Critical Reviews in Biotechnology, 2011; 31(2): 137–152

© 2011 Informa Healthcare USA, Inc.

ISSN 0738-8551 print/ISSN 1549-7801 online
DOI: 10.3109/07388551.2010.497961

REVIEW ARTICLE

Aerobic granular sludge: characterization, mechanism of

granulation and application to wastewater treatment
Dawen Gao1,2, Lin Liu1, Hong Liang1, and Wei-Min Wu3
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1
School of Forestry, Northeast Forestry University, Harbin, P. R. China, 2State Key Laboratory of Urban Water Resource
and Environment, Harbin Institute of Technology, Harbin, P.R. China, and 3Department of Civil and Environmental
Engineering, Stanford University, Stanford, CA, USA

Abstract
Aerobic granular sludge can be classified as a type of self-immobilized microbial consortium, consisting mainly of
aerobic and facultative bacteria and is distinct from anaerobic granular methanogenic sludge. Aerobic granular
technology has been proposed as a promising technology for wastewater treatment, but is not yet established as a
large-scale application. Aerobic granules have been cultured mainly in sequenced batch reactors (SBR) under hydraulic
selection pressure. The factors influencing aerobic granulation, granulation mechanisms, microbial communities
and the potential applications for the treatment of various wastewaters have been studied comprehensively on the
laboratory-scale. Aerobic granular sludge has shown a potential for nitrogen removal, but is less competitive for the
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high strength organic wastewater treatments. This technology has been developed from the laboratory-scale to pilot
scale applications, but with limited and unpublished full-scale applications for municipal wastewater treatment. The
future needs and limitations for aerobic granular technology are discussed.
Keywords:  Aerobic granular sludge, sequencing batch reactor, biological wastewater treatment

Introduction
Granular sludge technology is one of the great achieve-
ments in environmental biotechnology of the twentieth
century. Granular sludge is a self-immobilized microbial
consortium, with a high density and used in wastewater
treatment reactors. Granular sludge was first found in
anaerobic upflow anaerobic sludge blanket (UASB) reac-
tors to treat industrial wastewaters at the end of the 1970s
(Lettinga et al., 1980). Anaerobic granular sludge consists
mainly of methanogenic, syntrophic acetogenic and
various hydrolytical-fermentative bacteria and has been
widely applied in full-scale anaerobic reactors for waste-
water treatment since the 1980s (Hickey et  al., 1991).
Aerobic granular sludge is developed under aerobic
conditions and mainly used for the aerobic degradation
of organics and also for nitrogen removal under aerobic
and anoxic conditions (Liu et al., 2004). In addition, an-
aerobic or anoxic granular sludge consisting of anaerobic
ammonium-oxidizing (ANAMMOX) bacteria has also be
developed (Imajo et  al., 2004; Kartal et  al., 2010; Quan
et al., 2008; Shi et al., 2009;Viaeminck et al., 2008).
Aerobic granular sludge was first reported in an aerobic
upflow sludge blanket reactor by Mishima et al. (1991).
Morgenroth et al. (1997) used a sequencing batch reactor
(SBR) to develop aerobic granular sludge and the granu-
lar sludge was formed after 40 days of incubation. Since
that time, SBR has been utilized for aerobic granulation
(Peng et al., 1999; Beun et al., 1999) and identified to be a
proper reactor configuration (Liu et al., 2004). This is sig-
nificantly different from the anaerobic process using an
upflow reactor for anaerobic granulation. To date, most
research work on the development of aerobic granular
sludge or aerobic granulation has been conducted using
SBR. The research efforts have focused on the cultivation
conditions, factors influencing granulation, and the mi-
crobial community of the granular sludge. At present, the
research work on the mechanism of aerobic granulation
and its application for a wide variety of wastewater has

Address for Correspondence:  Dawen Gao, School of Forestry, Northeast Forestry University, Harbin, P.R. China. Tel.: 86-451-82165870; Fax:
86-451-82190385; E-mail: gaodw@nefu.edu.cn
(Received 31 March 2010; revised 28 May 2010; accepted 29 May 2010)

137
 138  Dawen Gao et al.
been conducted by research groups around the world,
especially in Singapore, the Netherlands and China.
The definition of aerobic granules is similar to the
definition of anaerobic granular sludge. Granular sludge
is a type of conglomerate formed by microorganisms
and has a granular appearance; their shape does not
rely on the presence of water and they can withstand a
certain amount of pressure (Dolfing, 1987). In 2005, the
International Water Association (IWA) held the first ses-
sion of aerobic granular sludge seminars at the Technical
University of Munich in Germany, and a clear definition
of aerobic granular sludge was established, i.e. granules
making up aerobic granular activated sludge are to be
understood as aggregates of microbial origin, which do
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not coagulate under reduced hydrodynamic shear, and
which settle significantly faster than activated sludge
flocs (de Kreuk et al., 2005). In our opinion, the biofilm
particles developed on an inert carrier (such as granular
activated carbon, sand, etc.) due to the attachment growth
in fluidized bed reactors and other reactors cannot be
considered as aerobic granules, although they appear as
granular shapes and have a high biomass density.
Laboratory research results have indicated that aerobic
granular sludge has the following advantages compared
to activated sludge which is in a flocculent form:
Aerobic granular sludge has high biomass retention.
This allows a reactor to have a high biomass concentra-
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tion and treat wastewater at a relatively high volumetric
loading rate;
(1) Aerobic granular sludge has a good settling prop-
erty. The settling velocity of aerobic granular sludge
depends on the size and density of the granules and
can reach 50–90 m/h.
(2) A long sludge retention time of the flocculent sludge
can be achieved, which is especially beneficial to
nitrifiers and ANAMMOX bacteria. Aerobic and
anoxic zones are present inside the granules that
can simultaneously perform different biological
processes in the same system, i.e. simultaneous
nitrification-denitrification (SND) could occur
(Beun et al., 1999; Lei et al., 2006). Partial nitritation
of ammonium to nitrite and the ANAMMOX reaction
were also observed in the granules developed in the
SBR (Shi et al., 2009; Viaeminck et al., 2008).
(3) Aerobic granular sludge can be used to treat various
wastewaters and is able to tolerate shock loading due
to the unique granular structure and high biomass
concentration in the reactor. In addition, no sludge
bulking was observed with the aerobic granular
sludge (Moy et al., 2002).
To date, several hundred papers related to aerobic gran-
ules have been published. In this review article, we have
summarized the updated major progress in the research
and application of aerobic granular technology. The re-
sults derived from these studies could be useful for the
understanding of the application potential of aerobic
granules.
Characteristics of aerobic granular sludge
This section summarizes studies on the characteristics
of granules including their physical properties, chemical
properties, microbial community and advanced tools for
the characterization of the granules.
Physical characteristics of aerobic granular sludge
Settling properties
The settling ability of the sludge is an important pa-
rameter which directly relates to the biomass retention
capacity and the solid-liquid separation in the reactor.
The settling velocity of aerobic granules in reactors var-
ied from 18 to 90 m/h, even up to 130 m/h (Zheng et al.,
2005; Zheng et al., 2006; Xiao et al., 2008; Shi et al., 2009).
These values were similar to those obtained for anaero-
bic granules and were much higher than the values from
flocculent activated sludge (7–10 m/h) (Schmidt et  al.,
1996). The sludge volume index (SVI), another parameter
of sludge settling ability, of aerobic granules is generally
below 80 mL/g, and even as low as 20 mL/g (Zheng et al.,
2005; Lin et al., 2005; Gao et al., 2010). Liu et al. (2005a)
developed a model for the settling ability of aerobic gran-
ules (as well as for anaerobic granules), which indicated
that the settling velocity of the granules was a function of
SVI, average size and biomass concentration. When the
size of the granules is small enough, the model becomes
the well-known Vesilind equation as follows:
V = V0 e − kX (1)
 S
where VS is the settling velocity, V0 is the initial settling
velocity, X is the sludge concentration, and k is the em-
pirical settling parameter.
Specific gravity and water content
Aerobic granules have a high density and compact struc-
ture, which helps in the biomass retention time and
solid-liquid separation. The specific gravity of aerobic
granules in SBR varied from 1.004 to 1.1 kg dry weight/m3,
which was much higher than that (1.002–1.006 g/m3) for
flocculent sludge (Shi et al., 2009; Li et al., 2009; Zheng
et  al., 2005). The water content of the granules (about
94–97%), was much lower than that (>99%) of flocculent
sludge (Lin et al., 2005).
Physical strength
Aerobic granules usually have enough physical strength
to maintain the stability of their granular form and struc-
ture during operation. The physical strength is an impor-
tant parameter used to characterize the granular sludge
for its structural features and capability to withstand high
abrasion and water shear force. The physical strength
of aerobic granules has been examined indirectly by
measuring the integrity coefficient (IC) as proposed by
 Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment  139
Tay et al. (2002). They determined the IC by measuring
the weight ratio of the residual granules versus the total
weight of the granular sludge after 5 min of shaking at
200 rpm on a platform shaker. Xiao et al. (2008) has sug-
gested the measurement of the integrity coefficient (ICt )
as function of shaking time,
ICt = (1 − SSt/SS0 ) × 100% (2)

where SS0 is the total amount of granular sludge and SSt
is the amount of the sludge solids in the supernatant after
t minutes of the vortex shear test. Although the IC is an
indirect measurement it provides an overall and practi-
cal indication of the shear strength of the sludge particles
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in a dynamic fluid condition. Tay et al. (2004a) reported
that the IC was 96.9% and 96% for granules developed
in a laboratory and a small pilot scale SBR, respectively,
suggesting that they were similar. The physical strength of
the granules formed by bacteria was different from those
mainly formed by fungi. The physical strength of the fun-
gal granules was weaker and more likely to be broken due
to the loosely packed microstructure (Xiao et al., 2008).
Another method measured the compressive strength
of aerobic granules as the resistance against the compres-
sion force (Ren et al., 2008). The compressive strength of
calcium-rich granules was measured to be within a range
from 0.16 to 0.42N/mm2 with a Ca-content from 86.8 to
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151.1 mg/gSS. The values varied within a range of 0.003–0
.04N/mm2 for the granules with a Ca-content from 2.5 to
40 mg/gSS. This demonstrated that the Ca content in the
granules had a higher compressive strength compared
with those that contained less Ca (Ren et  al., 2008; van
Hullebusch et al., 2007).
Morphology and granule size
Compared with flocculent sludge, the granules show a
clear outline with a nearly spherical or elliptical shape.
Research on aerobic granules has indicated that the
morphology can be affected by a number of operational
conditions and factors, such as organic loading rate,
substrate applied, microbial population and operational
strategy. The color of the aerobic granules was found to
be dependent on the chemical composition and micro-
bial population. The granules were mainly yellow but
were also black colored (Zheng et al., 2006). The size was
an important parameter in the physical characteristics
of the aerobic granules, and the average diameter of the
aerobic granules had a very wide size range from 0.2 up
to 16 mm (Beun et al., 1999; Toh et al., 2003; Zheng et al.,
2006; Li et al., 2008; Gao et al., 2010). The size distribution
of the aerobic granules was related to the operational
conditions. As shown in Figure 1, when a high organic
loading rate was applied this could stimulate the forma-
tion of a larger size granule, and smaller size granules
could be developed under high shear conditions or with
long starvation periods (Li et al., 2008; Gao et al., 2010).
If the particle size was smaller than 4 mm, the aerobic
granules would have a better settling ability, density and
intensity. However, they were larger than 4 mm, the in-
creasing diameter could lead to a deterioration of settling
ability, density, and intensity (Toh et al., 2003). The dis-
solved oxygen (DO) was a major limiting factor for meta-
bolic activity of the aerobic granules with a size larger
than 0.5 mm. The diffusion limitations of the substrate
could not occur for aerobic granules with a size less than
0.4 mm, and the substrate removal rate by the granules
with a size of 0.5mm was almost three times that by the
granules of a 1 mm size. Thus, the optimal size of aerobic
granule has been suggested to be less than 0.5 mm in the
sense of mass transfer (Lin et al., 2005; Li et al., 2008a).
Porosity
Porosity has been found to be closely related to both the
substrate transport and the dissolved oxygen limitations
in aerobic granules. If the pores are walled up or clogged,
diffusion limitation of the substrate, the DO would rise,
and the bioactivity of the aerobic granules would be
negatively affected. Research results have shown a posi-
tive correlation between the bioactivity and porosity of
aerobic granules (Tay et al., 2002a; Zheng et al., 2007). The
definition and measurements for the porosity of aerobic
granules have been discussed in the literature (Zheng
et al., 2007; Ren et al., 2008). Activated flocculent sludge
normally has porosity greater than 0.95, and the aerobic
granules have a relatively low porosity. Zheng et al. (2007)
indicated that the porosity of the aerobic granules varied
from 0.68 to 0.93 and decreased with size. Xiao et al. (2008)
indicated porosities ranging from 0.78 to 0.97 for fungal
granules and from 0.68 to 0.92 for bacterial granules, and
the porosity rose as the size of granules increased.
Chemical characteristics of aerobic granular sludge
Cell surface hydrophobicity
According to thermodynamic theory, the increase in the
cell hydrophobicity could lead to a decrease of the excess
R1 R2
R3 R4
Figure 1.  Granules developed under different operational
conditions (bar = 2 mm): R1: high organic loading rate; R2: short
settling time; R3: long starvation period; and R4: high shear force
(Gao et al., 2010).
 140  Dawen Gao et al.
Gibbs energy in the cell, and thereby promote cell-to-cell
self-aggregates. Aerobic granulation can be considered as
a process of a cell-to-cell self-immobilization, so cell sur-
face hydrophobicity should play a decisive role in sludge
granulation. Several analytical methods have been used
to characterize the cell hydrophobicity of aerobic gran-
ules, including contact angle measurements, bacterial
adherence to hydrocarbons and phenathrene adsorption
(Tay et al., 2005; Zheng et al., 2006; Li et al., 2008b).
The cell surface hydrophobicity of the aerobic granules
is much higher than that of the flocculent sludge. The
mean contact angle values were 35° and 46.3° for floc-
culent sludge and aerobic granules, respectively (Zheng
et al., 2005). The cell surface hydrophobicity, in terms of
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adherence to hydrocarbons, was reported to increase
from 43.2% to about 78.2% as the sludge changed from
flocculent to a granular form in a SBR reactor (Zhang
et al., 2007). Qin et al. (2004) also found that the cell sur-
face hydrophobicity of the granules was increased by 2.5
to 3.5 times as compared to that of flocculent sludge.
The cell surface hydrophobicity of aerobic granules
is influenced by the structure of the granules and the
­operating conditions. The shells of the aerobic granules ex-
hibited a higher hydrophobicity than the core of the gran-
ules (Wang et al., 2005). The aerobic granules which were
bacteria-dominated could be coupled to the increase in
the cell surface hydrophobicity, while the decrease in the
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hydrophobicity of the fungal-dominated granules might
be attributed to the hydrophilicity of the ­filamentous
forms (Zheng et  al., 2006). The aqueous chemical com-
position also influenced the ­hydrophobicity. An increase
in cell ­surface hydrophobicity of the aerobic granules was
observed when the loading rate was above 2.5 kg phe-
nol/m3 •d in an SBR fed with phenol (Jiang et al., 2004).
As the free ammonia concentration changed from 2.5 to
39.6 mg  N/L, the cell surface hydrophobicity declined
from 70.6 to 40.1% (Yang et al., 2004a). The cell hydropho-
bicity was significantly increased as the settling time and
the cycle time were shortened (Tay et al., 2002d; Lei et al.,
2004; Qin et al., 2004). The shear force and the ratio of the
extracellular proteins to the polysaccharides had a posi-
tive effect on the hydrophobicity, and the specific surface
hydrophobicity increased as the granule size increased
(Toh et al., 2003; Zhang et al., 2007; Tay et al., 2001a).
Extracellular polymeric substances (EPS) of aerobic granular
sludge
Extracellular polymeric substances (EPS) are organic
macromolecule polymers secreted by microorganisms
(mainly bacteria) under certain environmental condi-
tions, and form a three-dimensional matrix in which the
bacteria can embed. The major components of EPS are
the protein, polysaccharide, humic acids, nucleic acids,
lipids and other miscellaneous materials.
EPS could be utilized by the bacteria themselves, since
at least 50% of the polysaccharide (PS) and 30% of the
protein (PN) in the EPS produced by aerobic granules
are biodegradable. The EPS in aerobic granules include
two major components, i.e. biodegradable and non-
biodegradable EPS fractions. The average biodegrada-
tion rate of EPS in aerobic granules in terms of chemical
oxygen demand (COD) was found to be slower than that
of the acetate by five fold, but faster than that of the non-
biodegradable EPS by 50 times (Wang et al., 2007; Zhang
et al., 2003). In addition, the EPS content in the core of
the aerobic granules was nearly five times higher than
that in the outer shell of the granules. The majority of the
biodegradable EPS was in the core of the aerobic granule,
while the non-biodegradable EPS was mainly observed
on the outer shell of the granules. Thus, only the non-bio-
degradable EPS produced by the aerobic granules would
contribute to the structural stability of the aerobic gran-
ules (Wang et al., 2005). In the acetate-fed and phenol-fed
reactors, the EPS showed a different distribution in the
aerobic granules. The proteins and β-D-glucopyranose
polysaccharides were found in the core of the granules,
while α-D-glucopyranose polysaccharides accumulated
in the outer shell. In the phenol-fed reactor, the proteins
were found only in the core of the granules, while the α-
and β-D-glucopyranose polysaccharides were accumu-
lated at the outer layers (Chen et al., 2007).
The content of EPS in the aerobic granules was much
higher than that in the flocculent sludge and biofilm
(Adav et al., 2007a; McSwain et al., 2005; Tay et al., 2001;
Liu et al., 2004b). While other researchers have also indi-
cated that the decrease of the EPS content was concur-
rent with the formation of aerobic granules (Zheng et al.,
2005), di Iaconi et al. (2006) reported that proteins were
the main component of EPS in aerobic granules, and the
protein rich EPS may improve the structural stability of
the granules. McSwain et  al. (2005) also found that the
protein content became higher than the polysaccharide
content in the EPS during granulation. Zhang et al. (2007)
reported that the extracellular protein content increased
from 49.4 mg/g VSS in the seed sludge to 148.3 mg/g VSS
in the aerobic granules during the granulation, while
the extracellular polysaccharides remained almost
unchanged. Adav et  al. (2008c) compared seven EPS
extraction methods for aerobic granules and indicated
that formaldehyde plus NaOH as a chemical reagent
enhanced the efficiency of EPS extraction methods in
phenol-fed granules. The collected EPS revealed no
contamination by intracellular substances and consisted
mainly of proteins, polysaccharides, humic substances
and lipids. The quantity of extracted proteins showed
a weak correlation with the quantity of extracted poly-
saccharides, but no correlation with the quantity of the
extracted humic substances. The role of EPS in aerobic
sludge granulation has been investigated (Adav et  al.,
2007a; Chen et al., 2007; Zhang et al., 2007; Wang, 2006a)
and its influence on granulation has been considered to
be related to the hydrophobicity, cell surface charge and
bridging functions as discussed below.
(1) Cell-surface hydrophobicity. As mentioned previ-
ously, an increase in the cell-surface hydrophobicity
 Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment  141
reduces the Gibbs free energy on the cell-surfaces
and enhances the ability of coagulation among the
cells. Therefore, hydrophobic EPS may play a vital
role in the granulation process (Liu et  al., 2003b).
The EPS with protein and amino acids are more
hydrophobic, but polysaccharides are hydrophilic.
Research on anaerobic granular sludge has indi-
cated that the polysaccharide content in granular
sludge is limited (<2%) while about 80% of EPS was
protein-associated (Dolfing, 1987; Veiga et al., 1997).
A similar observation is likely to be found in aero-
bic granules. It was found that the outer shell of the
aerobic granules exhibited a higher hydrophobicity
than that of the interior core area, and the insoluble
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EPS present in the granule shells (Wang et al., 2005).
Thus, the hydrophobicity of EPS is likely to be one of
the factors influencing granulation.
(2) Cell surface charge. Cell surface charge plays an
important role in biological flocculation and in the
stability of bacterial flocs. The cell surface of bacte-
ria is negatively charged under neutral pH condi-
tions (Rouxhet et al., 1990). As there is electrostatic
repulsion, the cell finds it difficult to form a floc or
aggregate. The EPS bound to the cell would reduce
the charge on the cell surface and enhance aggrega-
tion. Tsuneda et  al. (2003) found that the EPS can
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increase the flexibility of the cell surface, and greatly
reduce the cell surface charge. Zhang et  al. (2007)
also indicated that there is a strong inverse corre-
lation between the value of the ratio of proteins/
polysaccharides and the surface charge of aerobic
granules. The increase in the ratio between proteins
and polysaccharides could decrease the surface
negative charge of the bacteria, thereby reducing the
electrostatic repulsion between bacteria, and further
enhance aerobic granulation.
(3) Bridging function. One hypothesis is that the
polysaccharides in EPS may serve as adhesives to
bind the bacteria together to form a micro-colony.
The polysaccharide polymers on the surface of a
small granular particle or aggregate could serve as
a bridge to trap or connect other small granules or
aggregates together to form larger-sized granules.
Research results have shown that the bridge func-
tion was enhanced when the content of EPS was
increased (Jiang et al., 2002; Liu et al., 2004).
Microbial characterization of aerobic granular sludge
Aerobic granules can be developed with mixed cul-
tures (such as activated sludge) or defined microbial
cultures, as well as with fungi. Aerobic granulation has
been observed in reactors fed with various organic sub-
strates (glucose, acetate, ethanol, phenol, etc.), indus-
trial wastewaters, landfill leachates (Cassidy et al., 2005;
Figueroa et al., 2008; di Iaconi et al., 2007; Liu et al., 2004;
Weber et al., 2007) and synthetic water with ammonium
(Viaeminck et  al., 2008). The microbial structure of
the granules was dependent on the inoculum, chemi-
cal composition, DO concentration and the size of the
granules. In general, a granule could contain organic-
degrading, nitrifying, and denitrifying bacteria as well
as anaerobic bacteria (ANAMMOX, methanogens, etc.)
if the granules were developed with sewage or a similar
wastewater. A conceptual aerobic granule is composed
of three microbial communities, i.e. heterotrophic,
aerobic bacteria grown on the outside, ammonium-
oxidizing bacteria in the middle and facultative and
anaerobic bacteria in the core of the granules.
The predominant microbial species of anaerobic
granular sludge are acetate-and hydrogen-utilizing
methanogens (Hickey et  al., 1991; Liu et  al., 2004) be-
cause methanogenesis is the final step of the anaerobic
degradation of organics. On the other hand, the micro-
bial community of aerobic granular sludge can vary from
limited species to extremely diversified species and is
strongly affected by substrate or wastewater composi-
tion, inoculum and environmental and operational fac-
tors. For example, aerobic granular sludge developed
with organic carbon compounds and nitrate has been
reported to contain bacterial strains belonging to the
genus Poterioochromonas, Epistylis, Geotrichum, and
Geotrichum klebahnii (Williams et  al.; 2006). Aerobic
granular sludge grown on ­ammonia had a dominance of
ammonia-oxidizing bacteria and nitrite-oxidizing bacte-
ria (Yang et al., 2004; Tsuneda et al., 2003a). In granular
sludge developed with brewery wastewater, the fila-
mentous bacteria were the predominant species which
mainly belonged to Thiothrix and Sphaerotilus natans
(Weber et al., 2007). Although the microbial community
structure is influenced by the type of substrate, a similar
evolutionary pattern of granulation was observed (Tay
et al., 2001). According to scanning electron microscopic
(SEM) observations, the granules fed with glucose con-
tained more filamentous bacteria, while those fed with
acetate had rod-like bacteria on the surface (Tay et  al.,
2001). In a one-stage oxygen-limited SBR for autotrophic
nitrification/denitrification, the granules had aerobic
and anoxic ammonium-oxidizing activities. Fluorescent
in-situ hybridization (FISH) ­demonstrated the presence
of both β-proteobacterial aerobic ammonium oxidizers
and Planctomycetes (anoxic ANAMMOX bacteria) in the
granules (Viaeminck et al., 2008).
Filamentous bacteria are considered to play an im-
portant role during the granulation process because they
may form the initial framework for small sludge particles,
which serve as cores or carriers. Some researchers have
tried to use filamentous fungi as cores to enhance aero-
bic granulation. Morgenroth et  al. (1997) reported that
microorganisms started growth together on carriers
formed by filamentous fungi and finally grew as aerobic
granular sludge from the framework of the filamentous
fungi. Other researchers reported that initially big par-
ticles were formed based on fungal cores, and then these
disintegrated into small particles due to mass transfer
limitations; these small granules grew continuously to
 142  Dawen Gao et al.
form an aerobic granular sludge (Peng et al., 1999; Beun
et  al., 1999). However, the enhancement with fungi is
likely to be unnecessary because aerobic granulation by
a bacterial inoculum can be achieved within one or more
months.
The microbial composition of aerobic granules devel-
oped for phenol degradation has been characterized by
several investigators. Yeast genera have been found in the
aerobic granules developed for phenol degradation. Phe-
nol- and phenolic-degrading Rodotorula, Trichosporon,
and Candida were isolated from activated sludge which
formed aerobic phenol-degrading granules (Jiang et al.,
2005; Margesin et al., 2005). Candida tropicalis, capable
of degrading phenol with a high tolerance to phenol
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toxicity, was isolated from aerobic granular sludge and
found to be a functionally dominant microorganism for
phenol degradation (Adav et  al., 2007). Proteobacteria
were isolated from aerobic granular sludge degrading
phenol (Heinaru et  al., 2000). The strains belonging to
γ-Proteobacteria and α-Proteobacteria were found to be
dominant; β-Proteobacteria were also found to be the
prevailing bacteria in the granular sludge. All of these
bacteria had a minimal phenol degradation capability
but a high propensity for self-aggregation (Jiang et  al.,
2006, 2007; Adav et al., 2008, 2008a).
The size of aerobic granular sludge may influence
the distribution of the microbial community structure
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in the granules. Lemaire et  al. (2008) reported that the
­Accumulibacter spp. was dominant in the outermost 200
μm region of the granule, while the Competibacter spp.
dominated in the cores of the granules. Tay et al. (2002e)
demonstrated that anaerobic bacteria were present in
the center and approximately 800 μm below the surface
of the granule and Nitrosomonas spp., which oxidizes
ammonia to nitrate, was the major species at 70–100 μm
below the surface. However, regardless of size, both live
and dead bacteria were densest in the outer layer of the
aerobic granules, which were about 0.6 mm thick. The live
cells appeared only in the peripheral zone, while dead
bacteria spread into the inner zone (Toh et al., 2003).
The DO concentration influences the microbial struc-
ture of the granular sludge. The deficiency of DO influ-
ences the growth of different filamentous bacteria dur-
ing granulation. For example, Thiothrix sp. is favored by
growth at low DO concentrations (Lee et al., 2003), while
other filamentous bacteria such as Microthrix parvicella
can grow over a wide range of DO concentrations (Ros-
setti et al., 2005).
Different temperatures may also result in different
dominant microbes. The microbial community struc-
ture at 25°C showed the least similarity with those at
other temperatures. Thermomonas sp. existed in a large
amount only at 25°C, and Curtobacterium ammoniigenes
was the major bacteria at 30°C. However, Ottowia sp.
could be found to prevail between 30°C and 35°C (Song
et al., 2009).
Defined microbial cultures have also been tested to de-
velop aerobic granular sludge for wastewater treatment.
Wang et al. (2006) used a nitrogen-fixing bacteria (Azoto-
bacter sp.) and white rot fungus (Pchrysosporium flora) as
a mixed culture for the development of aerobic granular
sludge to treat paper mill wastewater. Adav et al. (2008b)
developed a single culture aerobic granular sludge with
Acinetobacter calcoaceticus to degrade phenol. Granules
with sizes up to 2.3 mm were formed in the SBR after 49
days of operation. The granules had an excellent settling
ability and degraded phenol at 0.994 and 0.519 g phenol/
gVSS •day at 250 and at 2000 mg/L, respectively.
Finally, the microbial community is dynamic during
aerobic granulation. A polymerase chain reaction- dena-
turing gradient gel electrophoresis PCR-DGGE analysis
showed an evident shift of the microbial populations oc-
curring after the sludge in the SBR was developed from a
flocculent to the granular form. The microbial population
of aerobic granules was completely different from that of
the seed sludge (Liu and Tay 2008a; Li et al., 2008).
Innovative tools for characterizing
granulation
As aerobic granules have a unique structure and charac-
teristics, it is hard to measure the reaction process inside
the granules by conventional analytical methods or tools.
In recent years, several innovative methods and tools
have been developed or tested for the characterization of
aerobic granular sludge.
The measurement of the DO spatial distribution in-
side the granules has been considered as an important
approach to monitor the bioactivity, the bioreactions
and also to understand the reaction mechanisms. A
microelectrode was successfully fabricated using pho-
tolithography for the determination of DO distributions
inside an aerobic granule (Liu et al., 2007c). The test re-
sults showed that the DO distribution in aerobic granules
could be monitored with the microelectrode stably and
reliably. In addition, the microelectrode could be re-
newed several times. A gold microelectrode array (MEA)
was manufactured by microfabrication techniques and
used to monitor the DO distributions inside the aerobic
granules accurately. In addition, the MEA could be used
to determine constituents, profiles and functions in situ in
the small spaces of the aerobic granules (Liu et al., 2009).
The MEA included five gold microelectrodes which had
a good linear response to the DO with a lifetime of more
than 10 days.
The microstructure of the aerobic granules can be ob-
served using a confocal laser scanning microscope. This
method has been used to measure the porosity profiles
in aerobic granules (Tay et  al., 2003; Yang et  al., 2008;
Yarlagadda et al., 2008). Zheng et al. (2007) used size-ex-
clusion chromatography (SEC) to determine the porosity
and internal pore size distribution of aerobic granules.
Sheng et al. (2006) reported on the use of three-dimen-
sional excitation–emission matrix (EEM) fluorescence
spectroscopy to study the properties of EPS in aerobic
granules. This technique can distinguish the fluores-
 Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment  143
cence compounds present in the complex EPS mixtures
from various origins. Fluorescence properties could be
characterized by changing the excitation wavelength
and the emission wavelength simultaneously. According
to their results, three fluorescence peaks were found in
the EEM spectra of EPS of the granules; two peaks were
attributed to the protein-like fluorophores; and the third
one to the humic-like fluorophores. The results showed
that both pH and EPS content had an important effect on
EEM fluorescence spectra of EPS, but the ionic content
had a weak impression.
Storage and reactivation of aerobic granular
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sludge
Anaerobic granular sludge is stable in the granular form
and maintains bioactivity for the long term (from months
to years) under starvation conditions (Wu et  al., 1995)
but the bacterial aggregates formed by aerobic and fac-
ultative bacteria could be disaggregated after a relatively
short storage period. For the scale-up and long-term sta-
bility of aerobic granular sludge bioreactors, a number
of research projects have been conducted on the storage
and reactivation of aerobic granules. The storage stabil-
ity, physicochemical characteristics, and re-activation of
aerobic granules are crucial and essential factors for the
scale-up from laboratory to pilot and eventually to full-
For personal use only.
scale applications (Zeng et  al., 2007). To date, research
results have indicated that the stability and integrity of
aerobic granules can be maintained during a storage
or starvation period of weeks or months. The activity
and structural integrity of the aerobic granules during
the storage time depended on the storage temperature,
length of storage time, storage medium and the charac-
teristics of the granular sludge itself (Liu et al., 2004a).
Tay et  al. (2002c) investigated the aerobic granules
developed on glucose and acetate, and found the former
lost about 60% activity, but the latter lost 90% activity after
4 months storage in tap water at 4°C, with a decrease in
strength of 7–8% in comparison with the fresh granules.
The effects of different storage solutions on the stability
and activity of the aerobic granules were also investigat-
ed. After 8 weeks of storage at 4°C, the aerobic granules
stored in all the storage solutions became irregular and
smaller. The particle size of the granules in tap water
and a physiological solution decreased 34% and 22%,
respectively. Moreover, cell hydrolysis was observed
during the storage period, which resulted in the partial
disaggregation of granules. However, granules stored at
4°C in phosphate-buffered saline (PBS) had the smallest
activity change (Ng, 2002; Liu et al., 2004a).
The remaining activity of the aerobic granules in-
creased as the storage temperatures were lowered and
declined continuously as storage time elapsed. Adav
et al. (2007c) found that a low storage temperature and
the toxicity of phenol could delay hydrolysis or biotrans-
formation of proteins in the cores of the granules and en-
hanced the granule stability and activity. They also found
that granules stored either with 500 mg/L of phenol solu-
tion or at −20°C with any storage solution maintained a
high phenol degradation capability.
Zhu et  al. (2003) reported that aerobic granules fed
with glucose could be stored for up to 7 weeks without
losing the integrity of the granules and the metabolic
potential. Another report showed that the aerobic gran-
ules retained good integrity and activity after 2 months
of storage at room temperature and the strength of the
granules could be fully restored after 3 weeks of re-activa-
tion (Zhang et al., 2005). The particle size, settling ability
and structural integrity of phthalic acid (PA)-degrading
aerobic granules were found to be stable after 8 weeks of
storage at 4°C (Zeng et al., 2007). Aerobic granules grown
on a complex substrate could restore their activities to
previous levels after a 7 day operation.
Aerobic granules after storage can be used as seed
sludge to startup the bioreactors. Liu et al. (2005c) suc-
cessfully started a scaled-up aerobic granular sludge re-
actor (approximately 34 L in volume) with 2 liters of aero-
bic granules stored for 4 months. The microbial activity
recovered quickly and new granules started to grow after
day five. Wang et  al. (2008a) re-activated aerobic gran-
ules after a 7 month storage period and found that the
activities of the heterotrophs and nitrifiers were fully re-
covered within 16 days and 11 days, respectively. During
storage, the nitrifiers decayed more slowly and reinstated
more rapidly than the heterotrophs. The selection of
slow-growing bacteria was considered as an approach to
improve the stability of aerobic granules. Accumulation
of EPS was an effective strategy for maintaining the struc-
tural integrity of aerobic granules during long-term stor-
age (di Iaconi et al., 2007; de Kreuk and van Loosdrecht,
2004). However, the stability of aerobic granules for long-
term periods (>8 months) has not been reported. The
maintenance of the stability of aerobic granules is still an
operational issue for scale-up to full scale application.
The ANAMMOX-containing granules are considered
as a promising technology for nitrogen removal (Kartal
et al., 2010). To date, no report has been published about
the storage and stability of this granular sludge. Because
of the slow growth and long doubling time of ANAMMOX
bacteria (10–12 days), the granular type with ANOMMOX
bacteria could be much more stable than other aerobic
granules.
Factors influencing or controlling aerobic
granulation
To date, the published literature has indicated that aero-
bic granulation is mainly dependent on reactor configu-
ration, environmental, and operational conditions, so is
not a function of microbiological groups. As mentioned
previously, the pneumatically agitated reactors, includ-
ing bubble column and airlift reactors with sequencing
batch operation, are favorable for the development of
aerobic granules. The formation of aerobic granules is a
complex process influenced or controlled by several fac-
 144  Dawen Gao et al.
tors. The main factors except for reactor configuration are
summarized as follows.
The presence of divalent metal ions
Divalent metal ions have been proven to have a positive
effect on granulation. The metal ions, especially calcium
and magnesium are recognized to enhance granulation
in SBR (Yu et al., 2001; Jiang et al., 2003; Li et al., 2009; Ren
et al., 2008). The mechanisms of enhancement of granu-
lation may involve several functions: (1) inorganic ions
neutralize the negative charge on the surface of bacteria
and enhance aggregation (Morgan et al., 1990); (2) forma-
tion of precipitates by the inorganic ions serve as nuclei
to accelerate the microbial aggregation, especially in the
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Michigan State University on 03/24/11
case of Ca2+; and (3) inorganic ions form an ionic bond
on the surface of the particles and act as a cation bridge
to enhance granulation. After granulation, calcium also
binds to EPS and thereby forms the EPS-Ca2+-EPS cross
linkages to strengthen the granule structure (Yu et  al.,
2001; Ren et al., 2008).
The effect of Ca2+ augmentation on aerobic granula-
tion in sequential aerobic sludge blanket reactors was
tested by Jiang et  al. (2003). Augmentation with 100 mg
Ca2+/L significantly reduced the granulation period from
32 to 16 days. It was found that the Ca2+-fed granules were
denser, more compact with a better settling ability and
with a higher polysaccharide content. The augmentation
For personal use only.
of Mg2+ (10 mg/L) also significantly reduced the granula-
tion period from 32 to 18 days using glucose-containing
synthetic wastewater (Li et al., 2009). In addition the Mg2+
amendment resulted in larger sized granules with a high-
er EPS content. Our own research has confirmed that ad-
dition of either Ca2+ or Mg2+ accelerated the granulation
process. Calcium precipitates were observed inside the
granules, but no precipitates were found when Mg2+ was
used (Figure 2). The results suggested that the amend-
ments of both Ca2+ and Mg2+ are helpful, but may not be
essential for the granulation.
Aerobic starvation
The control of the aerobic starvation phase in SBR could
accelerate the granulation process. The mechanism is still
not clear, but several hypotheses have been discussed.
During SBR operation, the aeration stage is divided into
two phases i.e. the substrate degradation phase followed
by an aerobic starvation phase. The length of each phase
is the function of the concentration and degradation rate
of the organic substrates. A recent study showed that full
complete aerobic granulation in SBR can be accelerated
with a relatively long starvation time. However, aerobic
granules were also observed in SBR with a shorter star-
vation time, but the biomass was usually mixed with
flocculent sludge. The size of the aerobic granules with a
shorter starvation time was much bigger than those with
a longer starvation time. The large size granules also had
a low settling velocity and a high SVI, thereby showing in-
stability and a collapse of the operation (Liu et al., 2008a).
In addition, too short or too long a starvation duration
Figure 2.  Aerobic granules developed with added Ca2+ (left) and
Mg2+ (right) (bar is 2 mm) (Liu et al., 2010)
could make the ratio value of the polysaccharides/pro-
teins deviate from the optimum value, thereby leading
to a low biomass density and a weak settling ability (Liu
et al., 2007b). A longer aerobic starvation phase could in-
crease the production of EPS in the sludge. This suggests
that the longer starvation period selects microorgan-
isms which secrete more EPS, which would help aerobic
granulation (Tay et al., 2001; Li et al., 2006). In addition,
under starvation bacteria become more hydrophilic (Tay
et al., 2001). A longer starvation period had a significant
impact on hydrophobicity, zeta potential and EPS (Liu
et  al., 2008a). Thus, reasonable starvation duration was
necessary for the formation and stability of the aerobic
granules in SBR. Li et al. (2006) also indicated that dura-
tion of starvation influenced the initial stage of aerobic
granulation and suggested that the starvation provided
an anaerobic microenvironment, which favors the anaer-
obic metabolism of facultative microorganisms, which in
turn facilitates the granulation process.
Selection pressure
Hydraulic selection pressure is essential for aerobic
granulation. The selection pressure includes hydraulic
shear force and settling time. The effectiveness of the
selection pressure has been examined by a large num-
ber of researchers (Liu et al., 2005) and continues to be
a research topic, especially for scale-up. Hydraulic shear
force was found to have a significant impact on the for-
mation and structure of aerobic granular sludge and the
high shearing force is one of the key factors for the forma-
tion of aerobic granules (Dulekgurgen et al., 2008). Aero-
bic granular sludge did not develop at a low superficial
air gas velocity (0.008 m/s) in an SBR, but was observed
at a high velocity (0.025 m/s). Further study showed that
granules did not form when the gas flow rate was below
0.003 m/s, the level at which the sludge was retained as
flocs without a change of the surface hydrophobicity of
the cells. In addition, the shear force changed the EPS
content and the surface hydrophobicity of the cells. The
EPS and hydrophobicity was found to be increased in the
presence of a higher shear force (Tay et al., 2002b).
The settling time can be used to select the sludge par-
ticles with an improved settling ability and to remove
flocculent sludge from the reactor. In addition, it was
reported that short settling times selected the cells with a
higher surface hydrophobicity and enhanced the content
 Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment  145
of EPS in sludge. The settling time has been considered
to be another major factor controlling granulation (Lei
et  al., 2004). Studies have demonstrated that aerobic
granular sludge is formed with a short settling time, and
the granules with a larger diameter developed as shorter
settling times were applied (Qin et  al., 2004; McSwain
et  al., 2004). Lei et  al. (2004) indicated that granular
sludge was difficult to develop when the setting time was
greater than 15 min and large-sized granules were ob-
served with settling times of between 5 and 10 min. Our
tests confirmed this observation (Gao et al., 2010).
Organic loading rate
The control of the organic loading rate to certain levels
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could be helpful for granulation. The organic loading rate
could be adjusted between a famine and feast status in
the substrate to provide a driving force for either aggrega-
tion or granulation (Shi et al., 2007). Compared to anaero-
bic granulation, aerobic granulation could be achieved
across a very wide range of organic loading rates. Forma-
tion of the granules was observed at organic loading rates
varying from 2.5 to 15 kg COD/m3•day (Liu et al., 2004).
However, with acetate as the substrate, an extremely high
organic loading rate (>21 kg/m3 day) caused disintegra-
tion of the aerobic granules (Adav et al., 2010). Zheng et al.
(2006) reported that even at a relatively high rate of 6 kg
COD/m3 day, the aerobic granules formed were compact
For personal use only.
at the beginning, but gradually transitioned to large-sized
filamentous granules. The hydrophobicity and specific
gravities of the compact granules were higher than the
filamentous ones. Li et al. (2008) indicated that the aero-
bic granules formed at different organic loading rates had
different morphologies, structural properties and bacte-
rial species. A higher organic loading rate resulted in the
formation of larger and looser granules within a shorter
period, while a lower organic loading rate led to the for-
mation of smaller and more tightly packed granules over
a longer period. The reactor with the higher organic load-
ing rate had the lowest species diversity, by contrast, the
highest species diversity was found in the reactor with the
lowest organic loading rate. Further results showed that
the optimum COD loading rate for aerobic granulation in
the SBR was 2.52 kg/m3 day (Kim et al., 2008).
Temperature
Granulation at 8°C and 25°C was compared by de Kreuk
et al. (2005a). At 8°C, the sludge was aggregated as soon as
the aeration was stopped, but the granules formed were
unstable. With a startup at 20°C and lowering the temper-
ature to 15°C and 8°C, the granules formed were stable.
Song et  al. (2009) tested granulation at 25°C, 30°C and
35°C and reported that 30°C was the optimal temperature
for aerobic granulation. At 30°C, the granules formed
had a more compact structure, better settling ability and
higher bioactivity than at other temperatures. The oxygen
utilization rate was up to 1.64 g O2/g MLVSS·day with a
COD removal rate of 97% and a total P removal rate of
75%. Zitome et al., (2007) reported the formation of ther-
mophilic aerobic granules at 55°C, but flocculent sludge
was also found in the reactor. Due to the poor solubility
of oxygen at high temperatures, a high temperature or
thermophilic aerobic granular sludge process cannot be
considered as a practicable or cost-effective approach.
Dissolved oxygen
The effect of the DO concentration on aerobic granulation
in the SBR is not clear, but the DO concentration could
be influenced by maintaining a proper shearing force via
aeration. McSwain et al. (2008) operated two SBRs with
the same shear force but with two different DO levels.
They reported that aerobic granules could not form at a
DO concentration of less than 5 mg/L, even when sup-
plied with a higher shear force. This report has not been
supported by other researchers; since granules were
formed at lower DO levels and even aerobic conditions
followed by anoxic conditions (Viaeminck et al., 2008).
The applications of aerobic granules
Laboratory-scale tests
At present, aerobic granulation technology has been
mainly tested at the laboratory scale for the treatment of
various wastewaters, nutrient removal, organic toxic com-
pound degradation, dyestuffs and metal removal, etc.
Biological treatment of organic wastewater
Aerobic granules can be developed and used to treat
various organic wastewaters including dairy, brewery,
fish can, municipal and landfill leachates. Studies have
shown that the COD removal efficiency was stabilized
in order to treat high strength organic wastewater with
aerobic granules by a step-up increase in organic loading
from 6 to 15 kg COD/m3·day (Moy et al., 2002). The upside
of the COD loading rate using aerobic granules is likely to
be limited by the oxygen mass transfer. The formation of
the aerobic granules was found to be independent of the
substrate concentration. The granules can be success-
fully formed with influent COD concentrations from 500
to 3000 mg/L (Liu et al., 2003). Arrojo et al. (2004) devel-
oped aerobic granular sludge using industrial wastewater
from dairy products with a total COD of 1500–3000 mg/L,
soluble COD of 300–1500 mg/L and total nitrogen: 50–
200 mg/L in two SBRs. One was operated with a 10–30 min
anoxic condition, but the other was always under aerobic
conditions. The reactors were applied using organic and
nitrogen loading rates up to 7 kg COD/m3·day and 0.7 kg
N/m3·day with an N removal efficiency of 70%. In another
test of the treatment of dairy wastewater, the removal effi-
ciencies of 90% for COD, 80% for TN and 67% for TP were
achieved at a volumetric exchange ratio of 50% and cycle
duration of 8 h (Schwarzenbeck et al., 2005). Wang et al.
(2007a) developed aerobic granules with brewery waste-
water after nine weeks of operation. The granules had a
high and stable removal efficiency of 88.7% for COD and
88.9% for NH4+-N with a volumetric exchange ratio of 50%
and cycle duration of 6 h. The ­aerobic granules appeared
 146  Dawen Gao et al.
on day 19 in the reactor used to treat papermaking waste-
water (Wang et al., 2006). Jiang et al. (2004) reported that
a granular sludge reactor was operated at high-strength
industrial wastewaters containing influent phenol con-
centrations ranging from 333 to 833 mg/L. After about 2
months of operation, the reactor reached a steady state
and achieved stable biomass concentrations and con-
stant phenol removal efficiencies. In addition, aerobic
granular sludge developed with an acetate solution at
25°C and also worked well at higher temperature (35°C)
to treat low-strength domestic wastewater (Liu et  al.,
2007a). di Iaconi et al. (2007) reported using a laboratory
sequencing batch biofilter granular reactor using aerobic
granular sludge to treat municipal wastewater and land-
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fill leachate. During the treatment of municipal wastewa-
ter, at a maximum organic load of 7 kg COD/m3·day, the
effluent COD was lower than 50 mg/L. The total Kjeldahl
nitrogen (TKN) removal efficiency was higher than 87%
at an organic loading rate of 5.7 kg COD/m3·day and a
nitrogen loading rate of 0.8 kg of TKN/m3·day. During
the treatment of a landfill leachate, the all biodegrad-
able contents (i.e. about 80% of the COD content of the
leachate) was removed at an organic loading rate of 1.1 kg
COD)/m3·day.
When considering the operational costs for aeration
and mass transfer limitations, application of an aerobic
granular sludge reactor to treat high concentration waste-
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water at a high organic loading rate, e.g. > 5–7 kg/m3•day is
not likely to be a cost-effective approach, because high rate
anaerobic reactors such as the upflow anaerobic sludge
blanket (UASB) type work well without aeration and with
the recovery of methane gas as an energy resource.
Nitrogen and phosphorus removal
Aerobic granules have a great potential for the removal of
nitrogen. Within an aerobic granule, a DO gradient exists
and the redox profile of the granules can be divided into
three zones, i.e. an aerobic zone followed by a micro-ox-
ygen zone and an anoxic (or anaerobic) zone with a wide
range of micro-environments. The micro-environments
allow the growth of different trophic bacteria within gran-
ules with different metabolic functions (Figure 3) includ-
ing nitrifiers, denitrifiers and anaerobes (ANOMMOX and
even methanogens). Aerobic granules have been tested
for simultaneous organic matter and nitrogen removal.
Figueroa et al. (2008) studied the treatment of wastewater
from the fish canning industry with loading rates up to
1.72 kg COD/m3·day. Ammonium nitrogen was removed
via nitrification-denitrification up to 40% when the nitro-
gen loading rate was 0.18 kg N/m3·day. The formation of
mature aerobic granules occurred after 75 days of opera-
tion with a 3.4 mm of diameter, SVI of 30 mL/g VSS and a
granule density of around 60 g VSS/L. Wang et al. (2008)
used an aerobic granular sludge membrane bioreactor
(GMBR) to treat synthetic wastewater. At influent total
organic carbon (TOC) of 56.8–132.6 mg/L, the TOC re-
moval was 84.7–91.9%. The removal of the ammonium
nitrogen of 85.4–99.7% was achieved with an influent
Aerobic heterotrophic zone
(organic matter degradation)
Nitrification zone
(ammonia oxidation)
Anoxic/anaerobic zone
(denitrification, ANAMMOX, P removal,
anaerobic degradation etc.)
Figure 3.  Conceptual aerobic granular structure. Within the
granule, the profile of each zone varies and depends on the DO
concentration outside.
concentration of 28.1–38.4 mg/L and the total nitrogen
removal was 41.7–78.4%.
Aerobic granular sludge with partial nitritation and
ANAMMOX activity was developed in a one-stage oxy-
gen-limited laboratory SBR after 1.5 months. The nitro-
gen removal rate increased from 0.05 to 0.45 kg N/m3• day
in 2 months (Viaeminck et al., 2008). This confirmed that
ANAMMOX-containing granules can be developed at
low DO conditions for the purpose of nitrogen removal.
Aerobic granules have been tested for their enhanced
biological phosphorus removal. Phosphorus removal can
be achieved using biological phosphorus removal, denitri-
fying phosphorus removal and P precipitation with aero-
bic granules (Gilda et al., 2007). Dulekgurgen et al. (2003)
used aerobic granular sludge to remove phosphorus in
an SBR with activated sludge as the seed for granulation.
The concentration of phosphorus in the reactor inlet was
20.8 mg /L, while 0.1 mg/L in the outlet with P removal up
to 99.6%. Cassidy et al. (2005) obtained removal efficien-
cies for COD and P of 98% and for N and VSS of over 97%
when operating a granular reactor fed with slaughter-
house wastewater containing a total COD of 7685 mg/L;
soluble COD of 5163 mg/L; TKN of 1057 mg/L and VSS of
1520 mg/L. You et al. (2008) used acetate as a substrate to
grow aerobic granules for phosphorus accumulation. A
COD removal efficiency of 95% was reached with close to
100% phosphorus removal efficiency. The elemental com-
position and distribution of the P-accumulating granules
was highly related to the different substrate P/COD ratios,
and the accumulation of calcium and magnesium in the
granules was closely correlated with the poly-phosphate
content of the granules (Liu et al., 2005b).
Degradation of toxic substances
Aerobic granules have been tested to treat toxic organic
chemicals, especially phenolic compounds. Tay et al. (2004)
demonstrated that the aerobic granular sludge degraded
phenol at a specific rate greater than 1 g phenol/g VSS•day
at 500 mg/L of influent phenol, or at a reduced rate of 0.53 g
phenol /g VSS• day at 1900 mg/L of phenol in SBR. Removal
of 99% of phenol from a saline wastewater at an inlet phenol
 Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment  147
concentration of up to 1000 mg/L was reported using a total
cycle time of 17 h (Moussavi et al., 2010). Ho et al. (2010)
reported that the acclimated granules effectively degraded
phenol at a concentration of up to 5000 mg/L without any
severe inhibitory effects. Yi et al. (2006) studied the aerobic
biodegradation of p-nitrophenol (PNP) in the SBR. The
degradation rate increased as PNP concentration increased
up to 40.1 mg/L, to reach a maximum specific degradation
rate and then decreased as the nitrophenol concentration
continuously increased further.
Chlorinated phenolic compounds are characterized
by their extreme toxicity, even at low concentrations.
Aerobic granules have been employed in treating waste-
waters containing chlorinated phenolic compounds.
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Aerobic granules, grown on acetate as a carbon source,
had good resistance to high 4-chlorophenol concen-
trations in the influent even if the granules could not
acclimate in the short term. The degradation of 4-chlo-
rophenol by the granular sludge after adaption was com-
plete (­Carucci  et  al., 2009). Aerobic granules were also
developed for the biological degradation of 2,4-dichlo-
rophenol (2,4-DCP) with glucose as a co-substrate. With
a step-up increase in influent 2,4-DCP concentration,
stable granules with a diameter range of 1–2 mm were
obtained after 39 days. After granulation, high removal
efficiencies of the effluent 2,4-DCP and chemical oxygen
demand were up to 94% and 95%, respectively (Wang
For personal use only.
et al., 2007b).
Aerobic granules were cultivated at a volumetric ex-
change ratio of 50% and fed with two phenols (phenol
and p-cresol) at the rate of 2.0 kg/m3•day. After two weeks,
compact regular aerobic granules were observed. The re-
moval of COD and phenol concentration was observed up
to 93% and 97% respectively (Shams et al., 2008). Phenol-
fed granules effectively degraded pyridine at a concentra-
tion of 250–2500 mg/L. The presence of phenol limited
the pyridine degradation rate, but the pyridine concen-
tration had a minimal effect on the degradation of pyri-
dine. The DGGE analysis showed a microbial community
shift after the pyridine was introduced. Acinetobactor sp.
was dominant in the pyridine-phenol degrading aerobic
granules (Adav et al., 2007b). Aerobic granular sludge also
degraded 4-t-octylphenol (4-t-OP) at a much higher rate
than the anaerobic granules. The optimal pH was 9 for
the aerobic degradation. Microbial community analysis
revealed that the biodiversity and community structures
shifted and switched under different conditions. Bacillus
and γ-Proteobacteria were identified as the major species
in the granular sludge (Liu et al., 2008).
Aerobic granules were developed to degrade methyl
tert-butyl ether (MTBE) with ethanol as a cosubstrate
in SBR. The granules were observed after 100 days. With
effluent MTBE in the range of 15–50 μg/L, the removal
efficiency of MTBE exceeded 99.9%. The specific MTBE
degradation rate was observed to increase with increas-
ing MTBE concentrations from 25 to 500 mg/L with a peak
rate of 0.544 g MTBE/g VSS·day (Zhang et al., 2008).
Biosorption of dyestuffs and heavy metals
Aerobic granules have a large surface area and high po-
rosity and are considered to be used as a biosorbent for
dyestuff and heavy metal removal. Dyes and pigments
are used for coloring textiles, tannery, food, paper and
pulp, printing, carpet and mineral processing industries,
and could be serious pollutants in the environment if
discharged without proper treatment. Sun et  al. (2008)
studied the use of aerobic granules as a biosorption ma-
terial to remove a cationic dye, malachite green (MG).
The results indicated that an alkaline pH was favorable
for the biosorption of MG. The biosorption capacity was
found to be 56.8 mg of MG per gram of aerobic granules
at 30°C.
Research results have shown that aerobic granular
sludge has a high absorption capacity for the removal
of Cd2+ and Zn2+. The maximum adsorption capacity for
Cd2+ and Zn2+ has been reported to be 566 and 270 mg
/g, respectively (Liu et al., 2002, 2003c). The adsorption
efficiency of the granules was found to be related to the
concentration of the metal and granular sludge. When
the aerobic granular sludge concentrations remained at
100 mg /L, the amount of metal absorbed was directly
proportional to the concentration of metal ions. When
Cd2+ concentrations remained at 150 mg/L, the amount of
metal removed was proportional to the concentration of
sludge (Liu et al., 2003c). The biosorption of zinc (II) and
cobalt (II) in single- and binary-metal systems by aerobic
granules was examined at different pH values. The maxi-
mal adsorption capacity of the granules, (55.25 mg Co/g;
62.50 mg Zn/g ) compared with binary systems (54.05 mg
Co/g; 56.5 mg/g Zn) showed a reduction in the accumula-
tion of these metals onto aerobic granules. The maximal
adsorption capacity of the granules was 55.25 mg Co/g
at pH 7 and 62.5 mg Zn/g at pH 5 in the single systems.
Binary-metal addition induced a competitive sorption
among the metals with a reduction of the maximal ad-
sorption capacity by 1.2 mg/g and 6 mg/g for Co(II) and
Zn(II), respectively (Sun et al., 2008a). The mechanisms
of biosorption of Cd2+, Cu2+, Ni2+ and Cr3+ by the aerobic
granules were tested. The results showed that the remov-
al of these metal ions was due to ion exchange, binding
to extracellular polymers and chemical precipitation. It
was also found that K+, Mg2+ and Ca2+ were released into
the bulk solution during the Ni2+ uptake onto the aerobic
granules (Xu et al., 2006; Xu et al., 2008). The Ni2+ biosorp-
tion by aerobic granular sludge and the zeta potential of
the aerobic granules were studied at various initial pHs.
The Ni2+ and Cr3+ biosorption capacity increased with an
increase in pH and the optimal pH was 6 and 5, respec-
tively (Xu et al., 2006; Yao et al., 2009). The equilibrium,
thermodynamics and mechanisms of Ni2+ biosorption
were also investigated. The Ni2+ biosorption was associ-
ated with a significant release of Ca2+ ion from the aero-
bic granules. X-ray diffraction spectroscopy showed that
chemical precipitation of Ni2+ was not largely involved in
the Ni2+ biosorption by the aerobic granules and also that
 148  Dawen Gao et al.
the Ni2+ biosorption by aerobic granules was an endo-
thermic process (Liu et al., 2007a).
Modeling
Investigators have developed various mathematical
models to simulate the bioreactions within aerobic
granular sludge and granular sludge reactor systems.
Based on the thermodynamics of biosorption reac-
tions, a general model was developed to describe the
equilibrium biosorption of three heavy metals namely,
cadmium, copper and zinc by aerobic granules. The
biosorption capacity of individual Cd2+, Cu2+ and Zn2+
onto aerobic granules was estimated to be 172.7, 59.6
and 164.5 mg/g, respectively. This model provided good
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Michigan State University on 03/24/11
insights into aerobic granules-based biosorbers for the
removal of heavy metals (Liu et al., 2003a). A model was
developed to describe a granular sludge SBR for the si-
multaneous removal of COD, nitrogen and phosphate
(de Kreuk et al., 2007a). The optimum granule diameter
for maximum N- and P-removal at a DO of 2 mg/L and
at 20°C was between 1.2 and 1.4 mm with an optimum
COD loading rate of 1.9 kg COD/m3•day, followed by an
anoxic phase for further N-removal. A model of the si-
multaneous storage and growth processes in an aerobic
granular SBR fed with soybean-processing wastewater
was developed including the adsorption process, mi-
crobial maintenance, and substrate diffusion (Ni et  al.,
For personal use only.
2008). Another model was proposed to simulate the
formation of extracellular polymeric substances (EPS),
soluble microbial products (SMP), and internal storage
products (X-STO) in aerobic granular sludge (Ni et  al.,
2010). The results indicated that a higher substrate con-
centration resulted in a greater concentration of EPS,
SMP, and X-STO. An energy-dissipation based model
was used to quantitatively describe the shear stresses in
a granular sludge SBR (Ren et al., 2009). Gas bubbles and
collisions were found to be the main source of the shear
stresses at the granule surface.
When the overall reactor behavior was described
(substrate removal or sludge production), traditional
biofilm modeling can be used for aerobic granules (de
Kreuk et  al., 2007). Multiscale models were developed
by research groups at Universidade Nova de Lisboa in
Portugal, Waseda University in Japan and TU Delft in the
Netherlands. Wichern et al. (2008) developed a dynamic
mathematical model describing COD and nitrogen
removal from dairy wastewater in a granular SBR. The
modeling results were based on granules with an average
diameter of 2.5 mm and density of 40 g VSS/L. The opti-
mal operation conditions for nitrogen removal was a DO
concentration of below 5 mg O2/L. The lower DO concen-
trations led to thinner aerobic but thicker anoxic granular
layers with higher nitrate removal efficiencies. They sug-
gested that the total SBR-cycle times should be between
360–480 min. The reduction of the cycle times from 480
to 360 min with a 50% higher throughput resulted in an
increase of the peak nitrogen effluent concentrations by
40%. The volumetric loading rate should be higher than
4.5 kg COD/m3•day. Higher influent COD loads with a
COD volumetric loading rate of 9 kg COD/m3•day led to
an almost complete nitrogen removal.
Pilot applications
Aerobic granulation technology for wastewater treatment
has been mostly studied at a laboratory scale with a few at
the pilot scale (de Kreuk et al., 2007). To date, there is no
published data yet available about the operational perfor-
mance of full scale applications, although several full-scale
plants have been built in the Netherlands, Portugal and
South Africa (personal communication to Dr. Merle de
Kreuk). Only limited information about pilot studies has
been reported. A pilot plant was started up in September
2003 in Ede, the Netherlands. The plant consisted of two
parallel biological reactors with a height of 6 m and diam-
eter of 0.6 m, operated at a flow rate of 5 m3/h (volume of
1.5 m3). A sequencing batch biofilter granular reactor with
a volume of 3.1 m3 was developed to treat sewage at an Ital-
ian wastewater treatment plant. A company, EcoEngineer-
ing cultivated the granules on-site in small bioreactors for
the start up of a pilot plant with a volume of 2.7 m3 located
in a Hungarian pharmaceutical company. A pilot-scale
SBR for municipal wastewater treatment was constructed
in the Zhuzhuanjing Wastewater Treatment Plant, Hefei,
China. The SBR system had two column reactors, 6 m in
height, 0.5 m in diameter, and had a working volume of 1
m3 (Ni et al., 2009) as shown in Figure 4.
A previous feasibility study, both in laboratory and
pilot scale, showed that aerobic granular sludge tech-
nology is very promising (de Bruin et al., 2004; de Kreuk
et  al., 2007). Based on total annual costs, a granular
SBR (GSBR) with pre-treatment and a GSBR with post-
treatment proved to be more attractive than the reference
system of activated sludge alternatives (6–16%). A sensi-
tivity analysis showed that the GSBR technology was less
Figure 4.  Pilot-scale (1 m3) SBR with aerobic granules for
municipal wastewater treatment at Zhuzhuanjing WWTP, Hefei,
China.
 Aerobic granular sludge: characterization, mechanism of granulation and application to wastewater treatment  149
sensitive to land price and more sensitive to rain water
flow. The footprint of the GSBR variants was only 25%
compared to the reference because of the high allowable
volumetric load. However, the GSBR, with only primary
treatment, cannot meet the present effluent standards
for municipal wastewater, mainly because of the washout
of the less settleable biomass.
Perspectives
At present, although the research on aerobic granular
sludge has made significant progress, future study is
still needed in order to fundamentally understand the
granulation and also for development as a cost-effective
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Michigan State University on 03/24/11
technology. Future study needs can be summarized ac-
cording to the following aspects:
(1) Factors influencing granulation have been identi-
fied, including selection pressure (hydraulic shear
force and settling time), but the results have been
mainly obtained at a laboratory scale with glucose-
containing synthetic wastewaters. Further study is
needed using industrial or municipal wastewater to
achieve granulation at a large scale within a reason-
able start-up period.
(2) Stability of aerobic granular sludge has been studied
For personal use only.
for years. More research is still needed to understand
the stability for its long-term operation at largescale.
There are no research reports available about gran-
ules containing ANAMMOX bacteria. It is difficult
to apply aerobic granular sludge technology at full-
scale until the strategy for the maintenance of the
stability is better understood.
(3) The mechanism of aerobic granulation is based
on several hypotheses and is still not clear due to
the complexity of the target substrates and micro-
bial populations involved. At present, most of the
research results are based on several hypotheses
conducted under limited conditions and lack sup-
port by quantitative data. Therefore, the mechanisms
of aerobic granular sludge have to be explored fur-
ther. The study of the mechanisms in relation to
microbial community structures or dynamics should
also be addressed.
(4) Nitrogen and phosphorus removal by an aerobic
granular process appears promising and should
be investigated further and based on the microbial
ecology associated with a chemical engineering
approach. Research on granules with ANAMMOX
activity should be highly emphasized. The microbial
ecosystem of the granules should be addressed to
improve nitrogen and phosphorus removal.
(5) Research on the microbial community of aerobic
granular sludge using molecular biotechnology as
a tool is still limited, especially regarding the com-
munity structure dynamics during the granulation
process and various maintenance periods. The
further study of stability may require monitoring the
microbial community dynamics and proteogenom-
ics to understand the influence of microbial activity
on granulation and stability
Aerobic granular sludge has been tested for wastewa-
ter treatments mainly at a laboratoryscale and limited
pilotscale. To our knowledge, no reports on full-scale
application have been published, although several full-
scale plants have been built. Therefore, there is still a
lack of technical experience for the application and for
an economic analysis of the aerobic granulation process.
A large and full-scale demonstration must be conducted
to prove the technical and economical feasibility of the
aerobic granular process.
Declaration of interest
This research was supported by the Foundation for Au-
thor of National Excellent Doctoral Dissertation of the
P.R. China (abbr. FANEDD; No.200544), and the Research
Fund for the Doctoral Program of Higher Education, Min-
istry of Education of the P.R. China (20092302110059),
and the Scientific Research Foundation for the Returned
Overseas Chinese Scholars, Heilongjiang Province
(LC07C06), and the Scientific Research Foundation
for the Innovative Talents, Harbin City Government
(2007RFLXS002).
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