Review

Finding Knowledge Gaps in

Aerobic Granulation
Technology
Saurabh Jyoti Sarma,1 Joo Hwa Tay,1,* and Angus Chu1
This review identifies the knowledge gaps in aerobic granulation technology and
Trends
defines some problems for future studies. In particular, extracellular polymeric
Better settleability, a smaller footprint,
substances (EPSs) should be further characterized to understand the intermo- reduction in sludge volume, a minimum
lecular interactions among these polymers, the role of chelating agents in loss of active biomass, and an ability to
maintain a high biomass concentration
destabilizing EPS ionic bridges needs further elucidation, and early detection during wastewater treatment explain
of the quorum-quenching enzymes should be considered to avoid granule the popularity of aerobic granulation
segregation and process failure. Furthermore, the process should be supple- technology.

mented with volatile fatty acids as electron donors/carbon sources, and appro- Autoinducer molecules, quorum-
priate anoxic/anaerobic conditions should be provided for enhanced nitrogen quenching enzymes, and extracellular
polymeric substances are some of the
and phosphorus removal. Finally, the biodegradation, bioaccumulation, bio-
emerging research areas for investigat-
sorption, and mass transfer behaviors of the emerging contaminants within the ing the molecular mechanisms of gran-
granules need further investigation. ule formation.

Simultaneous biodegradation, bioac-

cumulation, and biosorption of the
Aerobic Granulation Technology emerging contaminants, which could
Aerobic granulation is a relatively new technology for wastewater treatment that has a number of not be achieved by conventional treat-
promising features (Box 1). Granule development is the backbone of the technology, but it is the ment, is a prospective research area of
least elucidated aspect. There are many theories to explain the granulation process; however, it this technology.

is difficult to support them with conclusive experimental evidence. Recently, a four-step granu- Long granule formation and maturation
lation mechanism has been proposed. It involves (i) initial cell–cell attachment to start the time, poorly understood molecular
process, (ii) development of microaggregates by these self-attached cells, (iii) extensive extra- mechanisms, granule disintegration,
unpredictable granule morphology,
cellular polymeric substance (EPS) biosynthesis by the aggregated microorganisms, and (iv)
and inefficient nutrient removal are
granule maturation in response to the external hydrodynamic parameters applied by the reactor some of the unresolved problems of
configuration and operating conditions [1]. A schematic overview of this mechanism is shown in this technology.
Figure 1. In general, microbial cell surfaces have negative charges. Repulsions among the similar
charges prevent the cells from attaching to each other without the help of another mechanism.
Neutralization of the microbial surface charges by divalent cations such as Ca2+ has been
considered as a possible mechanism facilitating initial cell–cell attachment. The van der Waals
force may also assist this cell–cell attraction.

Proton translocation (see Glossary) and the subsequent dehydration of the cell membrane is
another mechanism that may explain the initiation of granule formation. According to this
mechanism, fermentation of the substrate activates the proton pumps involved in proton 1
Department of Civil Engineering,
translocation across the cell membrane. The proton gradient established by this activity Schulich School of Engineering,
may protonate the cell surface, neutralize the negative charges, cause dehydration, and make University of Calgary, Calgary, AB T2N
the surface slightly hydrophobic. Thus, hydrophobic interaction among microbial cell surfaces 1N4, Canada

has been considered as a dominant mechanism of initial cell–cell adhesion of the granulation
process [2]. Therefore, there may be more than one mechanism responsible for cell–cell *Correspondence: jhatay@ucalgary.ca
attachment. (J.H. Tay).

66 Trends in Biotechnology, January 2017, Vol. 35, No. 1 http://dx.doi.org/10.1016/j.tibtech.2016.07.003

© 2016 Elsevier Ltd. All rights reserved.
 Box 1. Features of Aerobic Granulation Technology Glossary
Anaerobic granules were discovered in 1976, before aerobic granules. The first report on self-immobilized suspended- Autoinducer molecules: small lipid,
growth aerobic activated sludge appeared in 1991 [41,42]. Extensive research activities on aerobic granulation oligopeptide or borate ester
technology were mostly started during the period of 1998 to 2001. Aerobic granules are basically quorum sensing molecules secreted by microbial cells
mediated auto-immobilized microspheres of mixed microbial consortium and are typically 1–3 mm in diameter. Their as a part of a quorum sensing
outer surfaces are dominated by aerobic microorganisms, whereas the inner core regions may contain facultative and mechanism for intercellular
obligate anaerobic microorganisms as well as dead microbial biomass. They are almost exclusively produced in communication.
sequential batch reactors, and it may take one week to several months to develop mature granules with stable Biosorption: a physicochemical
COD removal efficiency. Unlike free or flocculated microbial cells in traditional wastewater treatment processes, compact process by which both living and
and heavier aerobic granules need very short settling times to be separated from the treated effluent. Large sludge dead microbial cells adsorb different
settling tanks are no longer required for this technology; therefore, a large amount of wastewater can be treated within contaminants without expending
one-fifth of the space occupied by a conventional treatment plant. Using this technology, it is possible to maintain around energy from metabolic activities.
three to five times more biomass per liter of the wastewater, which allows a larger water volume to be treated in a short Dewaterability: a characteristic of
period of time. Reduction in sludge volume is the other benefit of this technology. It can reduce the amount of biomass sludge that indicates how easily
released with the treated effluent, so cell-bound pollutants or nutrients are less likely to escape the system. water can be removed from a
particular type of sludge. Sludge may
During full-scale operation to treat real wastewater, aerobic granulation technology generates granular sludge with contain as much as 95% water, and
slightly different properties than conventional activated sludge. Their granular nature and crosslinked EPSs might affect for effective sludge management,
their biodegradability. The technology is actively being researched and developed, and the issues such as sludge removal of such water is desirable.
digestibility and dewaterability may not appear relevant now. However, during the technology's widespread real Emerging contaminants:
application, such issues may become problematic. environmental contaminants including
certain pharmaceuticals, personal
care products, and industrial
chemicals that may have negative
As shown in Figure 1, in the second step of the process, the attached cells gradually develop into
effects on human health or the
microaggregates of microorganisms by attaching to new cells. This step is followed by EPS receiving ecosystem, but without
production by aggregated microorganisms to develop immature granules. Granule maturation is strict guidelines on their
the final step of this process, where the granules become compact and reach their maximum environmental release and
monitoring.
diameter of around 1–3 mm. Anaerobic and facultative anaerobic microorganisms and dead Proton translocation: a
microbial cells constitute the core regions of the granules, whereas aerobic microorganisms phenomenon by which proton pumps
occupy the surfaces. The reactor geometry, type of substrate, and nucleating agents are some present in the biological membranes
other factors that influence the granulation process. Aerobic granules have been reported to (e.g., cell membrane, mitochondrial
membrane) transfer protons across
form in reactors with a wide range of height to diameter (H:D) ratios (0.39:10). Usually, a high H:D the membrane and may establish a
ratio is favorable for rapidly producing granules, whereas for the practical implementation of this proton concentration gradient.
technology in real wastewater treatment, a low H:D ratio is preferred. Similarly, between acetate- Rhizobia: a group of soil bacteria
capable of fixing nitrogen to plants.
and propionate-fed processes, granule development is slower in the propionate process, but it
They establish a symbiotic
produces stronger granules. Likewise, a nucleating agent such as granular activated carbon relationship generally with leguminous
particle can accelerate the granule formation process. These agents act as nuclei on which the plants and form root nodules. Inside
initial cell attachment takes place. These conclusions about the granulation process are these nodules, they convert
atmospheric nitrogen to ammonia by
observation-based; however, the actual molecular biological natures of these granules remain
using nitrogenase enzymes.
poorly understood. Selection pressure: in the present
context, a set of environmental
Even in the absence of complete information about the underlying mechanisms, efforts have factors such as temperature, DO,
COD and settling time that favor a
been made to optimize the granulation process. Engineering parameters such as the organic
certain group of microorganisms to
loading rate, shear stress, air flow velocity, hydraulic retention time, selection pressure, and dominate a microbial consortium.
microbiological parameters such as the type of dominant microorganisms, synthesis of EPS, Settleability: the speed at which a
and quorum sensing, have been investigated to establish their roles in the granulation process. particular type of sludge can settle
down during a wastewater treatment
However, all these data are not sufficient to develop a precisely predictable system. Therefore, process. More compact microbial
the purpose of this review is to highlight trends in recent research activities and to identify the aggregates have better settleability.
issues that merit further study. Quorum sensing: population-density
dependent gene expression
coordination observed in microbial
Granule Initiation and Development communities. In correlation with the
In order to gain popularity in real industrial or municipal wastewater treatment, the granule density of the microbial cells within a
development process should be predictable, in terms of the reproducibility of the results, and community, autoinducer molecules
are released to the surroundings. In
precisely controllable, by changing the process parameters (air flow rate and substrate con-
response to the concentration of
centration, among others). Although the installation of full-scale plants has been reported, these molecules, gene expression is
granulation mechanisms still need fundamental microbiology-level studies. For instance,

Trends in Biotechnology, January 2017, Vol. 35, No. 1 67

autotrophs are important parts of the nutrient removal process; however, their contributions to coordinated for a specific purpose
granule formation are not known. Some microorganisms can store excess nutrients in the form such as biofilm formation.
Quorum quenching: a phenomenon
of glycogen or polyhydroxyalkanoates (PHA). Whether they have any role in supplying energy for observed in microbial population
forming and maintaining the granules is not understood. The importance of microbial morphol- coordinated by a quorum sensing
ogy for granule formation has not been studied. By optimizing the process parameters, the time mechanism. As part of this
required for granule formation can be reduced (Table 1). Selection pressure has been consid- phenomenon, autoinducer molecules
involved in quorum sensing are
ered as one of the driving forces behind granulation. However, the molecular mechanisms hydrolyzed by certain microorganism-
underlying process parameter optimization or selection pressure are not known. Because secreted enzymes to disrupt the
granule formation is unpredictable, reproducing the process performance becomes difficult. molecular mechanism of quorum
sensing.
Therefore, further studies should be focused on developing more predictable processes based
on fundamental knowledge.

Liu et al. reviewed some probable mechanisms of anaerobic granule formation in an upflow
anaerobic sludge blanket (UASR) reactor in 2003 [3]. Various similar theories have been
proposed for aerobic granulation as well. Aerobic granules are mostly produced during waste-
water treatment in sequenced batch reactors (SBRs). The process conditions and microbial
communities are not the same in aerobic versus anaerobic systems, so further investigations to
compare the aerobic and anaerobic granule formation mechanisms would be useful to broaden
our understanding on initiation and development of granules. The common factors involved in
aerobic and anaerobic granule formation should be identified as the primary factors, or basic

Mechanisms: Mechanisms: Mechanisms:

(a) Proton translocaon
(b) Surface charge neutralizaon
(c) Cell surface hydrophobicity
(d) Van der waals force
(a) Quorum sensing (a) Effect of hydrodynamic forces
(e.g., air and water flow)
(b) Environmental
signals (e.g., (b) EPS synthesis
nitrogen starvaon) (c) Formaon of ionic-bridges
(d) Selecon pressure
(e) Growth of immobilized
biomass

EPS Aerobic zone

Anoxic/
anaerobic zone

Core zone with

dead cells

Step 2: Micro-
Step 1: Cell- aggregate Step 3: EPS Step 4:
Microbial cells cell adhesion formaon synthesis Maturaon Mature granule

0-30 days 30-60 days 60-200 or more days

Figure 1. The Aerobic Granule Formation Process. Schematic representation of the aerobic granule formation process and the mechanisms involved in each step.

68 Trends in Biotechnology, January 2017, Vol. 35, No. 1

Table 1. A Summary of Optimum Process Conditions and Granulation Time Reported for Sequencing Batch
Reactors
Granulation Mature Study Medium COD Reactor Temperature DO/air Refs
started granules duration operation (8C) flow rate
(days) (days) cycle

2 daysa, – 40 Synthetic 400– 4h 25 20 L/min [43]

5 daysb wastewater 800 mg/L

6 days – 240 Synthetic 4.2–5.5 mg 4h 17–22 5 L/min [44]

medium COD/L/day

7 days 15 – Synthetic 6.0 mg 4h 25 2–6 L/min [45]

wastewater COD/L/day

7 days 50 50 Real 200– 4h 18–30 – [46]

wastewater 600 mg/L

7 days – 85 Synthetic 8 mg 3h – Superficial [47]

wastewater COD/L/day air velocity
25 cm/s

17 days 56 60 Palm oil 2.5 mg 3h – Superficial [48]

mill effluent COD/L/day air velocity
2.5 cm/s

20 days 30 90 Synthetic – 4h 25 3.5–4 mg/L [49]

wastewater

30 days – 200 Real 600 mg/L 6h 16–27 >4 mg/L [50]

wastewater +
synthetic
medium

35 days 50 60 Synthetic 500 mg/L 6h 25 – [51]

medium

40 days 70 160 Synthetic 400 mg/L 4–8 h – >2 mg/L [52]

medium

After 130 330 Seafood 2–5 mg 3h 15–20 4–8 mg/L [53]

90 days industry COD/L/day
effluent

a
Stored granules revived.
b
When stored granules were used as seed.

requirements, of granulation. For instance, EPS synthesis, quorum sensing, cell surface hydro-
phobicity, and ionic bridges are the primary factors responsible for both aerobic and anaerobic
granule formation. Any factors that are not common in both aerobic and anaerobic granule
formations should be identified as secondary factors. Such secondary factors might decide the
quality or appearance of the granules, but granulation may occur even in their absence. The type
of substrate is one such factor. For instance, the morphological properties of granules prepared
by an acetate-fed process are slightly different from those of a propionate-fed process.
However, each of these substrates can support granule formation in the absence of other.
Thus, in order to understand the basic mechanisms, research and development activities should
be focused on the primary factors.

Extracellular Polymeric Substances (EPSs)

The EPS is one of the primary factors responsible for granule formation, and its role in the
process has already been established [4]. It consists of polysaccharides, proteins, lipids, nucleic
acids, and humus, among other components, where proteins and polysaccharides are the
major constituents [5,6]. Usually, the protein/polysaccharide ratio of the EPS matrix is approxi-
mately 3.4:6.2 [5,6]. b-Polysaccharides are mostly responsible for mechanically stabilizing the

Trends in Biotechnology, January 2017, Vol. 35, No. 1 69

granules [4]. As shown in Figure 1, the third and fourth steps of the granulation process are
dominated by EPS synthesis by the aggregated microorganisms. There are different inade-
quately explained aspects of EPSs that require detailed investigation. For instance, whether
some particular species, or almost all of the microorganisms present in the granules, are
responsible for EPS production is still largely unknown. Polysaccharides such as alginate might
play an important role in granule formation [7,8]. Therefore, research should be concentrated to
identify the metabolic pathways involved in the production of these polymers. Exopolysacchar-
ide synthesis in rhizobia is determined by external parameters such as the carbon source,
availability of nitrogen and phosphorus, and ionic strength of the medium; this synthesis is
controlled by transcriptional and post-transcriptional regulations [9]. Sinorhizobium meliloti and
Rhizobium leguminosarum are the two species of rhizobia where molecular mechanisms of
exopolysaccharide synthesis have been investigated to the greatest extent [9]. In the future, it
would be advantageous to consider such well-studied microorganisms for investigating the
molecular biology involved with EPS synthesis. However, being soil bacteria, these species are
less likely to be actively involved in aerobic granule formation. Therefore, the presence and
activities of these organisms in aerobic granules should be determined before considering them
as model organisms for studying EPS synthesis.

A synthesis of the literature on EPS characterization is provided in Table 2. It suggests that

previous characterization has mostly been limited to the extraction and identification of the
polymers as proteins and polysaccharides. Individual polymers are not precisely identified;
therefore, some novel molecules might emerge as the most important for the granulation
process. In this context, prior to any genetic- or molecular-level study, properly identifying
the EPS components is necessary. Furthermore, the polymer–polymer interaction in the EPS
matrix and the roles of already-identified polymers such as bacterial cellulose and algal lignin
should be studied.

Quorum Quenching
Quorum sensing is considered to be one of the molecular-level events involved in aerobic
granule formation and is largely understood as beneficial for the process [10–12]. It is another
primary factor responsible for granule formation. The process of quorum sensing includes the
population-dependent synthesis and release of autoinducer molecules and their detection by
surrounding microorganisms in order to coordinate their gene expressions [13]. N-Acyl homo-
serine lactones and autoinducer 2 have been identified as the major autoinducer molecules
responsible for quorum sensing [14,15]. Alternatively, quorum quenching is an opposite
phenomenon responsible for quenching or negating the quorum-sensing activities [16]. One
of the mechanisms of quorum quenching is the deactivation of autoinducer molecules [17].
There are different quorum-quenching enzymes that can break down the autoinducer molecules
[18], including N-acylated homoserine lactone (AHL) lactonase, AHL acylase, AHL oxidoreduc-
tase, decarboxylase, deaminase, and paraoxonase, among others [19]. Thus, the production of
quorum-quenching enzymes can reduce the quorum-sensing activities that affect granule
formation.

Quorum-quenching enzymes have been studied for their potential application in therapeutic
purposes [16,20]. For such applications, the production of the quorum-quenching enzymes is
enhanced to reduce quorum-sensing activities, and virulence or biofilm formation could there-
fore be minimized. Therefore, most of the research on quorum-quenching enzymes has been
focused on the enhanced production of these enzymes or on their applications in reducing
quorum-sensing activities [21–24]. Contrary to these applications, quorum-quenching enzymes
are undesirable for granulation technology. By interfering with quorum sensing, which is other-
wise considered to be beneficial, quorum-quenching enzymes may delay the granulation
process or destabilize mature granules. Therefore, it will be interesting to explore the possible

70 Trends in Biotechnology, January 2017, Vol. 35, No. 1

 Table 2. A Synthesis of Different Reports on Aerobic Granule EPS Characterization
Granule origin EPS extraction EPS analysis EPS components Conclusions Refs

Phenol-rich synthetic Formamide and NaOH (protein Granule hydrolyzed with b-Polysaccharides, b-Polysaccharides are mostly [4]
wastewater-fed SBRa and carbohydrate), methanol proteinase K, lipase, /-polysaccharides, lipids, responsible for stability
and chloroform (lipid) /-amylase and b-amylase proteins

Sodium acetate-rich synthetic Grounded, extracted with TOC, humus, protein, and Polysaccharides, proteins, EPSs of large granules have [54]
wastewater-fed GMBRa ultrapure water carbohydrate analysis humus, TOC lesser amounts of
polysaccharides and proteins

Glucose-, sodium acetate-, Phosphate-buffered saline, Protein and carbohydrate Polysaccharides, anionic Molecules of less than 1 kDa to [55]
sodium propionate-, and sonication, Tween 20, EDTA analysis, size exclusion proteins 1000 kDa detected
ethanol-fed SBR chromatography, anionic
exchange chromatography

Glucose- and peptone-fed Cation-exchange resin, NaOH TOC, protein analysis, Polysaccharides, proteins Noncellular proteins were [56]
SBR and heat extraction carbohydrate analysis, dominant in the cores
fluorescent staining

Sodium acetate-rich synthetic Cation-exchange resin Protein analysis using b-Polysaccharides, proteins, EPSs have five times more [5]
wastewater-fed SBR fluorescent dyes binding, nucleic acids protein than polysaccharides
carbohydrate analysis

Phenol-rich synthetic Ultrasound + formamide Protein, carbohydrate, humic Polysaccharides, proteins, Protein: [6]
wastewater-fed SBR + NaOH and nucleic acid analysis humus, lipids polysaccharide = 3.4:6.2

Phenol-rich synthetic Ultrasound + phosphate- Protein and carbohydrate Polysaccharides and proteins Protease activity breaks bonds [57]
wastewater-fed SBR buffered saline analysis between proteins and Mg2+

a
Abbreviations: SBR, sequencing batch reactors; GMBR, granular membrane bioreactor.
Trends in Biotechnology, January 2017, Vol. 35, No. 1
71
correlations between the activities of quorum-quenching enzymes and the initiation, develop-
ment, and stability of aerobic granules. If any correlation is observed, the process parameters
could be optimized to minimize their effects on granule formation and stability. If these enzymes
are found to be detrimental for the stability of the granules, detecting them early in the process
would be a useful early warning tool for granule destabilization and process failure.

Before using quorum-quenching techniques on aerobic granule-based wastewater treatment

technology, it would be more logical to better understand quorum-sensing mechanisms. There
is a strong correlation between the attachment ability of aerobic granular sludge and the
concentration of quorum-sensing autoinducer molecules such as AHLs [12]. However, their
role in regulating gene expression to bring out phenotypic changes leading toward granule
formation is not elucidated. Similarly, how environmental parameters trigger the synthesis of
quorum-quenching enzymes is also not known. Autoinducer molecules are the common links
between the two phenomena. Therefore, fundamental investigations on autoinducer molecules
might shed light on both quorum sensing and quorum quenching.

Ion Chelating Agents

Multivalent cations such as Ca2+, Mg2+, and Fe2+ are considered to be essential for granulation.
Ca2+ ions may crosslink anionic polysaccharides such as alginate molecules present in the EPS,
and they play a role in granule formation and stability. These ionic bridges are one of the primary
factors responsible for granule formation. In this context, it has been observed that sodium
alginate microspheres, prepared using CaCl2 as the crosslinking agent, were completely
dissolved within 24 hours in presence of the phosphate ions [25]. Phosphate ions are recog-
nized as ion chelating agents and used to dissolve calcium-alginate gel [25,26]. This observation
indicates that the presence of chelating agents such as phosphate ions (PO43–) in the waste-
water may destabilize the aerobic granules by chelating the multivalent cations involved in the
ionic bridges of the EPS.

This hypothesis suggests that the release of soluble phosphate (PO43–), either by solubilizing
inorganic phosphate precipitates such as Ca3(PO4)2 by organic acids present in the wastewater,
or by the microorganisms during biological phosphorus removal, might be related to the
destabilization of aerobic granules. Around 60–70% of the phosphorus removed by granular
biomass is in the form of calcium phosphate precipitated within the granules [27]. During this
process, a portion of the Ca2+ ions involved in the ionic bridges of the EPS would also be
precipitated as calcium phosphate, which, in turn, may destabilize the granules.

Apart from the phosphate ion, wastewater may contain other ion-chelating agents such as
amino acids, peptides, organic acids, and others. These potential chelating agents can be both
released and taken up by the microorganisms present in the wastewater. Therefore, whether
the alternating ‘feast and famine’ conditions applied for granulation are at all related to the
uptake and release of these agents and stability of the granules should be investigated. The
cores of the granules have different microorganisms, which can produce proteases to degrade
extracellular proteins [28]. Protein hydrolysis will release amino acids and peptides, not all of
which may be subsequently consumed by the surrounding microorganisms. Therefore, they
may be responsible for destabilizing the granule by chelating the cations; this aspect needs
further investigation.

Granule Segregation
At present, how long the healthy granules could be maintained in a wastewater treatment
process is unpredictable. The exact molecular mechanism behind the disintegration of aerobic
granules has not been elucidated [29]. In fact, the factors influencing the instability of the granules
have not been clearly identified.

72 Trends in Biotechnology, January 2017, Vol. 35, No. 1

An increase in their size due to microbial growth is a probable factor responsible for the instability
of the granules. The presently available information is not sufficient to predict the growth rate of
the microorganisms within the granules; therefore, their size and diameter remains largely
unpredictable. In general, stable granules with a diameter of 1–3 mm are effective in maintaining
a large number of viable cells. However, there is no reliable method to keep the diameters of the
granules constant. The presence of phosphorus-accumulating microorganisms is another
probable reason for granule segregation; however, the mechanism is not clearly understood
[30]. As discussed in the previous section, one possible reason is that under anaerobic
conditions, phosphorus-accumulating microorganisms can release soluble phosphorus
(PO43–) into their surroundings. Such phosphorus might be precipitated as calcium or magne-
sium phosphate, making these multivalent cations unavailable for crosslinking the polymers of
the EPS matrix. A study suggests that granule segregation is favorable for anaerobic ammo-
nium-oxidizing bacteria [31]. Therefore, granule segregation could be an outcome of different
types of factors, ranging from process parameters to extensive microbial growth, availability of
free multivalent ions, and competition among different groups of microorganisms. It may not be
possible to completely stop granule segregation. Alternatively, optimizing the process for the
concomitant segregation and regeneration of granules could be a better approach to deal with
this problem.

Selective Enrichment of Microorganisms for Nutrient Removal

The biological nitrogen removal process has two major steps: nitrification and denitrification.
During nitrification, ammonia is oxidized to nitrite and nitrate, whereas denitrification involves
reduction of nitrite or nitrate to molecular nitrogen. Denitrification of nitrite is 1.5–2 times faster than
that of nitrate [32]. Autotrophic ammonia-oxidizing bacteria convert most of the ammonia to nitrite,
but nitrate production is negligible. Therefore, granules dominated by autotrophic ammonia-
oxidizing bacteria are more suitable for nitrogen removal than those dominated by nitrite-oxidizing
bacteria. However, no further information on their taxonomic position and metabolic pathways is
available, which makes it difficult to identify and optimize the process parameters to prepare
granules dominated by these bacteria. Likewise, two different unclassified groups of denitrifying
Accumulibacter have been found in aerobic granules [33]. One of these bacteria can drive the
denitrification reaction by coupling phosphorus uptake with nitrate reduction, whereas the other
cannot. Selective enrichment of the group capable of simultaneous phosphorus removal and
denitrification would enable the same process to remove both nutrients. Thus, the process
conditions promoting such enrichment should be identified. In this context, the colors of the
granules could be used as qualitative indicators of the dominant microorganisms (Box 2).

Reports of nitrogen and phosphorus removal by aerobic granules are summarized in Table 3. At
high initial nitrogen concentrations of around 250 mg/L or above, these removal processes fail to

Box 2. Colors of Aerobic Granules and their Significance

Microbial granules can be of different colors, such as white, yellow, yellowish brown, gray brown, black, carmine, and red,
although they are mostly either yellow or yellowish–brown. The coexistence of microbiologically different white and yellow
granules has been reported for biological phosphorus removal processes. White granules are dominated by Candidatus
Accumulibacter phosphatis, whereas yellow granules are dominated by Candidatus Competibacter phosphatis. Micro-
organisms are homogenously distributed within the white granules, and they are comparatively more compact and
smooth. It has been postulated that yellow granules are formed by aggregation of various microcolonies, whereas each
white granule is developed from only one such colony [30]. Therefore, if the process could be acclimatized in such a way
that it has more white granules dominated by phosphorus-accumulating bacteria, then better phosphorus removal could
be expected. In fact, the metabolic activities and microbial population of aerobic granules are largely malleable, and
further investigation is needed to develop proper protocols for acclimatizing the granules for specific purpose. Although
precise molecular biological techniques will be more effective in optimizing the process parameters for enriching desired
microorganisms, the colors of the granules could be good qualitative indicators of their metabolic activities. For example,
brownish-yellow aerobic granules gradually turning gray-brown may indicate the transition from aerobic to anaerobic
metabolism.

Trends in Biotechnology, January 2017, Vol. 35, No. 1 73

Table 3. A Synthesis of Different Reports on Nutrient (N and P) Removal by Aerobic Granulation Technology
Target Permitted Initial Treated effluent Removal Aerobic/ Refs
nutrient effluent concentration concentration efficiency anoxic/
concentration (%) anaerobic

Nitrogen 6 mg/La 50  12 (N-NH4+), 26 mg/L (NO3–N), >97 Airflow rate [58]

1057  63 mg/L 2  2 mg/L (TKN) 100 L/h
(TKN)

30 mg/L 0.008 mg/L 94 Low DO (20%) [59]

(N-NH4+), 1.7 mg/L
(NO3–N), ˂0.01 mg/
L (NO2–N)

50 mg/L (N-NH4+) ˂1 mg/L (nitrate-N), 95 Alternative high [60]

0.4 mg/L (N-NH4+) and low DO

257.4  69.3 mg/L 20.8  28.1 (mg/L) 92.3  8.5 Airflow rate [61]
(N-NH4+) 120 L/h

48.70 mg/L 5.6 mg/L 88.5 Airflow rate [62]

390 L/h

 35 mg/L (N-NH4+) – 70–85 Airflow rate [63]

240 L/h, DO
over 90%

1079.3  91.3 mg/ 601.9  54.6 mg/L 44.1  4.4 Airflow rate [61]
L (N-NH4+) 120 L/h

Phosphorus 0.25 mg/La 10–15 mg/L ˂1.0 mg/L >90 Airflow rate [62]
390 L/h

217  38 mg/L 4  2 mg/L >98 Airflow rate [58]

100 L/h

33.6 mg/L 0.6 mg/L 98.3 Nitrogen [64]

sparging
(150 L/h)
after feeding

14 mg/L – 80 Airflow rate [50]

240 L/h

20 mg/L 2.4 mg/L 88.3  7.4 High DO (not [65]

controlled)

0.4 mg/L 98.3  0.2 Constant

low DO
(30%)

80 mg/L (PO43–P) 1.2 mg/L (PO43–P) 98.5 Airflow rate [66]

18  6 L/h

a
Kelowna wastewater treatment facility limitsi.

meet the effluent quality requirement (6 mg/L of total nitrogen in the final effluent). Similarly, they
are unable to meet the effluent phosphorus level (0.25 mg/L) of some municipalities (Kelowna
wastewater treatment facility, British Columbia, Canada). The previous reports suggest a
tendency toward better nitrogen and phosphorus removal at low dissolved oxygen (DO)
concentration. A better response could be expected by integrating the technology with an
anoxic/anaerobic treatment to create favorable conditions for heterotrophic denitrifying (anoxic)
and phosphorus-accumulating (anaerobic) microorganisms.

Biodegradation, Bioaccumulation and Biosorption

The most promising feature of aerobic granules in treating emerging contaminants is that they
can simultaneously treat pollutants by three environmentally friendly methods: biodegradation,
bioaccumulation, and biosorption. The amount of pollutant biodegraded, bioaccumulated, or

74 Trends in Biotechnology, January 2017, Vol. 35, No. 1

adsorbed to the granules should be separately estimated to identify the dominant mechanism of
removal. Depending on the type of pollutant, the dominant mechanism may be different. Being
composed of both aerobic and anoxic/anaerobic zones, these granules are likely to provide the
advantages of both aerobic and anaerobic biodegradation. Similar to any other mixed microbial
culture, the microorganisms in aerobic granules can share the biodegradation pathways, where
a degradation product produced by one organism can be utilized by others to achieve complete
degradation.

Conventional activated-sludge processes suffer from solid/biomass loss due to bulking. Sludge
bulking is a situation where sludge fails to settle down to leave a clear supernatant on the top of

Table 4. A Synthesis of Different Reports on Treatment of Emerging Contaminants by Aerobic Granulation

Technologya
Target compound Concentration Medium Removal efficiency Refs

Phenol 250 mg/L Phenol-rich synthetic Complete degradation [67]

wastewater within 29 h

Phenol 650 mg/L Phenol-rich synthetic Complete removal [68]

wastewater within 240 min

Phenol 1000 mg/L Phenol-rich synthetic 49 mg phenol/g
VSS/h [69]

wastewater

Phenol 2500 mg/L Phenol-rich synthetic 1 g phenol/g
VSS/d [70]

wastewater

2-Fluorophenol 0.44 mM Medium containing Complete degradation [71]

5.9 mM acetate

Aniline 0–2000 mg/L Phosphate buffer 111.63 mg/g
VSS/h [72]

2-Chloroaniline 0–2000 mg/L Phosphate buffer 41.72 mg/g
VSS/h [72]

Cresol isomers 1500 mg/L Synthetic medium Complete degradation [73]

within 60–80 h

Paracetamol 500–5000 mg/L Synthetic medium Complete degradation [74]

with paracetamol within 28–48 h
as the only carbon
source

Acid red 18 50 mg/L Synthetic wastewater 53–55% removal [75]

Endocrine Estrone (32 ng/L) Primary effluent of 62.2% removal [76]

disrupter municipal WWTP
17b-Estradiol 68% removal
compounds
(23.6 ng/L)

Bisphenol A 91.8% removal

(103.4 ng/L)

4-tert-Octylphenol 77.9% removal

(35.2 ng/L)

2,4,6-Trichlorophenol 20 mg/L Synthetic wastewater Complete removal [77]

containing sodium
acetate (420–1000 mg/L)

Dibutyl phosphite 3 mM Mineral salt media Complete degradation [78]

containing 516.9 mg/L of within 8 h
sodium acetate

Polybrominated 4 mg/L Synthetic wastewater 95% removal [79]

diphenyl ether
(BDE-209)

a
Abbreviations: VSS, volatile suspended solids; WWTP, wastewater treatment plant.

Trends in Biotechnology, January 2017, Vol. 35, No. 1 75

the settling tank. This effect happens mainly due to the presence of a large number of Outstanding Questions
filamentous microorganisms. Additionally, activated sludge cannot completely degrade emerg- Is there any relationship between quo-
ing contaminants, and effluents are often found contaminated with excreted drugs and endo- rum-quenching enzymes and granule
formation and stability? If so, what are
crine-disrupting agents [34–36]. Aerobic granules have superior settleability, and because they the environmental signals that initiate
do not rely only on biodegradation, they could be a better option for removing persistent organic the synthesis of these enzymes?
pollutants and nanomaterials by means of bioaccumulation and adsorption. Persistent organic
pollutants with high octanol–water partition coefficients (KOW) would tend to adsorb onto the Which polysaccharides are present in
extracellular polymeric substance
surfaces of the granules, and they could be subsequently removed. Similarly, nanomaterials
matrix of aerobic granules? Which
such as carbon nanotubes can be evaluated for their bioaccumulation within aerobic granules. polysaccharide is most important for
granule formation, and what intermo-
As summarized in Table 4, until 2006, phenol was the only toxic organic pollutant to be treated lecular interactions exist among these
polymers?
by aerobic granulation technology. Subsequent studies were focused on other pollutants, such
as polyacrylamide, paracetamol, dibutyl phosphite, bisphenol A, 17b-estradiol, acid red 18, and
Are ion-chelating agents involved in
polybrominated diphenyl ether, among others. However, the contributions of different micro- granule segregation by destabilizing
organisms to the degradation of organic pollutants within the granules are mostly unknown. the multivalent cation bridges of the
Likewise, the mass transfer behaviors of these granules have not been investigated. Due to the EPS matrix? If so, which ion-chelating
agent is most responsible?
differences in their chemical properties, each pollutant might encounter different levels of mass
transfer resistances within the granules. Pores and channels in the granules might be essential,
Will the stability of the granules be
and their roles in mass transfer need further investigation. affected if a particular microbial com-
munity is enriched by process optimi-
Aerobic granulation has been considered as a promising technology for removing heavy metals zation for a specific purpose, such as
denitrification?
such as nickel, cadmium, copper, and zinc [37–39]. Wastewater may contain heavy metals at a
very low concentration, and if this technology can deal with these pollutants at such low Why is aerobic granulation technology
concentrations, it would give it an advantage over conventional activated sludge processes. not as effective as enhanced biological
Sometimes, one heavy metal is better removed than other metals [40]. However, the reasons phosphorus removal processes for
behind such observations are not known. Fundamental investigations into such variations in removing phosphorus from wastewa-
ter? Is it possible to improve the phos-
adsorption pattern may reveal new information on adsorption mechanisms. The pH can phorus removal efficiency of this
determine the overall surface charges of the granules to influence their adsorption behavior. technology? Is phosphorus precipita-
Even within a granule, a pH gradient might exist due to the production of organic acids in the tion within the granules good for the
technology?
inner anoxic zone, and this possibility needs further investigation from a biosorption perspective.
Is it possible to remove a complex mix-
Concluding Remarks ture of different emerging contaminants
The interplay between quorum sensing and quorum quenching certainly has a broader role in from real wastewater samples by aer-
granulation. Autoinducer molecules may provide a vital lead in investigating this phenomenon. obic granules? Is it possible to simulta-
neously optimize biodegradation,
An elaborate characterization of the EPS matrix by identifying the individual proteins and
bioaccumulation, and biosorption of
polysaccharides might broaden the overall understanding of intermolecular interactions involved these pollutants?
in granule formation. New biomass synthesis is necessary for granule formation, so it may not be
possible to completely eliminate a long granulation period characterized by low chemical oxygen
demand (COD) removal efficiency. Therefore, for a real full-scale application, such as treating a
few hundred million liters of wastewater per day, a backup option must be in place to deal with
partially treated wastewater generated during this period. If any technique can be developed to
give an early warning of granule segregation, engineering strategies such as reseeding with
stored granules could be pursued to minimize its impact on the wastewater treatment process.

Aeration is necessary for aerobic granule development, but it reduces the nitrogen and phos-
phorus removal efficiency of the technology. Therefore, granule development and wastewater
treatment should be optimized as two separate processes. Additionally, volatile fatty acids could
be used as carbon sources and electron donors for enhanced phosphorus and nitrogen
removal, respectively. Phosphorus precipitation within the granule is a unique feature. However,
it might have a negative impact on phosphorus removal as it may interfere in phosphorus release
and uptake cycle of biological phosphorus removal process. This possibility needs further
investigation.

76 Trends in Biotechnology, January 2017, Vol. 35, No. 1

The removal of emerging contaminants is one of the areas where this technology might be
superior to the conventional activated-sludge process. Compared to sludge in traditional
process, aerobic granules tend to be washed out with the effluents less; therefore, this
technology could be more effective in removing pollutants that bioaccumulate or adsorb on
to the granules. Additionally, aerobic granulation technology allows relatively more biomass to be
used to treat the same volume of wastewater. It increases the chance of eliminating emerging
contaminants. Thus, the knowledge gaps identified during this study (see Outstanding Ques-
tions) should be addressed for further improving this technology.

Acknowledgments
Natural Sciences and Engineering Research Council - Industrial Research Chair (NSERC-IRC) of Canada, City of Calgary,
and University of Calgary has been acknowledged for financial support.

Resources
i
www.eocp.ca/wp-content/uploads/2012/10/plants-Kelowna.pdf

References
1. Zhang, Q. et al. (2016) Aerobic granular processes: current
research trends. Bioresour. Technol. 210, 74–80
2. Liu, Y. et al. (2002) Anaerobic granulation technology for waste-
water treatment. World J. Microbiol. Biotechnol. 18, 99–113
3. Liu, Y. et al. (2003) Mechanisms and models for anaerobic granu-
lation in upflow anaerobic sludge blanket reactor. Water Res. 37,
661–673
4. Adav, S.S. et al. (2008) Extracellular polymeric substances and
structural stability of aerobic granule. Water Res. 42, 1644–1650
18. Lin, Y.H. et al. (2003) Acyl-homoserine lactone acylase from Ral-
stonia strain XJ12B represents a novel and potent class of quo-
rum-quenching enzymes. Mol. Microbiol. 47, 849–860
19. Czajkowski, R. and Jafra, S. (2009) Quenching of acyl-homoserine
lactone-dependent quorum sensing by enzymatic disruption of
signal molecules. Acta Biochim. Pol. 56, 1–16
20. Chen, F. et al. (2013) Quorum quenching enzymes and their
application in degrading signal molecules to block quorum sens-
ing-dependent infection. Int. J. Mol. Sci. 14, 17477–17500

5. Miksch, K. and Beata, K. (2012) Distribution of extracellular poly- 21. Chu, Y.-Y. et al. (2013) A new class of quorum quenching mol-
meric substances and their role in aerobic granule formation. ecules from Staphylococcus species affects communication and
Chem. Process Eng. 33, 679–688 growth of gram-negative bacteria. PLoS Pathog. 9, e1003654

6. Adav, S.S. and Lee, D.-J. (2008) Extraction of extracellular poly-
meric substances from aerobic granule with compact interior
structure. J. Hazard. Mater. 154, 1120–1126
7. Lin, Y. et al. (2008) Bacterial alginate role in aerobic granular
bioparticles formation and settleability improvement. Sep. Sci.
Tech. 43, 1642–1652
22. Grandclément, C. et al. (2016) Quorum quenching: role in nature
and applied developments. FEMS Microbiol. Rev. 40, 86–116
23. Adak, S. et al. (2011) Quorum quenching–an alternative antimi-
crobial therapeutics. In Science against Microbial Pathogens:
Communicating Current Research and Technological Advances
(Méndez-Vilas, A., ed.), pp. 586–593, Formatex

8. Lin, Y. et al. (2010) Characterization of alginate-like exopolysac- 24. Mani, A. et al. (2012) Assessment of quorum quenching activity of
charides isolated from aerobic granular sludge in pilot-plant. Water Bacillus species against Pseudomonas aeruginosa MTCC 2297.
Res. 44, 3355–3364 Global J. Pharmacol. 6, 118–125

9. Janczarek, M. (2011) Environmental signals and regulatory path- 25. Sarma, S.J. and Pakshirajan, K. (2012) Pyrene biodegradation by
ways that influence exopolysaccharide production in rhizobia. Int. Mycobacterium frederiksbergense using an encapsulated oil sys-
J. Mol. Sci. 12, 7898–7933 tem. Polycycl. Aromat. Compd. 32, 457–468

10. Ren, T.-T. et al. (2010) The quorum-sensing effect of aerobic
granules on bacterial adhesion, biofilm formation, and sludge
granulation. Appl. Microbiol. Biotechnol. 88, 789–797
11. Zhang, S.-H. et al. (2011) Effect of interspecies quorum sensing on
the formation of aerobic granular sludge. Water Sci. Technol. 64,
1284–1290
12. Lv, J. et al. (2014) The microbial attachment potential and
quorum sensing measurement of aerobic granular activated
sludge and flocculent activated sludge. Bioresour. Technol.
151, 291–296
26. Mater, D.D. et al. (1995) Effect of gelation temperature and gel-
dissolving solution on cell viability and recovery of two Pseudo-
monas putida strains co-immobilized within calcium alginate or k-
carrageenan gel beads. Biotech. Tech. 9, 747–752
27. Filali, A. et al. (2012) Stability and performance of two GSBR
operated in alternating anoxic/aerobic or anaerobic/aerobic con-
ditions for nutrient removal. Biochem. Eng. J. 67, 10–19
28. Adav, S.S. et al. (2009) Proteolytic activity in stored aerobic gran-
ular sludge and structural integrity. Bioresour. Technol. 100, 68–
73

13. Schauder, S. et al. (2001) The LuxS family of bacterial autoin-
ducers: biosynthesis of a novel quorum-sensing signal molecule.
Mol. Microbiol. 41, 463–476
14. Steindler, L. and Venturi, V. (2007) Detection of quorum-sensing
N-acyl homoserine lactone signal molecules by bacterial biosen-
sors. FEMS Microbiol. Lett. 266, 1–9
15. Rickard, A.H. et al. (2006) Autoinducer 2: a concentration-depen-
dent signal for mutualistic bacterial biofilm growth. Mol. Microbiol.
60, 1446–1456
29. Liu, Y.Q. et al. (2005) Relationship between size and mass transfer
resistance in aerobic granules. Lett. Appl. Microbiol. 40, 312–315
30. Barr, J.J. et al. (2010) Granule formation mechanisms within an
aerobic wastewater system for phosphorus removal. Appl. Envi-
ron. Microbiol. 76, 7588–7597
31. Winkler, M.K.H. et al. (2011) Segregation of biomass in cyclic
anaerobic/aerobic granular sludge allows the enrichment of anaer-
obic ammonium oxidizing bacteria at low temperatures. Environ.
Sci. Technol. 45, 7330–7337

16. Dong, Y.-H. et al. (2007) Quorum-quenching microbial infections: 32. Shi, X.-Y. et al. (2009) Characteristics of aerobic granules rich in
mechanisms and implications. Philos. Trans. R. Soc. Lond. B Biol. autotrophic ammonium-oxidizing bacteria in a sequencing batch
Sci. 362, 1201–1211 reactor. Chem. Eng. J. 147, 102–109

17. Fetzner, S. (2015) Quorum quenching enzymes. J. Biotechnol. 33. Gonzalez-Gil, G. and Holliger, C. (2011) Dynamics of microbial
201, 2–14 community structure of and enhanced biological phosphorus

Trends in Biotechnology, January 2017, Vol. 35, No. 1 77

 removal by aerobic granules cultivated on propionate or acetate.
Appl. Environ. Microbiol. 77, 8041–8051
34. Lee, H.-B. and Peart, T.E. (2000) Determination of bisphenol A in
sewage effluent and sludge by solid-phase and supercritical fluid
extraction and gas chromatography/mass spectrometry. J. AOAC
Int. 83, 290–297
35. Fuerhacker, M. (2003) Bisphenol A emission factors from industrial
sources and elimination rates in a sewage treatment plant. Water
Sci. Technol. 47, 117–122
36. Miao, X.-S. et al. (2004) Occurrence of antimicrobials in the final
effluents of wastewater treatment plants in Canada. Environ. Sci.
Technol. 38, 3533–3541
37. Xu, H. et al. (2006) Effect of pH on nickel biosorption by aerobic
granular sludge. Bioresour. Technol. 97, 359–363
38. Liu, Y. et al. (2003) A general model for biosorption of Cd2+, Cu2+
and Zn2+ by aerobic granules. J. Biotechnol. 102, 233–239
39. Xu, H. et al. (2004) Removal of dissolved copper (II) and zinc (II) by
aerobic granular sludge. Water Sci. Technol. 50, 155–160
40. Wang, L. et al. (2015) A comprehensive comparison of bacterial
and fungal aerobic granules: formation, properties, surface modi-
fication, and biosorption of metals. RSC Adv. 5, 104062–104070
41. Bathe, S. et al., eds (2005) Aerobic Granular Sludge, IWA
Publishing
42. Mishima, K. and Nakamura, M. (1991) Self-immobilization of aer-
obic activated sludge–a pilot study of the aerobic upflow sludge
blanket process in municipal sewage treatment. Water Sci. Tech-
nol. 23, 981–990
43. Liu, Q. et al. (2005) Startup of pilot-scale aerobic granular sludge
reactor by stored granules. Environ. Technol. 26, 1363–1370
44. Li, J. et al. (2014) Structure analysis of aerobic granule from a
sequencing batch reactor for organic matter and ammonia nitro-
gen removal. Int. J. Environ. Res. Public Health 11, 2427–2436
45. Tay, J.-H. et al. (2001) The effects of shear force on the formation,
structure and metabolism of aerobic granules. Appl. Microbiol.
Biotechnol. 57, 227–233
46. Li, J. et al. (2014) Aerobic sludge granulation in a full-scale
sequencing batch reactor. Biomed. Res. Int. 2014, 268789
47. Li, Z. et al. (2006) Aerobic granular sludge: a promising technology
for decentralised wastewater treatment. Water Sci. Technol. 53,
79–85
48. Abdullah, N. et al. (2011) Aerobic granular sludge formation for
high strength agro-based wastewater treatment. Bioresour. Tech-
nol. 102, 6778–6781
49. Dangcong, P. et al. (1999) Aerobic granular sludge–a case report.
Water Res. 33, 890–893
50. Jungles, M. et al. (2014) Sequencing batch reactor operation for
treating wastewater with aerobic granular sludge. Braz. J. Chem.
Eng. 31, 27–33
51. Linlin, H. et al. (2005) The formation and characteristics of aerobic
granules in sequencing batch reactor (SBR) by seeding anaerobic
granules. Process Biochem. 40, 5–11
52. Morgenroth, E. et al. (1997) Aerobic granular sludge in a sequenc-
ing batch reactor. Water Res. 31, 3191–3194
53. Val del Río, A. et al. (2013) Stability of aerobic granular biomass
treating the effluent from a seafood industry. Int. J. Environ. Res. 7,
265–276
54. Xuan, W. et al. (2010) The EPS characteristics of sludge in an
aerobic granule membrane bioreactor. Bioresour. Technol. 101,
8046–8050
55. Caudan, C. et al. (2012) Extracellular polymeric substances (EPS)
from aerobic granular sludges: extraction, fractionation, and
anionic properties. Appl. Biochem. Biotechnol. 166, 1685–1702
56. McSwain, B.S. et al. (2005) Composition and distribution of extra-
cellular polymeric substances in aerobic flocs and granular sludge.
Appl. Environ. Microbiol. 71, 1051–1057
57. Yu, G.-H. et al. (2010) Extracellular polymeric substances (EPS) and
extracellular enzymes in aerobic granules. Dry. Tech. 28, 910–915
78 Trends in Biotechnology, January 2017, Vol. 35, No. 1
58. Cassidy, D.P. and Belia, E. (2005) Nitrogen and phosphorus
removal from an abattoir wastewater in a SBR with aerobic gran-
ular sludge. Water Res. 39, 4817–4823
59. De Kreuk, M. et al. (2005) Simultaneous COD, nitrogen, and
phosphate removal by aerobic granular sludge. Biotechnol. Bio-
eng. 90, 761–769
60. Lochmatter, S. et al. (2014) Nitrogen removal over nitrite by aera-
tion control in aerobic granular sludge sequencing batch reactors.
Int. J. Environ. Res. Public Health 11, 6955–6978
61. Wei, Y. et al. (2012) Organic and nitrogen removal from landfill
leachate in aerobic granular sludge sequencing batch reactors.
Waste Manag. 32, 448–455
62. Jiang, X. et al. (2016) Enhanced biological phosphorus removal by
granular sludge in anaerobic/aerobic/anoxic SBR during start-up
period. Desalin. Water Treat. 57, 5760–5771
63. Bassin, J.P. et al. (2011) Effect of elevated salt concentrations on
the aerobic granular sludge process: linking microbial activity with
microbial community structure. Appl. Environ. Microbiol. 77,
7942–7953
64. Yilmaz, G. et al. (2008) Simultaneous nitrification, denitrification,
and phosphorus removal from nutrient-rich industrial wastewater
using granular sludge. Biotechnol. Bioeng. 100, 529–541
65. Lochmatter, S. et al. (2013) Optimized aeration strategies for
nitrogen and phosphorus removal with aerobic granular sludge.
Water Res. 47, 6187–6197
66. Kishida, N. et al. (2009) Simultaneous nitrogen and phosphorus
removal from high-strength industrial wastewater using aerobic
granular sludge. J. Environ. Eng. 135, 153–158
67. Jiang, H.-L. et al. (2006) Enhanced phenol biodegradation and
aerobic granulation by two coaggregating bacterial strains. Envi-
ron. Sci. Technol. 40, 6137–6142
68. Khan, F. et al. (2010) Degradation profile of phenol in sequential
batch reactor. Environ. Int. J. Sci. Tech. 5, 123–135
69. Adav, S.S. et al. (2007) Degradation of phenol by aerobic granules
and isolated yeast Candida tropicalis. Biotechnol. Bioeng. 96,
844–852
70. Tay, J.-H. et al. (2004) High-rate biodegradation of phenol by
aerobically grown microbial granules. J. Environ. Eng. 130,
1415–1423
71. Duque, A.F. et al. (2011) 2-Fluorophenol degradation by aerobic
granular sludge in a sequencing batch reactor. Water Res. 45,
6745–6752
72. Zhu, L. et al. (2012) Reaction kinetics of the degradation of
chloroanilines and aniline by aerobic granule. Biochem. Eng. J.
68, 215–220
73. Lee, D.-J. et al. (2011) Degradation of cresols by phenol-accli-
mated aerobic granules. Appl. Microbiol. Biotechnol. 89, 209–215
74. Hu, J. et al. (2012) Biodegradation of paracetamol by aerobic
granules in a sequencing batch reactor (SBR). Adv. Mater. Res.
441, 531–535
75. Sadri Moghaddam, S. and Alavi Moghaddam, M.R. (2016) Aerobic
granular sludge for dye biodegradation in a sequencing batch
reactor with anaerobic/aerobic cycles. CLEAN–Soil, Air, Water
44, 438–443
76. Balest, L. et al. (2008) Removal of endocrine disrupter compounds
from municipal wastewater by an innovative biological technology.
Water Sci. Technol. 58, 953–956
77. Milia, S. et al. (2016) Performance and characteristics of aero-
bic granular sludge degrading 2,4,6-trichlorophenol at different
volumetric organic loading rates. CLEAN–Soil, Air, Water 44,
615–623
78. Reddy, G.K.K. et al. (2014) Aerobic granular sludge mediated
biodegradation of an organophosphorous ester, dibutyl phos-
phite. FEMS Microbiol. Lett. 359, 110–115
79. Ni, S.-Q. et al. (2014) Interaction of polybrominated diphenyl ethers
and aerobic granular sludge: biosorption and microbial degrada-
tion. Biomed. Res. Int. 2014, 1–10