steady levels of DO
(0.3–0.5 mg/L) which inhibited the proliferation of
NOB and by consequence resulted in nitrite accumu-
lation. AOB domination in the reactor could be
implied from the significant decrease of VSS concen-
trations observed in the first days of operations which
is most likely referred to the extent loss of NOB. It was
suggested that DO concentrations (0.5–0.8 mg/L) and
pH (8.0–8.5) are significant key parameters for the
NOB inhibitions. On average, 82% of the ammonium
was converted to nitrite in this MBR rector. Although,
increasing ammonium loading rates resulted in nitrite
accumulation higher levels of nitrate were observed
after increasing the loading rates to a certain level
(when the ammonium concentrations in the influent
were raised to higher than 400 mg/L).
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Media in suspension
Membrane
(a) modules
(b)
Effluent Effluent

Influent

RAS
Influent
WAS

(c)

Effluent Effluent
Downer

Downer
Riser

Influent Excess sludge

Fig. 5 Biofilm reactor configurations. a MBR; b MBBR; c BAS

Moreover, the effect of different nitrogen loading
rates on AOB community was studied in a MBR
treating anaerobically digested swine wastewater
which is characterized by its high ammonium con-
centration and low carbon to nitrogen ratio (Sui et al.
2014). The reactor was divided into two different
zones a biofilm zone and a membrane zone with a
recycle ratio of 400% between both zones. The biofilm
zone was not aerated and was filled with polyethylene
carriers of 95% porosity and 500 m2/m3 surface area
with a filling ratio of 50%. A PVDF membrane of
0.1 lm pore size and 0.14 m2 was installed in the
membrane zone which was physically cleaned with
tap water and then submerged in NaClO3 solution for
the recovery of the flux. Water temperature was
controlled at 25 °C while pH was not controlled but it
was found to be in the range of 7.52–8.51 in the
effluent. The reactor was operated under 4 stages with

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different total nitrogen loading rate 0.27, 0.11, 0.06
and 0.06 kg N/(m3 d) respectively and different COD
to TN ratio and BOD to TN that ranged from 1.78 and
0.3 in lower ammonium loading rates to 8.76 and 3.02
in higher ammonium loading rate respectively. The
corresponding HRT for the four stages was 8, 8, 5 and
3 days respectively. SRT was maintained at 90 days
on average by discharging an amount of the SS from
the membrane zone every day. The biofilm zone was
not aerated and low DO concentration was detected
due to the recycle flux for the membrane zone while
DO concentrations was high in the membrane zone
([ 5.0 mg/L) for membrane scouring purposes. It was
noted that ammonium removal rate were higher at
lower loading rates reaching 99.63% in stage IV most
likely due to the increase in COD/TN and BOD/TN
ratios to 8.76 ± 0.30 and 3.02 ± 0.09 respectively
which was in the optimal range reported by Zhang
et al. (2013) and Kishida et al. (2003) for achieving
highly efficient removal of TN for swine wastewater
treatment (3.0–4.5 for BPD/TN) (Kishida et al. 2003;
Zhang et al. 2013). On the other hand, FA concentra-
tions were lower than the range reported in the
literature. Moreover, the ammonium nitrogen concen-
trations had a significant influence on the AOB
diversity. Nitrosomonas eutropha and Nitrosomonas
sp. OZK11 were the dominant AOB species during the
experiment. At high ammonium concentrations, N.
eutropha was the dominant AOB species while with
the decrease of ammonium concentrations and the
increase of C/N ratio the AOB community diversity
decreased where N. eutropha started to disappear and
Nitrosomonas sp. OZK11 became the dominant AOB
species and played a significant role in oxidizing
ammonium. The aforementioned effect could be
referred to the low affinity for ammonium of N.
eutropha which allowed it to perform under high
ammonium concentration but when the ammonium
concentration in the influent decreased it was gradu-
ally eliminated and only Nitrosomonas sp. OZK11
with high affinity for ammonium had high conversion
efficiency.
5.1.1.2 Fixed bed bioreactor (FBB) The
performance of an up-flow fixed film bioreactor
treating low C/N synthetic wastewater in partial
nitrification was investigated under oxygen limiting
conditions (Bagchi et al. 2009). The FBB was filled
with a media of uniformly sized pieces of refractory
305
bricks. The reactor was seeded with biomass from an
activated sludge process treating nitrogenous coke
wastewater and fed with synthetic wastewater devoid
of any organic carbon. The reactor was initially
operated under fed batch mode at HRT of 12 h for the
development of the biofilm for 90 days then switched
to continuous feeding mode for the rest of the
experiment. The reactor temperature and pH were
controlled at 30 ± 2 °C and 7.5 ± 0.2 respectively.
The reactor was operated under three consecutive
stages. During stage I (startup period), the reactor was
fed with ammonia and nitrite at a ratio of 1:2 and the
DO in the feed was 4.2 ± 0.3 mg/L. Complete
nitrification was achieved and no nitrite
accumulation occurred in the reactor. During stage
II, ammonia loading rate was increased and nitrite
loading rate was decreased gradually till it was
eliminated in the feed to reduce the substrate for
NOB and to make the entire DO in the feed available
for AOB and by consequence washout NOB. As a
result, ammonia oxidation rate increased however
nitrite oxidation rate also increased even after the
elimination of nitrite loading in the feed which
indicates the occurrence of complete nitrification.
That was attributed to the arrangement of AOB and
NOB in the biofilm. AOB located in the outer layer
consumes ammonia from the feeding and converts it
into nitrite which diffuses into the inner layers of
biofilm where NOB are located which by its turn
convert it into nitrate. Stage III aimed to achieve
partial nitrification through reduce DO concentrations
in the feed. Firstly, DO was reduced from 4.2 ± 0.3 to
2.2 ± 0.3 mg/L which resulted in the increase of
nitrite accumulation rate from 11 to 65% of the total
nitrite and nitrate however a significant amount of
nitrate was still present in the effluent. Therefore, the
feed DO concentration was further reduced to
1 ± 0.1 mg/L. A dramatic fall in ammonia oxidation
rate was noted with the reduction of DO but after
3 months of operation it recovered gradually. This
could be referred to a reduction in AOB and NOB
activity due to the sudden DO limiting conditions but
after a period of operation AOB got adapted to the
limitation of DO and recovered its activity.
Subsequently, the effect of HRT under DO limiting
conditions was investigated and HRT of 18 h was
revealed to be the optimum. In the end of stage III,
ammonia oxidation rate efficiency reached more than
90% and a nitrite accumulation rate of 85% was
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reached in the final effluent. It can be revealed from
this study that DO concentration has a crucial effect in
the proliferation of AOB and washout of NOB.
Moreover, Liang et al. (2011) conducted their
experiment to optimize partial nitrification in a fixed
bed biofilm reactor treating synthetic wastewater
(Liang et al. 2011). The reactor was packed to 80%
capacity with a mixed carrier material of hollow
polyhedron polyethylene ball of 460 m2/m3 specific
area and sponge rectangle cubes of 60–80 pores per
inch at a 1:1 volumetric ratio. The reactor was seeded
with sludge from an ethanol wastewater treatment
plant and the carrier media was inoculated in that seed
for 48 h. The reactor was fed from the top of the mixed
culture with synthetic wastewater with NH4Cl as main
substrate along with other minerals devoid of any
organic matter except for the start-up period. Tem-
perature was controlled at 30 ± 1 °C. The experiment
was run under three stages. The first stage (startup
period) aimed to cultivate the nitrifying biofilm on the
surface of the media. To accelerate the biofilm
formation, glucose was added to the feed to stimulate
heterotrophic biofilm formation and by consequence
providing nitrifying biofilm a surface to form on.
Moreover glucose is biologically metabolized and
produce carbon dioxide which is used by nitrifiers as
carbon source. As expected, glucose stimulated the
nitrifying biofilm formation and after a short period a
thin layer of biofilm was observed on the media
surface. Subsequently, ammonia concentration in the
feed was increased gradually from 41.6–262.6 mg/L
at the end of the stage. DO concentration and pH were
maintained during the whole stage at 0.5–0.8 mg/L
and 7.8 ± 0.2 respectively. At ammonia concentra-
tion in the range of 41.6–103.5 mg/L and HRT of
12 h, no nitrite accumulation occurred. After raising
ammonia concentration to the range of
115.8–163.2 mg/L and decreased the HRT to 10 h,
nitrite started accumulation and reached 83% of the
total nitrite and nitrate produced. A further increase of
ammonia concentration to 206.4–262.6 mg/L resulted
in the conversion of the majority of ammonia to nitrite
which its accumulation rate was above 85% of the total
nitrite and nitrate in the effluent. The second stage
aimed to investigate the effect of HRT and alkalinity
on partial nitrification. To evaluate the HRT effect, the
reactor was operated under 3 HRT of 16, 12 and 8 h
through increasing influent volume with an ammonia
concentration of 130 mg/L. A maximum ammonia
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oxidation to nitrite of 75% was reached at HRT of 16 h
while decreasing the HRT resulted in lower nitrite
accumulation. Moreover, the reactor was operated
under enough, half and zero alkalinities in the influent.
It was revealed that alkalinity has a significant effect
on nitrite accumulation. At significant alkalinity,
ammonia removal efficiency reached 80% and nitrite
to ammonia ratio reached 3.4 whereas decreasing
alkalinity to half resulted in a drop in ammonia
removal to 58% as well as a decrease in nitrite to
ammonia ratio in the range of 1.06–0.97. Furthermore,
zero alkalinity leaded to a sharp drop in ammonia
removal to reach 33%. This effect could be referred to
the decrease of pH in the reactor which accompanied
the decrease of alkalinity and inhibited the AOB
activity.
5.1.2 Suspended carriers reactors (particulate
biofilm)
The alternate system for the growth of the biomass is
to be formed attached to carriers that are kept in
suspension (particulate biofilms). This suspension
cannot be attained using mechanical mixing due to
the high shear forces around the stirrer blades that may
damage the biofilm formation (Van Loosdrecht and
Heijnen 1993). Some examples for these types of
reactors are Biofilm fluidized bed reactors (BFB),
biofilm upflow sludge blanket (USB), biofilm airlift
suspension (BAS) and moving bed biofilm reactors
(MBBR). IN BFB and USB biofilm are kept in
suspension through the upflowing influent while in
BAS suspension is maintained through pumping air to
the system. For anaerobic processes, BFB and USB are
most commonly used whereas in aerobic processes
BAS and MBBR are most feasible.
5.1.2.1 Moving bed biofilm reactor
(MBBR) Moving Bed Biofilm (MBBR) reactors
(Fig. 5b) have been increasingly adopted for
nitrogen removal in wastewater treatment. The
MBBR process is a biofilm process that combines
advantages of both suspended growth systems and
conventional fixed film reactors. The biomass can
either reside in suspended microbial assemblages as
flocs or as biofilm attached to the media. Another
advantage of MBBR is that the reactor liquid is totally
mixed which eliminate the presence of unused space
and the need for recycled sludge. The filling fraction of
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carriers inside the reactor may be controlled but it is
recommended that it does not exceed 70% to allow the
carriers to move freely inside the reactor (Rusten et al.
2006).
For the evaluation of MBBRs performance, partial
nitrification–denitrification process has been used for
synthetic wastewater treatment in two MBBRs in
series one anoxic for partial denitrification process and
the other aerobic for partial nitrification process
followed by a settler (Zafarzadeh et al. 2010). An
average removal efficiency of total nitrogen and
ammonia of 98.23 and 99.75% respectively was
reached during high ammonium load and low oxygen
concentration. Polyethylene carriers of 0.95 g/cm3
density were used with a 50% filling ratio in the
aerobic reactor which allowed a specific biofilm
surface area of 250 m2/m3 and a total biofilm surface
area of 2.5 m2. Complete mixing was provided by a
mechanical stirrer and temperature was kept at
28.5 ± 1 °C using a water bath. The system was
seeded from a municipal wastewater treatment plant
and fed with synthetic wastewater with COD, NH4 and
PO4–P concentrations of 300–2000, 25–250 and
5–50 mg/L respectively. For startup period, the reac-
tor was operated at low ammonium loading rate
(25 mg-N/L) and DO concentration at the range of
1.0–1.5 mg/L at HRT of 20 h. A gradual increase of
ammonium was noticed which represented an indica-
tion for the growth of nitrifying bacteria. To test the
effect of DO concentration and ammonia loading rate
on partial nitrification, DO ranged in the aerobic from
0.5 to 3.3 mg/L and ammonium loading rate from 0.1
to 4.43 g N/(m2 d). The maximum ammonium
removal rate was 2.98 g N/m2 d and was reached
when the influent NH4–N concentration on the biofilm
surface area in the aerobic reactor was 4.43 g NH4–N/
(m2 d) at ammonium loading rate of 250 mg–N/L and
DO concentration in the range of 1.0–1.5 mg/L. At the
aforementioned conditions, nitrite accumulation rate
reached 83% in the aerobic reactor. It was noted that
although increasing ammonium loading rate resulted
in an increase in ammonium removal rate, at some
point increasing ammonium loading rate caused a
deterioration in nitrification rate. The aforementioned
deterioration is referred to the increase in free
ammonia (FA) and free nitrous acid (FNA)
concentrations.
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5.1.2.2 Biofilm airlift suspension (BAS) Airlift
reactors is usually composed of two connected parts,
a riser and a downcomer (Fig. 5c). Gas is pumped
from to the bottom and moves upward till it exists from
the top through riser. Then air recirculate through the
downcomer and provide aeration inside the reactor.
The difference in density between the riser and the
downcomer is what makes the liquid circulate between
the two parts (Nicolella et al. 2000). BAS reactors are
preferred in aerobic processes over aerated Fluidized
Bed Bioreactors (FBBR) reactors due to its simple
configuration for providing aeration inside the reactor.
In FBBR, large amounts of oxygenated water must be
recirculated over the bed which usually causes
hydraulic problems beside extra pumping costs.
Choi and Ahn (2014) compared the performance
of a biofilm airlift suspension reactor and a
suspended growth CSTR in partial nitrification
treating wastewater produced from the dewatering
of anaerobically digested sludge (Choi and Ahn
2014). The suspended growth reactor (SG) was
designed as CSTR without recycling whereas the
attached growth reactor (AG) with crumbled tires
and activated carbon media with 1500 m2/m3
surface area and 0.0958 g of average particle weight
at a filling ratio of 50%. The two reactors were
seeded with activated sludge from a municipal
wastewater treatment plant with a VSS to TSS ratio
of 75%. Aeration was provided from the bottom of
the reactor through air diffuser at 200 mL/min.
Temperature was controlled in both reactors at
30 ± 2 °C whereas pH and DO were not controlled.
The alkalinity to ammonia ratio was around 3.9 mg
CaCO3/mg NH4–N in the influent. Both reactors
were run at three phases with different nitrogen
loading rate ranging from 394 ± 12 to
1188 ± 9 mg N/L d for SG reactor and 788 ± 24
to 2376 ± 18 mg N/L d for AG rector. HRT was
controlled for to three phases at 24, 16, and 8 h for
the SG reactor and 12, 6 and 4 h for the AG reactor
respectively. During the three phases, the nitrogen
production rate was 58–255 mg NO2-N/L (nitrite
accumulation rate (NAR) = 14.7–64.7%) for SG
reactor and 145–290 mg NO2-N/L for AG reactor
(NAR = 36.7–73.7%) while the nitrate production
rate for both reactors was 3–49 mg NO3-N/L and
10–63 mg NO3-N/L for SG reactor and AG reactor
respectively. The maximum nitrite accumulation rate
for SG reactor was 64.7 ± 12.3% and occurred at
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NLR of 394 ± 12 mg N/(L d) and HRT of 24 h
whereas the maximum nitrite accumulation rate was
73.7 ± 4.5% and occurred at NLR of 788 ± 24 mg
N/(L d) and HRT of 12 h. The pH and DO in the
effluent of AG reactor were in the range of
6.07 ± 0.23–7.89 ± 0.11 and 3.90 ± 0.18–6.11 ±
0.51 mg O2/L respectively which was lower than
that in SG reactor which was in the range of
7.15 ± 1.07–8.51 ± 0.09 and 5.92 ± 0.21–6.19 ±
0.29 mg O2/L for pH and DO respectively. It was
noted that a stable biofilm formation occurred in the
AG reactor a higher AOB biomass retention than of
that of SG reactor which resulted in more ammonia
and bicarbonate alkalinity in AG reactor than in SG
reactor. On the other hand, it was noted that AG
reactor produced 66% less biomass in the effluent
than the SG reactor. From the aforementioned
results, it was deduced that AG reactor provided a
higher nitrite accumulation rate than SG reactor
under same configuration most probably due to the
higher biomass retention, higher substrate rate and
mass transfer. It also was suggested that the
optimum nitrogen loading is 0.42 g N/(L d) for the
SG reactor and 0.76 g N/(L d) for the AG reactor.
5.2 Granular processes
Aerobic granular sludge can be used as an alternative
technology to conventional activated sludge processes
in wastewater treatment. Aerobic granulation was first
reported by Mishima and Nakamura (1991) in an
aerobic upflow sludge blanket reactor treating munic-
ipal wastewater (Mishima and Nakamura 1991).
Aerobic granules are denser aggregates with higher
diameter and density compared to conventional flocs
which allow a faster settling and by consequence a
higher level of biomass retention. The aforementioned
characteristics lead to a reduction of capacity require-
ments and the ability to treat wastewater with higher
loading rates without the need of external settler due to
the ease of biomass separation in the same reactor.
However, granular reactors have some drawbacks in
the stability of long term operations. Aerobic granules
can be a promising technology to achieve partial
nitrification if appropriate configuration is to be
developed. Aerobic granulation for achieving partial
nitrification and accumulating AOB could be operated
in either SBR reactors or CSTR reactors.
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5.2.1 SBR granular reactors
Recently, achieving partial nitrification through using
aerobic granulation technology in SBR have been
adopted by several studies. Li et al. (2013a, b) adopted
aerobic granulation technology to cultivate granule
sludge for accumulating AOB in a SBR reactor (Li
et al. 2013a, b). The SBR reactor had a cycle of 12 h
consisted of 1 min of feeding, 11 h and 53–57 min of
aeration, 1–5 min of sludge settling depending on
sludge settling properties and amount of biomass
required to be discharged and 1 min of effluent
withdrawal. After each cycle slow settling flocs were
discharged to avoid the competition between these
small sludge flocs in suspended growth and dense
granules for substrate uptake and make the substrate
more available for uptake by the attached growth
dense granules, hence stimulating the granulation. The
amount of biomass discharged was calculated to
maintain a VSS concentration of 2000 mg/L in the
bioreactor. A 30 min of settling after flocs discharging
was allowed for the remaining sludge to settle before
effluent withdrawal. The bioreactor was seeded with
nitrifying activated sludge from a fermentation pro-
cess and fed with synthetic wastewater with ammo-
nium and phosphate concentration of 400 mg N/L and
30 mg P/L. Furthermore, filtered clean seawater was
added to the medium to increase its salinity to 1% to
supply inorganic salts for the biomass and no organic
substrate was added in the feed. DO concentration, pH
and temperature were controlled at 2–4 mg/L, 7.5 and
20–22 °C respectively. After 2 weeks of operation,
the granulation formation was clear and the mean size
of the sludge increased from 181 to 250 lm and it
continued to increase gradually till it reached around
330 lm which was probably due to the selective
discharge of loose sludge flocs. Moreover, F/M ratio
was adjusted during different stages of cultivation, it
was increased in the first stage to fasten granules
formation then reduced to allow stabilization of
smaller granules. It was noted that before granules
formation complete nitrification took place in the
bioreactor with low level of nitrite in the effluent. With
the granules formation, partial nitrification was
achieved with over than 90% nitrite accumulation in
the effluent. The larger size of granules led to a lower
DO concentration within the granules which promoted
AOB accumulation. The overall ammonia removal
rate was 99% with only 4 mg N/L of ammonia
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concentration in the effluent. The results of this
experiment revealed that aerobic granulation could
lead to nitrite accumulation through the selective of
slow settling sludge flocs and high ammonia loading in
the influent.
5.2.2 CSTR granular reactors
Regarding the poor stability of aerobic granulation
processes in long term operation, continuous flow
process could be preponderant than SBR in partial
nitrification. Wan et al. (2014) applied a novel strategy
to achieve stable partial nitrification in a continuous
flow reactor using aerobic granules after being culti-
vated in a SBR reactor. First, granules were cultivated
in a SBR reactor ay high COD of 3:1 acetate:
propionate. The reactor was inoculated with seed
sludge collected from a recycled sludge with SS of
6000 mg/L and fed with synthetic wastewater that
contains 200 g/L NH4Cl as ammonia source. The
reactor was run for 6 cycles a day, each cycle consisted
of 3 min feed, 227 min aerobic reaction and settling,
5 min decanting and 5 min idle. After 16 days of
cultivation, aerobic granules were inoculated to a
continuous flow reactor with SS of 820 ± 30 mg/L.
The reactor with same feed as the parent reactor but
with variable COD during operation. DO, pH, HRT
and temperature were kept at 7 mg/L, 7.2 ± 0.1, 12 h
and 28 ± 1 °C respectively. The CSTR were operated
in 3 stages of 20 days each at influent COD of
1500 ± 100, 750 ± 50 and 350 ± 50 mg/L. During
first stage, ammonia removal rate increased gradually
till it reached 60% with nitrite accumulation of
88–96% of the total nitrite and nitrate produced. In
stage II, decreasing C/N ratio from 28/1 to 14/1
resulted in enriching AOB and washing out NOB
however partial nitrification was not enhanced. After
decreasing COD is stage III, 400 ± 50 mg/L HCO3
was added as inorganic source of carbon to maintain
total carbon to nitrogen ratio at 14/1 which resulted in
an increase in partial nitrification to reach 85–90%
after 6 days. In cultivation stage in SBR reactor,
almost no AOB was present but it started to accumu-
late after a period of operation in continuous flow
reactor. High COD in the initial period of operation
stimulated the granule formation then decreasing its
concentration and adding instead inorganic carbon
source resulted in loosing part of the granules but
enriched AOB and inhibited NOB in the remaining
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granules which led to achieving partial nitrification in
a short period of 52 days (16 days in SBR and 36 days
in CSTR.
6 Full scale side-stream partial nitrification
systems
Over the last decade, numerous partial nitrification and
anammox technologies have been studied and some of
them have been implemented at full scale facilities as
an economical and environmental friendly way for
removing nitrogen from wastewater. Up till now, more
than 110 full scale shortcut BNR systems have been in
operation worldwide treating wastewater with high
nitrogen content such as reject water, landfill leachate
and industrial wastewater using different configura-
tions including Completely Autotrophic Nitrogen
removal Over Nitrite (CANON) process, Single
reactor for High activity Ammonia Removal Over
Nitrite (SHARON) process, Deammonification
(DEMON) process, Oxygen Limited Autotrophic
Nitrification Denitrification (OLAND) process, Single
stage Nitrogen removal using Anammox and Partial
nitrification (SNAP) process, and others (Mao et al.
2017).
The main difference among these configurations is
the number of stages used (whether it is a one-stage
system or two-stage system). In two-stage systems,
partial nitrification and Anammox (or partial denitri-
fication) are physically separated in two different
bioreactors controlled at different conditions such as
DO, temperature, pH and SRT. The first bioreactor is
designed at conditions that simulate the growth of
AOB to conduct partial nitrification with complete
ammonia oxidation to nitrite if its effluent is being fed
to a downstream partial denitrification system where
the nitrite is denitrified to nitrogen by heterotrophs or
50% partial nitrification with only half of the ammonia
oxidized to nitrite in the case of a downstream
anammox system where ammonia is oxidized anaer-
obically to nitrogen gas using autotrophic Anammox
bacteria. Examples of two-stage system include
SHARON and partial nitrification–anammox pro-
cesses. In term of reactor configuration, in full scale
two-stage systems typically the first partial nitrifica-
tion bioreactor is designed as a CSTR without sludge
retention (chemostat) such as the SHARON process or
a SBR while the second bioreactor is configured as an
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upflow reactor or a moving bed biofilm reactor
(MBBR) to decouple the HRT and the SRT in order
to maintain high concentration of the slow growth
anammox bacteria (Jaroszynski and Oleszkiewicz
2011).
On the other hand, in one-stage systems both partial
nitrification and anammox reaction are occurring in
the same bioreactor, however maintaining the coex-
istence of two different functional bacteria such as the
aerobic AOB and the anaerobic AOB (anammox
bacteria) is a challenging step towards the success of
these systems. In this sense, partial nitrification and
anammox reactions can be achieved in one stage by
alternating the aeration conditions using a SBR reactor
such as DEMON process or by using biofilm environ-
ment in a granular or attached growth system such as
CANON process (Muñoz-Palazon et al. 2018). In the
first case of SBR reactors, partial nitrification and
anammox occurs subsequently by cycling the aeration
between aeration and no aeration to enable the aerobic
ammonia oxidation to nitrite by aerobic AOB and
anaerobic remaining ammonia oxidation to nitrogen
by anammox bacteria, respectively. Whereas in
attached and granular case, layered biofilm structure
enable aerobic AOB to grow on the outside layer
where oxygen is abundant and protect anammox
bacteria by letting them grow in an oxygen deficient
environment in the inner layer resulting in partial
nitrification and anammox reactions occurring in the
same time Examples of one-stage system include
DEMON, OLAND and SNAP and commonly is
performed in a SBR or MBBR configuration
(Jaroszynski and Oleszkiewicz 2011).
Each of the aforementioned two configurations
comprises some advantages as well as drawbacks. A
one-stage system provides lower construction cost,
ability of treating high loads and avoids the accumu-
lation of high nitrite concentrations which might have
an inhibitory effect on the biomass as both partial
nitrification and anammox are occurring simultane-
ously, however, it needs longer start-up periods and
lower ability to recover from disturbance due to the
complexity of the microbial population in the system.
In contrast, a two-stage system is easier to operate,
provides the flexibility of controlling each process at
different conditions and has higher potential of
recovery in less time, but on the other hand on a
commercial point of view it might not be the better
option as it includes higher capital costs (Jaroszynski
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and Oleszkiewicz 2011; Joss et al. 2009; Veys et al.
2010; Wyffels et al. 2004). Early full scale implemen-
tations were towards two-stage system due to its ease
of operation and control over partial nitrification
which was considered as the limiting step in the
shortcut BNR as well to make used of the already
existing partial nitrification bioreactors such as
SHARON reactors, however recently this interest
has shifted to one-stage systems (Lackner et al. 2014).
This section intends to provide a summary of the
existing full scale partial nitrification and anammox
systems that have been implemented in the last
decade, a discussion on the economic evaluation of
these technologies as well as some of the remaining
challenges facing the operation of the full scale
systems.
6.1 A survey on the existing implementation
of partial nitrification in full-scale
The first full-scale partial nitrification implementation
worldwide was attempted in 2002 at the Rotterdam
Dokhaven WWTP to treat rejected water from the
anaerobic digester (Mulder et al. 2001). Dokhaven
WWTP was originally designed to serve a population
of 47,000 in a two-stage process, each stage compris-
ing an aeration tank and a clarifier. The main objective
of the two stage process was BOD removal and
Nitrogen removal through conventional nitrification
and denitrification, however there was a shortage in
the carbon source required for denitrification which
resulted in poor performance. Moreover, the tanks
were constructed underground in the city center and
there was no room for extension. After performing a
study on the nitrogen compounds sources entering the
aeration tanks, it was found that a big portion of the
influent nitrogen concentration was coming from the
sludge digestion (rejected water), thus it was sug-
gested that treating this load separately would
decrease the influent concentrations and enhance the
WWTP’s performance. Meanwhile, the SHARON
process has been successfully developed and tested in
a lab scale in Delft University (Hellinga et al. 1998).
Thus, it was decided to scale up the technology
directly to full scale without the need of constructing a
pilot scale in order to treat the rejected water of
Dokhaven WWTP after performing a model based
studies (Hellinga et al. 1999). The detailed up-scale
process design was performed by the company
Rev Environ Sci Biotechnol (2018) 17:285–321
Grontmij Water and Waste Management in co-oper-
ation with the Water Authority ZHEW. The SHARON
reactor was constructed in the place of a post-thickener
with a total volume of 1800 m3 and was designed as a
single stage Partial Nitrification/Denitrification (PN/
D) process through intermittent aeration. The average
influent ammonia concentration was around
1230 mg N/L with a maximum of 1530 mg N/L
corresponding to a nitrogen load of 520 kg N/day
which exceeded the design value of 1000 mg N/L.
However, the reactor was able to achieve an ammonia
removal efficiency of 90% with effluent ammonia
concentration of 100 mg N/L. It was confirmed that
the ammonia removal was via the nitrite pathway
through the methanol demand for denitrification, the
theoretical ratio between the methanol added and the
nitrogen removal (including the biomass yield) is
3.5 g CH3OH/g N in the case of conventional nitrifi-
cation and 2.2 g CH3OH/g N in partial nitrification
(Hellinga et al. 1999). At the steady state, this ratio
was calculated and found to be 2.4 g CH3OH/g N
implying that denitrification occurred mainly via
nitrite. The results from the first full-scale SHARON
process proved the success of the scale-up of the
partial nitrification process.
Moreover after the discovery of the ANAMMOX
process in a denitrifying fluidized bed reactor treating
effluent from a methanogenic reactor (Mulder et al.
1995), lab-scale tests were performed in a 10 L
granular sludge SBR in order to scale up the technol-
ogy for application (Van Dongen et al. 2001). The
process was designed as a two-stage Sharon–Anam-
mox process, in which a total nitrogen load of
1.2 kg N/m3/d is treated in a Sharon reactor without
pH control to produce an effluent of 1:1 ammonia to
nitrite ratio suitable for the subsequent Anammox
reactor. The results showed that more than 80% of the
ammonia was oxidized to N2 and qPCR tests proved
that Planctomycete-like bacteria dominated the mixed
culture in the Anammox reactor with a low percentage
of aerobic AOB implying the success of the process.
Thus, the full scale implementation of the process was
initiated by Delft University and assigned to Paques to
scale it up in Rotterdam Dokhaven WWTP (Abma
et al. 2007). The Anammox reactor was constructed
with a total volume of 70 m3 and combined to the
already existing Sharon reactor. The operation of the
Sharon reactor was adjusted by the end of 2004 to
switch the process from a one-stage Partial
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nitrification/denitrification to a two-stage partial nitri-
fication/anammox (PN/A) by halting the methanol
addition to stop the partial denitrification and inhibit
the partial nitrification at 50% ammonia conversion to
nitrite in order to provide a suitable influent for the
subsequent Anammox reactor. A lamella separator
was added between the two reactors to remove
residual sludge and prevent any AOB accumulation
in the Anammox reactor. The Anammox reactor was
seeded with nitrifying sludge from the main line
reactor as there were no available anammox seed for
the inoculation of the reactor unlike the future
anammox reactors. Thus, the start-up period took
3.5 years which was even 1.5 years longer than
expected due to some problems with the operational
stability, biomass loss, high nitrite concentrations
causing toxicity, and mixing problems causing dead
zones. The influent load was increased gradually and
exceeded the design load of 500 kg N/d (7.1 kg N/m3/
d) to reach over 10 kg N/m3/d. The system was able to
maintain high ammonia removal efficiency of 90–95%
since the beginning of 2006 with effluent nitrite
concentrations as low as 5–10 mg N/L and effluent
ammonia concentration ranging from 60 to 130 mg N/
L indicating the success of the full scale anammox
granular sludge technology.
On the other hand, the first successful full scale one-
stage Partial nitrification/Anammox (PN/A) system
was the pH controlled deammonification system
(DEMON) applied at the WWTP Strass, Austria in
2004 (Wett 2006). The process consisted of a 500 m3
SBR treating reject water through pH controlled
intermittent aeration system that was built to replace
a functioning partial nitrification/denitrification (PN/
D) system which was operated since 1996 in order to
eliminate the need of external carbon addition (Wett
et al. 1998). The SBR had a total cycle duration of 8 h
consisting of filling, decanting, settling and 6 h
reaction where the partial nitrification and the anam-
mox reactions occur simultaneously. The length of the
aeration intervals during the reaction time were
controlled through the pH in a manner that when the
H? ions produced from nitritation consume all the
alkalinity and the pH starts to drop reaching the lower
set point, the aeration stops to allow the anammox
bacteria to start the anaerobic ammonia oxidation.
Afterwards, during the anammox reaction some of the
alkalinity is restored combined with the continuous
feeding of the alkaline reject water, the pH starts to rise
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again until it reaches the upper set point switching the
aeration on again for the partial nitrification reaction
(Wett and Rauch 2003). The SBR was controlled at
low DO concentrations of 0.3 mg/L during the aera-
tion period to suppress NOB activity and halt the
ammonia oxidation at the nitrite stage. The start-up of
the process took 2.5 years comprising 2 years of
enrichment period and 6 months of actual start-up of
the full scale SBR. The enrichment was performed
through as stepwise strategy using a 4 L seed from a
pilot scale in Zurich in a 300 L reactor until the
biomass reached a specified mass then it was used to
seed a 2.4 m3 reactor which was afterwards used to
seed the 500 m3 SBR. The influent ammonia concen-
tration was around 1800 mg N/L corresponding to a
loading rate of 340 kg N/d. The daily measurements
results showed that the reactor was able maintain an
ammonia removal efficiency and a total nitrogen
removal rate of 89.3 ± 1.2 and 83.9 ± 1.8%, respec-
tively indicating that the SBR was able to remove
approximately 300 kg of nitrogen per day (Wett
2007). In 2009, a hydrocyclon has been installed after
the SBR to control the SRT by separating the slowly
growing Anammox bacteria and AOB allowing the
system to wash out NOB benefiting from the higher
aggregate density of the anammox bacteria (Wett et al.
2010). Moreover, a 500 kg of TSS was taken from the
SBR to seed another 400 m3 SBR Demon system at
the WWTP Glarnerland, Switzerland in 2007 (Joss
et al. 2009). The transfer of the anammox enriched
inoculation resulted in a decrease in the start-up period
to 50 days and afterwards the SBR was able to reach
the target effluent ammonia concentrations of 50 mg/
L (Wett et al. 2010).
6.2 Life cycle assessment (LCA) studies applied
to full-scale partial nitrification systems
Recently, there has been a paradigm-shift in the look
to WWTPs from energy intensive facilities used to
remove organics, nutrient and harmful substances into
energy sufficient or even energy producing facilities.
Moreover, broader interests have been oriented to the
environmental sustainability of these WWTPs. Thus,
new technologies such as partial nitrification and
anammox need to be assessed with regards to their
economic evaluation, energy consumption, and envi-
ronmental impacts. A very useful tool to evaluate
these emerging technologies is life cycle assessment
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Rev Environ Sci Biotechnol (2018) 17:285–321
(LCA) which has been widely used since their first
application in WWTPs in 1995 (Emmerson et al.
1995).
Schaubroeck et al. (2015) have performed an LCA
study on Strass WWTP, Austria to compare between
partial nitrification/denitrification PN/D (scenario 1)
and partial nitrification/anammox PN/A (scenario 2)
in terms of electricity consumption and environmental
impacts (Schaubroeck et al. 2015). The results showed
that the electricity consumption for scenario 1 and 2
were 2.66 and 1.5 kWh/Kg N removed, respectively
indicating that anammox would result in around 44%
savings in energy compared to nitritation/denitritation
due to lower aeration requirements and avoided COD
consumption. The results were in accordance to what
have been reported by Lackner et al. (2014) that the
energy demand of anammox side-stream treatment
systems ranged from 0.8 to 2 kWh/kg N (Lackner
et al. 2014). Compared to conventional nitrifica-
tion/denitrification (N/D) systems which have been
reported to have an energy demand of 4 kWh/kg N,
PN/D implies 34% savings in energy consumption
against 63% for PN/A (Schaubroeck et al. 2015; Wett
et al. 2010). However, the LCA study on Strass
WWTP showed that N2O emissions produced from
both PN/D and PN/A scenarios were 7.8 and 1.3%
which are higher than those produced from conven-
tional N/D systems (lower than 0.01% of the N load).
The previous high N2O emissions can be referred to
the high nitrite accumulation—that is considered as a
precursor for N2O production—in the PN/D scenario
compared to PN/A and conventional systems (Chan-
dran et al. 2011; Soliman and Eldyasti 2016). In terms
of climate change effects, both scenarios had lower
overall greenhouse gas emissions about 18.5 and
5.5 kg CO2 equivalent/PE/year for scenario 1 and 2,
respectively compared to average reported WWTPs
applying conventional N/D of around 12–80 kg CO2
equivalent/PE/year due to the prevention of fossil CO2
emissions (Clauwaert et al. 2010; Hospido et al. 2008).
Overall, the LCA studies performed on Strass WWTP
proved that these new technologies are reliable in
terms of energy savings and environmental impacts
(climate change and eutrophication), however N2O
emissions should be decreased through better process
control.
Another LCA study was performed on the full scale
Dokhaven WWTP, Rotterdam over its complete life
cycle to compare the environmental impacts between
Rev Environ Sci Biotechnol (2018) 17:285–321
three different scenarios for treating reject water, (1)
two-stage PN/A (sc. 1), (2) one-stage PN/D (sc. 2), and
(3) recirculation of the reject water to the mainline to
be treated through conventional N/D (sc. 3) (Hauck
et al. 2016). In terms of overall performance, the plant
had a total nitrogen removal efficiency of around
85–90% for both the first two scenarios compared to
60% in sc. 3 which led to a decrease in the marine
eutrophication by 16%. However, climate change
impacts were higher for PN/A system by 9% com-
pared to conventional N/D due to higher N2O emis-
sions from the partial nitrification reactor and even
slightly more for PN/D due to the additional methanol
consumption. These results are in contrast to what
have been reported for single stage PN/A which can be
explained as in the latter case partial nitrification and
anammox reaction are occurring at the same time
which prevent nitrite accumulation and by conse-
quence decrease N2O emissions. In terms of economic
evaluation, the PN/D process gave an estimation of
1.1–1.7 $/kg N removed comparable to the two-stage
PN/A as it turned out that the cost of the second
Anammox construction balanced the costs of the
methanol addition and the extra aeration requirement
(Van Dongen et al. 2001). On the other hand,
conventional N/D gave an estimation of 2.8–5.6 $/
kg N removed whereas other physical–chemical pro-
cesses values are 5.60–14 $/kg N removed. The
previous results elucidate that PN/D and two-stage
PN/A implies savings of around 40–80 and 70–90%
when compared to conventional N/D and physi-
cal/chemical processes, respectively.
In general LCA studies confirmed that the imple-
mentation of partial nitrification and anammox tech-
nologies in the sidestream have induced numerous
benefits towards environmental benefits, however it
should be noted that further LCA studies should be
developed for better and more comprehensive
assessment.
7 AOB’s challenges and opportunities
7.1 AOB’s challenges
As previously discussed, several partial nitrification
systems have been reported during the last decades,
however there are some challenges encountering these
systems and hindering the implementation of partial
313
nitrification into full scale municipal wastewater
treatment plants. Those challenges are mostly associ-
ated with the suppression of NOB specially in
mainstream conditions where NOB growth are higher
than those of AOB such as low temperature and low
ammonia concentration. Moreover, mainstream
wastewater is characterized by its high COD:N ratio
which will promote the competition between hetero-
trophic bacteria and AOB. Other than the challenge of
applying partial nitrification to the mainstream line,
some more issues need to be extensively studied and
addressed towards the scale-up of the process such as
the filamentous bulking problems resulting from the
low aeration requirements of partial nitrification
systems and the low phosphorus removal resulting
from the inhibition of FNA on phosphate accumulat-
ing organisms (PAO).
7.1.1 Mainstream conditions challenge
Partial nitrification has been implemented success-
fully in more than 30 full-scale wastewater treatment
plants, however it was applied to the sidestream lines
to treat high strength nitrogen streams devoid of any
organic carbon (Vlaeminck et al. 2012). However,
sidestream represents only less than 30% of the total
ammonia in a WWTP, thus recent studies should be
focused more on partial nitrification at mainstream
conditions of low temperature and high COD: N ratio.
As discussed previously, one of the main strategies
to favor AOB growth over NOB was running the
system at high temperature to shift the equilibrium
between ammonium and ammonia towards free
ammonia which has an inhibitory effect on NOB at
lower values than that of AOB and consequently
facilitating the suppression of NOB. Thus, low influent
temperature represents a crucial challenge towards
maintaining stable nitrite accumulation, however
recently some studies reported achieving partial
nitrification at low and ambient temperature. Kouba
et al. (2017) reported successful partial nitrification of
real municipal wastewater in a SBR running at 12 °C
through using an aerobic duration control strategy
(Kouba et al. 2017). This strategy depended on
adjusting the air supply based on the actual oxygen
consumption rate using a PID controller, in a manner
that when the oxygen consumption rate drops below
20% of the maximum consumption the aerator was
switched to off automatically. In that way, it was
123
314
ensured that the DO concentration were only used to
oxidized to ammonia to nitrite and whenever all the
ammonia was oxidized no more oxygen is provided to
NOB to oxidize the produced nitrite. Using the
previous strategy, an ammonia removal percentage
of 57% was reached with nitrite accumulation rate of
92% producing an effluent suitable for the following
anammox process. However, it is noteworthy the seed
used in the study was taken from a partial nitrification
SBR containing previously enriched AOB culture and
not conventional activated sludge. Moreover, the real
wastewater feed was pretreated in lab-scale anaerobic
membrane bioreactor resulting in as COD:N ratio of
0.77 which is much lower than the average ratio in the
mainstream lines, thus the previous study cannot be
considered as a mainstream partial nitrification how-
ever it is a step forward towards achieving successful
mainstream PN.
7.1.2 Phosphorus removal in partial nitrification
Nitrogen (N) and Phosphorus (P) are the key nutrients
leading to eutrophication in water streams, thus
coupling both N and P removal in one stage is an
aspect of great importance. However, partial nitrifica-
tion systems are characterized by the abundance of
nitrite which has been reported as a main inhibitor on
PAO metabolism which affects the phosphorus
removal efficiency in those systems (Yarbrough
et al. 1980). As a matter of fact, a study on the nitrite
effect on PAO reported that nitrite inhibits both the
aerobic and anaerobic phosphate uptake leading to a
deterioration in the biological phosphorus removal
rates (Saito et al. 2004). Moreover, results from a
series of batch experiments on activated sludge
obtained a biological phosphorus removal process
and exposed to various concentration of nitrite showed
that nitrite concentration higher than 8 mg NO2-N/L
completely inhibits the anaerobic phosphate uptake
rate as well as the aerobic phosphate uptake rate but at
lower extent (Meinhold et al. 1999). Thus, it can be
concluded from the previous studies that nitrite might
be detrimental to biological phosphorus removal
(BPR), however a study on the effect of nitrite on
BPR in an anaerobic/aerobic/anoxic/aerobic SBR
demonstrated that PAO with the higher relative anoxic
activity can tolerate more nitrite exposure that those
the lower relative anoxic activity suggesting that BPR
dominant with high anoxic activity PAOs might have a
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Rev Environ Sci Biotechnol (2018) 17:285–321
stable performance even at the presence of excess
nitrite concentration (Yoshida et al. 2006). In addition,
it was reported that nitrite has a much less inhibitory
effect on glycogen accumulating organisms (GAO)
which are the main competitors of PAO for substrate
in the anaerobic phase providing GAO a higher
advantage and consequently lowering phosphorus
removal (Ye et al. 2010). Therefore, further investi-
gation should be conducted to address the inhibitory
effect of nitrite on BPR towards coupling P removal
into partial nitrification systems.
7.2 AOB’s opportunities
AOB accumulated in partial nitrification systems
might be a crucial source for energy and resource
recovery from wastewater treatment plants. One of the
main potential of AOBs is the methanol production
through methane oxidation via the nonspecific action
of the membrane bound ammonia monooxygenase
(AMO). As discussed previously, ammonia is oxi-
dized to nitrite through two steps: in the first step
ammonia is oxidized to hydroxylamine catalyzed by
the membrane bound ammonia monooxygenase
(AMO) while in the second step hydroxylamine is
further oxidized to nitrite catalyzed by the hydroxy-
lamine oxidoreductase (HAO). It has been found that
AMO which resembles to methane monooxygenase
(MMO) in methane oxidizing bacteria (MOB) has the
ability to oxidized methane to methanol (Hyman and
Wood 1983; Jones and Morita 1983). Thus, develop-
ing bio-based systems able to produce methanol using
AOB represents a potential step towards reducing the
energy use and by consequence achieving a net-zero
consumption or even net-zero production wastewater
treatment plants. Methanol can also be attractive not
only as gasoline blends, but also for use in fuel cells,
combined with long-chain fatty acids and lipids to
form biodiesel, or chemically dimerized to dimethyl
ether (DME, also a fuel). Additionally, methanol is
considered to be one of the most widely used
chemicals for enhancing denitrification in wastewater
treatment. Recently, Taher and Chandran, (2013) used
the AOB in a fed-batch sequential bioreactor and
reported up to 60 mg COD/L/mg of CH3OH produced
within an incubation time of 7 h, approximately ten
times the yield previously obtained by pure cultures
(Taher and Chandran 2013). Despite these positive
results, CH4 oxidation to CH3OH by AOB can be
Rev Environ Sci Biotechnol (2018) 17:285–321
inhibited by NH3 (the primary substrate for the
oxidative enzyme AMO) as well as the production of
CH3OH itself. Further, reducing equivalent supply can
be a limiting step in the production of methanol from
methane (Ge et al. 2014). In order to overcome the
aforementioned problem, Lebrero et al. (2016) sug-
gested using hydroxylamine (NH2OH) as an electron
donor and a source of depletion of accumulated NH3
(Lebrero et al. 2016). However. NH2OH is reported to
be an expensive, unstable inorganic compound, and
explosive at high temperature. Therefore, further
studies should focus on testing alternative electron
donors for methane oxidation via AOB.
In addition, AOB can be used for the removal of
pharmaceutical residues which have become an
emerging threat to the aquatic system in the last
decades. Pharmaceutical are present in wastewater in
trace concentrations (sometimes ng/L) which hinders
their utilization as sole carbon source and by conse-
quence their biodegradation by different microorgan-
isms. However, it has been found that AMO has the
ability to degrade various aromatic compounds
through cometabolic biodegradation such as hydro-
carbons (Ge et al. 2014; Keener and Arp 1994; Rasche
et al. 1990; Xu et al. 2016). Moreover, the degradation
of five selected pharmaceuticals ibuprofen (IBP),
ketoprofen (KTP), carbamazepine (CBZ), dexametha-
sone (DXM) and iopromide (IOP) by AOB was
reported under ammonia starvation conditions
(Dawas-Massalha et al. 2014). Furthermore, another
study revealed that N. europaea and mixed ammonia-
oxidizing bacteria in nitrifying activated sludge were
able to degrade triclosan and bisphenol A (2 wastew-
ater micropollutants) while ibuprofen was only
degraded by mixed culture proving that AOB has the
ability of removing pharmaceutical residues whether
in pure or mixed cultures (Roh et al. 2009).
8 Conclusions
There is no doubt that nitrogen has become an
important key parameter in the evaluation of the
effluent of the WWTPs. While conventional BNR was
the dominant process to remove the nitrogenous
compounds, recently SBNR implying more time and
economic savings is recognized as the main approach
towards better nitrogen removal. The first step of
SBNR whether anammox or nitrite shunt process is the
315
successful partial nitrification by halting the nitrifica-
tion step at the nitrite stage through promoting the
AOB growth and inhibiting the activity of NOB. This
paper reviewed all the AOB characteristics including
the factors that affect their growth and assure their
ability to outcompete NOB as well as the reported
technologies towards achieving and maintaining high
partial nitrification performance.
Furthermore, this work elucidated that several
partial nitrification and anammox technologies have
been successfully implemented in the side lines of full-
scale WWTPs with different volumetric nitrogen
loading rate reaching a NLR higher than 10 kg N/
m3/d at the Rotterdam Dokhaven WWTP. However, it
was evident that most of the experimental studies
focused on wastewater with high ammonium concen-
trations or low to C/N ratio and were performed at high
temperature which suit the conditions of sidestream
lines only and hinders the implementation of SBNR
process to the main stream of municipal WWTPs.
Recent advances have been made to overcome some of
the process limitation such as the high temperature,
low C/N ratio and low phosphorus removal perfor-
mance, however a more in depth studies on the
complete application of SBNR to the mainstream is
still missing. Thus, future studies should be directed to
overcome the bottlenecks for the scale-up of main-
stream partial nitrification and Anammox processes
which are: (1) the successful selection of AOB over
NOB at low temperature and low ammonia concen-
tration (no FA and FNA inhibition on NOB), (2) The
competition between heterotopic denitrifies and slow
growing Anammox bacteria at the presence of abun-
dant organic carbon and insufficient alkalinity
reserves (high COD:N ratio), (3) The slow start-up
periods due to the slow activity of Anammox bacteria
at low temperatures, and (4) residual nitrate, nitrite and
ammonia in the effluent. In general, successful main-
stream implementation depends upon maintaining the
balance between AOB and Anammox bacteria while
suppressing NOB and heterotrophic denitrifies, a very
good candidate to do so is attached growth systems
involving granules or biofilm carriers which showed a
high ability in marinating high solids retention times
needed for the accumulation of both bacteria.
Moreover, AOB accumulated in the partial nitrifi-
cation systems might be used as a contributor towards
reducing the energy use of WWTP and by conse-
quence achieving a net-zero consumption or even net-
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316 Rev Environ Sci Biotechnol (2018) 17:285–321

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