International Biodeterioration & Biodegradation 112 (2016) 1e11

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Effect of reactor height/diameter ratio and organic loading rate on

formation of aerobic granular sludge in sewage treatment
Nik Azimatolakma Awang, Md Ghazaly Shaaban*
Department of Civil Engineering, Faculty of Engineering, University of Malaya, 50603, Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Although a high volume of research on aerobic granular sludge exists in the body of related literature, the
Received 8 January 2016 determination of biokinetic parameters of aerobic granules using a linear plot for biodegradation of real
Received in revised form wastewater has rarely been discussed. Therefore, this study aims to determine the biokinetic parameters
20 April 2016
of aerobic granules that develop in a sequencing batch reactor (SBR) with sewage as a substrate, using a
Accepted 23 April 2016
linear plot. The difference in biokinetic parameter (k, Ks, Y, Kd and mmax) values gathered from five aerobic
granules batches were tested as whether it is linearly correlated with the variable organic loading rate
(OLR) and reactor height/diameter (H/D) ratio. The R2 value of 0.97 and 0.82 was obtained from the linear
Keywords:
Aerobic granular sludge
correlation between mmax and reactor H/D ratio, and Y and OLR, respectively. This demonstrates that
Biokinetic parameters biomass production (Y and mmax) proportionally depends on the increase in OLR and reactor H/D ratio.
Sewage This might be because the effectiveness of the aerobic granules measured by Ks are greater in high H/D
Stability ratio reactors with values over 315 mg/L as compared to less than 276 mg/L in low H/D ratio reactors.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Aerobic granular sludge (AGS) is a novel and promising tech-
nology within a compact wastewater treatment plant based on
simultaneous organics and nutrient removal in one sequencing
batch reactor (De Kreuk, 2006). Unlike conventional activated
sludge systems, AGS technology has a regular, dense and strong
microbial structure, good settling ability, high biomass retention, as
well as the ability to withstand shock and toxic loadings (Wang
et al., 2007a). Liu et al. (2014) mention that a reasonable organic
loading rate (OLR), high reactor height/diameter (H/D) ratio,
sequencing batch operation and settling time are essential factors
for a successful granulation process at full scale operation.
There exists only a small number of related works that describe
pilot and full scale operations of AGS technology for sewage or
domestic wastewater treatment. At a pilot scale, Ni et al. (2009) and
Su et al. (2012) achieve a complete aerobic granulation after 200
and 45 days respectively. In terms of full scale operation, Liu et al.
(2014) and Pronk et al. (2015) achieve mature and compact aero-
bic granules after 337 days and 5 months respectively. A longer
period to achieve mature aerobic granules that develop in sewage is
* Corresponding author.
E-mail address: ghazaly@um.edu.my (M.G. Shaaban).
expected, since pilot and full scale reactors with high working
volumes will yield a low reactor H/D ratio and variable OLR.
Moreover, the stability of AGS has been questioned by a several
recent achievements. Liu and Tay (2012) suggest controlling the
OLR in a specific optimal range to benefit the growth of aerobic
granules over flocculent sludge during long term operation. How-
ever, controlling the OLR in the operation of real wastewater is
challenging, mainly due to the variation in influent parameters.
Furthermore, Long et al. (2015) demonstrate the collapse of the
aerobic granule system after 66 days operation, during which the
OLR increased from 15 to 18 kg/m3 d.
The stability of aerobic granules can be defined as the ability of
microbial granules in resisting hydrodynamic and mechanical
shear force (Sheng et al., 2010). The state of aerobic granule stability
is measured in terms of bioactivity, microbial community compo-
sition and aerobic granule characteristics (Chen et al., 2007). The
bioactivity of aerobic granules has often been quantified based on
specific oxygen uptake rate (SOUR) and biokinetic parameters.
Biokinetic parameters aid in describing the rate of substrate utili-
zation and biomass production, or growth by microbial (Muda
et al., 2011), which are important to process the design and opti-
mization of system performance (Chen et al., 2008). The biological
treatment system design from biokinetic parameters is expected to
be superior over other empirical methods that concern fewer
empirical factors (Kaewsuk et al., 2010). Currently, comprehensive

http://dx.doi.org/10.1016/j.ibiod.2016.04.028
0964-8305/© 2016 Elsevier Ltd. All rights reserved.
2 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11

reviews and research works with regards to aerobic granule char- L). Substituting Eq. (2) into Eq. (3), we obtain
acteristics (Adav et al., 2008; Sheng et al., 2010; Xiao et al., 2008)
and microbial community (Kong et al., 2015; Liang et al., 2015; Zhou ðSo  Se Þ kS
U¼ ¼ (4)
et al., 2014) are well reported. However, there exist only a few re- tX Ks þ S
ports that consider the bioactivity of aerobic granules in treating
real wastewater, especially in the case of sewage. where U represents the specific substrate utilization rate (mg CODs/
Chen et al. (2008), Liu and Tay (2007) and Muda et al. (2011) mg VSS d). In double reciprocal form, Eq. (4) can be changed to the
mainly focus on the determination of endogenous decay coeffi- following form
cient (Kd), synthesis yield coefficient (Y) and maximum specific
tX Ks 1 1
microbial growth rate (mmax) for aerobic granules that develop in ¼ þ (5)
ðSo  Se Þ k S k
synthetic wastewater, directly from equations, and not from a
linear plot. Meanwhile, Asadi et al. (2014), Kaewsuk et al. (2010) A liner plot of (tX/So  Se) versus 1/So yields a positive slope of
and Mizzouri and Shaaban (2013) provide details for the determi- Ks/k and intercept of 1/k. Thus, KS and k can be obtained.
nation of biokinetic parameters using a linear plot for the biodeg-
radation of real wastewater in reactors other than a sequencing 2.1.2. Microbial growth
batch reactor (SBR). Under steady state, the mass balance for microbial growth in
Therefore, this study aims to determine the biokinetic param- SBR can be expressed as follows
eters of aerobic granules that develop in SBR with various config-
urations, with the use of sewage as a substrate. Considering the fact dX
rg ¼ (6)
that the performance of aerobic granules is significantly influenced dt
by OLR and reactor H/D ratio (Awang and Shaaban, 2015), to the
best of the researcher's knowledge, this study is the first to describe Eq. (6) can also be expended as:
the relationship between change in biokinetic parameters with OLR
dX Q ðXo  Xe Þ
and reactor H/D ratio. The outcome of this study may be useful for rg ¼ ¼ (7)
better design and operation of SBR, which utilizes aerobic granule dt V
technology in practical applications.
where rg represents the microbial growth rate (mg VSS/L d), Q
represents the influent flow rate (L/d), V represents the reactor
2. Materials and methods working volume (L), Xo represents the influent biomass concen-
tration (mg VSS/L), and Se represents the effluent biomass con-
2.1. Development of kinetic equation for biokinetic parameters centration (mg VSS/L).
The relationship between the rate of microbial growth and the
The proposed equations for substrate utilization and microbial rate of substrate utilization in SBR can be expressed as follows
growth are based on the studies conducted by Han and Levenspiel
(1988), Kaewsuk et al. (2010), Liu et al. (2005a), Metcalf and Eddy rg ¼ Yrsu  kd X (8)
et al. (2003) and Okpokwasili and Nweke (2005).
Substituting Eq. (7) into Eq. (8), we obtain
2.1.1. Substrate utilization
Under steady state, the mass balance for substrate utilization in Q ðXo  Xe Þ ðSo  Se Þ
¼Y  kd (9)
SBR can be expressed as follows VX tX
dS where Y represents the synthesis yield coefficient (mg VSS/mg
rsu ¼ (1) CODs), Kd is the endogenous decay coefficient (1/d) and X is the
dt
biomass concentration in reactor (mg MLVSS/L). The inverse of the
Eq. (1) can also be expanded to term on the left-hand side of Eq. (9) is defined as the sludge
retention time (SRT). Thus, Eq. (9) can be written as follows
dS Q ðSo  Se Þ ðSo  Se Þ
rsu ¼ ¼ ¼ (2)
dt V t 1 ðSo  Se Þ
¼Y  kd (10)
SRT tX
where rsu represents the substrate utilization rate (mg CODs/L d), Q
is the influent flow rate (L/d), V is the reactor working volume (L), So A liner plot of 1/SRT versus (So  Se/tX) yields a positive slope of
is the influent substrate concentration (mg CODs/L), Se is the Y and an intercept of kd. Thus, kd and Y can be obtained. The
effluent substrate concentration (mg CODs/L), and t is the hydraulic maximum specific microbial growth rate (mmax) can be determined
retention time (d). from the multiplication of k and Y.
Based on the assumption that the rate of substrate (sewage)
utilization is generally proportional to the biomass concentration, 2.2. Reactor setup
and dependent on the substrate concentration in bioreactor char-
acteristics (first order reaction), Eq. (2) can also be expressed using Several practical experiments were conducted in three 3 open,
a modified Monod equation as follows column-type SBRs that are constructed identically from plexiglass,
with a working volume of 4.5 L. The schematic diagram of the basic
dS kSX SBR operation is illustrated in Fig. 1. Influent was fed into the reactor
¼ (3) from the bottom of the column, whilst an effluent standpipe was
dt Ks þ S
fixed for a 50% volumetric exchange ratio. The rate and volume of
where S represents the concentration of substrate in the reactor influent and effluent during the static feeding and decanting phase,
(mg CODs/L), X is the biomass concentration in the reactor (mg respectively, were controlled by peristaltic pumps. Mixing during
MLVSS/L), k is the maximum specific substrate utilization rate (mg the reaction phase was accomplished by the aeration from a porous
CODs/mg VSS d), and Ks is the half saturation coefficient (mg CODs/ stone at the bottom of each reactor.
 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11
Fig. 1. Schematic diagram of SBR operation.
Air was supplied to the system by an air pump, and the rate was
controlled by a flow meter with an air flow rate of 4 L/min, resulting
in an SUAV of 0.7e1.33 cm/s. Control of aeration, feeding and
decanting pumps associated with each SBR operating strategy were
programmed into and controlled by a digital timer. The dissolved
oxygen and pH of the system were monitored without further
adjustment.
Fig. 2 illustrates three different SBR configurations used in this
study, namely, SBR1, SBR2 and SBR3. SBR1 and SBR2 were counted
as bubble column type reactors with a difference in terms of the H/
D ratio, which was 11.3 and 4.4 respectively. Moreover, SBR3 had
the exact configuration of SBR2, except for the draft tubes within it
(airlift type reactor). Thus, in order to keep the working volume at
4.5 L, there was a slight difference in working height for both re-
actors due to the volume added by the draft tubes, which were
48 cm and 49 cm for SBR2 and SBR3 respectively. The volumetric
exchange ratio for SBR2 and SBR3 were counted as approximately
50%. The tabulated data for the reactor setup as are listed in Table 1.
2.3. Sewage and seed preparation
Sewage (substrate) for this study was collected from Pantai 1
STP and Damansara STP (KLR354). Pantai 1 STP was designed based
on the activated sludge system to treat sewage from a contributing
population equivalent to 377, 000. On the other hand, Damansara
STP was constructed to treat sewage for a population of 100,000
using oxidation ditches process. Sewage from Pantai 1 STP was used
as a substrate for the formation of Control Batch, Batch1 and
Batch2, while Batch3 and Batch4 used sewage from Damansara STP
as a substrate. The location for the sewage collection was shifted
due to the low influent intake in Pantai 1 STP, which at that time
was sharing the collection inlet with the new Pantai 2 STP. Sewage
3
was taken directly from the primary clarifier (after coarse
screening) and stored at 5  C for a maximum of 18 days. During this
time, the settled suspended solids from sewage collection tanks
were removed. Influent tank for reactors were replaced every 2 or 3
days.
Activated sludge collected from Pantai 1 STP was used as a seed
for the formation process for Control, Batch1, Batch2 and Batch3.
Instead of activated sludge, the seed for Batch4 comprised aerobic
granules, which were stored for 8 months at 5  C. Stored aerobic
granules were used with the aim of proving that the stability and
ability of aerobic granules were to be reformed within a short
period compared to normal conditions. Before the start-up, the
seed is aerated for a one-day period without the addition of
external carbon sources. The volume of active sludge added to the
SBRs prior to the formation process is 50% from the working vol-
ume and 1.0 L for Batch4.
2.4. Procedure layout
The operating conditions applied in the formation of all batches
are listed in Table 2. The operating conditions for Control Batch
remained the same until the end of the formation process, with the
exception of cycle time. The reaction, settling and feeding time for
Batch1, Batch2, Batch3 and Batch4 were changed according to the
performance of effluent quality and sludge volume index (SVI). The
aeration rate for all batches was fixed at 4 L/min, which resulted
with dissolved oxygen within the range of 7.0e9.0 mg/L. It is
important to note that during formation of all batches, OLR was
variable throughout the formation process. Once aerobic granules
went through the formation-breakage cycle, and steady-state was
achieved, the reactor was turned off. Reactor maintenance was
carried out only when excessive proliferation of filamentous mi-
croorganisms on the reactor wall occurred.
2.5. Analytical method
Parameters such as mixed liquor volatile suspended solids
(MLVSS), soluble COD (CODs), total COD, suspended solids (SS),
volatile suspended solids (VSS) and sludge volume index (SVI) were
analysed according to APHA standard methods (2005). Ammo-
nium, nitrite and nitrate were determined by the photometric
determination using a Merck test kit. A spectrophotometer with a
wavelength in the range of 320e1100 nm was used to measure the
photometric absorbance. Measurements of all the parameters were
carried out two to three times per week.
3. Results
3.1. Substrate characterization
Substrate characterization is important in understanding the
bioactivity of aerobic granules, especially when equivalent oper-
ating conditions are applied to the SBR system. The repetition of an
identical experiment would not lead to the same results, mainly
due to the variability in substrate constituent, particularly real
wastewater. The characteristics of raw sewage collected from Pantai
1 STP and Damansara STP are shown in Table 3, with all parameters
measured in mg/L. The results suggest that the concentration of all
parameters (except for SS and VSS) was almost two times higher in
Damansara STP than Pantai 1 STP. Sewage characteristics, especially
SS, highly depend on the weather during the time of collection. This
might be associated with the system collection point in the sewer.
Rubbish such as plastic bags and dry leaves were found in the
primary clarifier during the rainy season at Pantai 1 STP.
Analysis on the relationship between total COD and soluble COD
4 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11

Fig. 2. Schemes of SBRs with 3 different configurations.

Table 1
Reactor setup for formation of aerobic granular sludge.

Reactor setup Y Batches /

Control Batch1 Batch2 Batch3 Batch4

Influent Pantai 1 Pantai 1 Damansara Damansara Damansara

Reactor SBR1 SBR1 SBR2 SBR3 SBR1
H/D ratio 11.3 11.3 4.4 4.4 10
Working volume (L) 4.5 4.5 4.5 4.5 4.0
OLR (kg CODs/m3 d) 0.20e0.48 0.12e0.49 0.12e0.49 0.26e0.81 0.26e0.81
SUAV (cm/s) 1.33 1.33 0.7 0.7 1.33

Table 2
Operating conditions applied for formation of aerobic granular sludge.

Operating conditions Y Batches /

Control Batch1 Batch2 Batch3 Batch4

Phase 1 2 1 2 1 2 1 2 1 2
HRT (h) 8 6 8 6 8 6 8 6 8 6
Cycle (h) 4 3 4 3 4 3 4 3 4 3
Reaction (min) 195 135 195e229 135 195e220 150e160 185 125e145 170 110e145
Settling (min) 30 30 30e15 30 30e5 15e5 15 15 30 30e15
Feeding(min) 6 6 6 6 6 6 30 30e10 30 30e10
Idle (min) 3 3 3 3 3 3 4 4 4 4
Decanting (min) 6 6 6 6 6 6 6 6 6 6

(CODs) with SS, as shown in Fig. 3, exhibit a huge increment for
total COD compared to CODs when the SS value is increasing. A high
SS value contributes to an increase in the COD value, since more
organic and inorganic materials were oxidized by potassium di-
chromate. The results showed that the total COD values for influent
were exceptionally high due to the increase in SS when sewage was
collected during the rainy season. Thus, CODs was used as a
parameter to assess the quality of effluent instead of the total COD.
Based on Table A.3 from Guidelines for Developers, Volume IV
(Ministry of housing and local government, 1998) and the total COD
 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11 5

Table 3 aerobic granule system was in a steady state. In this study, the
Characteristics of raw sewage. steady state period is defined as a condition in which the SBR
Parameter (mg/L) Pantai 1 STP Damansara STP Standardc system consists of mature aerobic granules that could easily adapt
Min Max Averagea Min Max Averageb A B
and stabilize in variable substrate concentrations and changes in
operating parameters. The results from MLSS, MLVSS, F/M ratio and
BOD5 41 60 53 54 140 97 20 50
high quality of effluent were used as a benchmark to determine the
Total COD 60 230 180 163 470 270 120 200
Soluble COD 39 120 83 86 231 150 60 100 steady state period. The performance of each batch and data which
SS 58 195 97 32 136 80 50 100 had been used to plot the linear graphs is presented in Table 4. Eqs.
VSS 40 145 78 26 122 78 e e (5) and (10) were used to compute the kinetic coefficients of k, Ks, Y,
NHþ4 13 33 20 22 53 41 5 5
Kd and mmax during steady state through the plotted linear graph, as
NO2 0.02 0.08 0.05 0.01 0.07 0.01 e e
NO3
0.30 0.40 0.38 0.1 0.2 0.11 20 50 shown in Figs. 4 and 5, and the results are shown in Table 4.
PO3-
4 3.3 20.8 10.0 7.2 19.5 15.4 e e The R2 value for substrate utilization fit line (Fig. 4) is above 0.90,
a
Average of 12 samples collected from April 2013 till March 2014.
which indicates that the kinetic coefficients of k and Ks are accurate
b
Average of 6 samples collected from Jan 2015 till April 2015. in this study. In contrast, the R2 reading for the microbial growth fit
c
Acceptable conditions of sewage discharge of Standard A and B from line (Fig. 5) was lower: 0.52 for Batch2 AGS, and above 0.70 for
Environmental Quality (Sewage) Regulation 2009, Malaysia.

Fig. 3. Relationship between total COD and CODs with SS increment. Data was obtained between June 2013 and November 2013.

average values, sewage from Pantai 1 STP and Damansara STP could
be classified as low and medium respectively. The BOD/CODs ratio
for Pantai 1 STP and Damansara STP, which were over 0.5, indicated
that sewage could easily be treated by biological means.
As showed in Table 3, the average carbon to nitrogen to phos-
phorus ratio (C:N:P) in this study is far from the ideal ratio of
100:5:1 for aerobic treatment. However, in this study, sewage was
treated as is, without the addition of nutrients, with the aim to
assess the competency of aerobic granules in treating the sewage
itself. According to Lotito et al. (2012), real wastewater should be
treated as is, without the addition of micro- or macro-nutrients.
This aids in identified the effective treatability of wastewater, and
verifies the capability of biomass in the reactor to stress conditions.
Moreover, depending on the purpose of treatment, the ratio of
C:N:P can always be modified for both real and synthetic waste-
water by adding external carbon sources from alcohols (Wang et al.,
2013a), or by enhancing the production of short-chain fatty acid
(Wang et al., 2013b; Zhao et al., 2015) for biological phosphorus
removal.
3.2. Determination of process kinetic coefficients
The performance of each batch is discussed here, since the main
aim of this study is to determine the relationship between change
in kinetic coefficients with OLR and reactor H/D ratio. The results
presented for each batch were selected at conditions when the
Control, Batch1, Batch3 and Batch4. Asadi et al. (2014) reported an
R2 reading of 0.62 for the determination of mmax and Ks, and sug-
gested the lack of fit was due to the low biodegradability of COD.
Indeed, although the biodegradability of sewage for all samples
based on BOD/CODs ratio is over 0.5, it is the lowest for the steady
state CODs/NHþ 4 ratio for Batch2, which is 3.3, followed by Batch3,
Batch4 and Batch1, which had values of around 4 and over 6.3 for
Batch1. Therefore, this explains the reason the fit line for substrate
utilization is greatly higher than for microbial growth. Further-
more, a low CODs/NHþ 4 ratio also had resulted in lower CODs, SS
and NHþ 4 removal efficiency. This is supported by Yang et al. (2004)
and Wang et al. (2007b), who suggested that the aerobic granula-
tion and nitrification processes are closely related to the COD/N
ratios applied to the reactors, in which a COD/N ratio lower than 10
will inhibit both heterotrophic and nitrifying bacteria.
3.3. Effect of reactor H/D ratio and OLR on kinetic coefficients values
As shown in Table 4, the values of k, Ks and mmax are excep-
tionally high for Batch1, which are 27.55 1/d, 3133 mg/L and 5.63 1/
d, respectively. This could be attributed to the active competition
between the aerobic granules present in the reactor, with newly
added microbial from the activated sludge on day 33, which is 16
days just before Batch1 in steady state. Activated sludge was added
to Batch1 due to the excessive wash out of biomass at a low OLR of
0.13 kg COD/m3 d (Awang and Shaaban, 2015). This result is in line
6 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11

Table 4
Corresponding kinetic coefficient of substrate utilization and microbial growth for this study.

Batch Perioda Average OLRa (kg CODs/m3 Removala (%) X (mg MLVSS/ S0 (mg CODs/ Se (mg CODs/ SRTa Kinetic coefficient
(days) d) L) L) L) (d)
CODs SS NHþ 4 k (1/ Ks (mg/ Y (mg/ Kd (1/ mmax (1/
d) L) mg) d) d)

Control 55e71 0.32 80 70 100 1050 73 3 20 2.74 946 0.17 0.01 0.46
e e e e e e
97 97 1130 100 14 35
Batch1 49e63 0.43 80 57 73 360 109 12 4 27.55 3133 0.20 0.03 5.63
e e e e e e e
90 89 84 570 122 23 7
Batch2 89e103 0.41 71 66 71 340 85 12 5 0.91 228 0.13 0.12 0.11
e e e e e e e
90 90 91 460 118 23 8
Batch3 41e67 0.63 55 60 75 720 126 51 5 0.34 276 0.47 0.02 0.16
e e e e e e e
75 78 83 890 203 61 8
Batch4 40e83 0.63 49 57 71 610 126 52 5 0.27 315 0.68 0.07 0.18
e e e e e e e
69 76 83 1390 203 75 9
a
During steady state.

Fig. 4. Linear plot of substrate utilization for determination of k and Ks coefficient during steady state.

with Kaewsuk et al. (2010) for a high Ks value, which demonstrated
the activeness of aerobic microbial. However, Asadi et al. (2014)
found that a process or system with a high value of Ks is less effi-
cient. Thus, a plot of mmax versus Ks was plotted (Fig. 6) to determine
the activeness of aerobic granules produced in SBR with various H/
D ratios.
The result suggests that the activeness of aerobic granules
produced in SBR with an H/D ratio of 4.4 (Batch2 and Batch3) is
lower than in SBR with an H/D ratio of 10 (Batch4) and 11.3 (Control
and Batch1). This explains the reason that, at equal OLR and oper-
ational conditions, the period to achieve steady state in SBR1 with
high H/D ratio is shorter than in SBR2, which has a low H/D ratio.
 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11 7

Fig. 5. Linear plot of microbial growth for determination of Y and Kd coefficients during steady state.

Fig. 6. Relationship between maximum specific microbial growth rate (mmax) and half saturation coefficient (Ks); Control ¼ :, Batch1 ¼ C, Batch2 ¼ *, Batch3 ¼ þ, Batch4 ¼ -.

A review by Liu and Tay (2002) stresses that a column-type
upflow reactor with higher H/D ratio can provide a longer circular
flowing trajectory, which in turn creates an effective hydraulic
attrition for microbial aggregation. This circular hydraulic attrac-
tion force helps to shape the microbial aggregates into regular
granules that have high cellular surface hydrophobicity and mini-
mum surface Gibss energy. Thus, it can be concluded that the
period to achieve a steady state is governed by the reactor H/D
ratio; in which microbial aggregation is more productive in reactors
with a high H/D ratio. In comparison to Batch4, the use of divided
draft tubes for SBR3 proves to be beneficial in reducing the time
period of setup for Batch3. The performance of SBR with low H/D
ratio appears to have a higher resistance to variable and low OLR by
providing a short settling distance for biomass in facing unfavor-
able circumstances (Awang and Shaaban, 2015).
Based on the study by Kaewsuk et al. (2010), in the determi-
nation of kinetic coefficients of Mixed-PnSB using dairy wastewater
as a substrate, the value of Ks was determined to be 174 mg/L
instead of 8000 mg/L, as reported by Kantawanichkul (1990), who
using coconut cream. Kaewsuk et al. (2010) confirmed that higher
organic substance, and oil and grease concentration, in coconut
cream compared to dairy wastewater and acetate had caused an
8 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11

increase in the value of Ks. This thus shows that substrates with
complicated structures of organic matter will subsequently have a
high Ks value compared to those with simple structures.
However, as shown in Table 5, the Ks value in this study is higher
compared to the results by Asadi et al. (2014), Kaewsuk et al. (2010)
and Mizzouri and Shaaban (2013), which employ a substrate with
more a complicated structure than sewage. Besides, the Ks value in
this study is significantly higher than that reported by Metcalf and
Eddy et al. (2003), and is equal to the study by Pala and Bo € lükba
(2005), who both consider sewage as a substate. Hence, this
shows that the reactor type, configurations, system and scale all
influence the value of Ks.
A plot of OLR versus Y is shown in Fig. 7 to determine the in-
fluence of OLR on the development of aerobic granules. Considering
the differences in sewage composition (sewage collected at Pantai
STP and Damansara STP) and operating conditions applied, the
reading for the R2 linear fit line of 0.82 is shown in Fig. 7, and can be
considered as reliable. Thus, this indicates that biomass production
proliferates with the increase in OLR, and this fits well with the
earlier assumption of the first order reaction characteristics of
sewage.
However, a report by Chen et al. (2008) claims that Y in a steady
state decreases with an increase in OLR from 6.0 to 12.0 kg COD/
m3 d. Since the increase in OLR for this study is small
(0.32e0.63 kg CODs/m3 d) compared to the work by Chen et al.
(2008), it is hard to decide which value is true. Moreover, it has
been reported several times (Long et al., 2015; Ma et al., 2013) that
an increase in OLR results in the disintegration of aerobic granules.
In this case, the comparison should be distinguished between high
and low strengths of wastewater. The result also reveals that at
similar OLR, the production of biomass described by Y values will
always be greater in reactors with a high H/D ratio, which dem-
onstrates the increase in Ks values that facilitate the microbial
aggregation activity.
Additionally, Y in this study is almost always within the reported
range for sewage (Table 5). This value is considered 0.3e0.6 mg/mg
by Metcalf and Eddy et al. (2003) and 0.7 mg/mg by Pala and
Bo€lükba (2005). It appears that values of Y are higher for sewage
than other types of substrates (Table 5), although the concentration
of influent COD is much lower in sewage (maximum 231 CODs mg/L
in this study). Liu et al. (2005b) mentions that the low Y value was
caused by the diversity of microorganisms in completing the
oxidation process from complicated food chains. Thus, it can be
concluded that substrate utilization and bacterial growth are highly
dependent on the characteristics of the substrate itself.
This is in agreement with the work by Zhou et al. (2014), which
demonstrated that microbial selection pressure is not a prerequisite
for aerobic granulation from both the dynamic granulation steps
and the molecular biology aspects. Instead, the dominance of a
particular microbial is governed by the substrate composition (type
of wastewater) and concentration (OLR). Kong et al. (2015) recently
investigated microbial identification using a polymerase chain re-
action (PCR) coupled with next-generation sequencing (NGS)
method, with Illumina Miseq as platform. The result by Kong et al.
(2015) show that aerobic granules exposed to cefalexin consists of
an abundance of Bacteroidetes and Chloroflexi instead of Proteo-
bacteria. Papadia et al. (2011) reported that a direct correlation
exists between reactor configurations and microbial biomass,
which is mostly composed of Pseudomonas, Stenotrophomonas and
Ochrobactrum genera. The study by Kong et al. (2009) showed that
the reactor H/D ratio has no effect on the physical characteristics
and microbial community structure of aerobic granules.
Comparing the data in Table 5 with the results in Table 4, the
values of Kd in this study are normally within the reported range of
conventional activated sludge (Metcalf and Eddy et al., 2003) and
SBR-AGS system (Chen et al., 2008; Liu et al., 2005a; Muda et al.,

Table 5
Corresponding kinetic coefficients of substrate utilization and microbial growth from other studies based on Monod equation.

Reactor System Substrate COD (mg/L) K (1/d) Ks (mg/L) Y (mg/mg) Kd (1/d) mmax (1/d) References

SBR-AGS Batch Sewage form Pantai 1 39 0.91 228 0.13 0.01 0.11 This study
e e e e e e
120 27.5 3133 0.20 0.12 5.63
SBR-AGS Batch Sewage from Damansara 86 0.27 276 0.47 0.02 0.16 This study
e e e e e e
231 0.34 315 0.68 0.07 0.18
SBR-AGS Batch Acetate 2000 e e 0.142 0.065 0.075 Chen et al., 2008
e e e e
4000 0.231 0.069 0.027
SBR-AGS Batch Glucose 3342 13.2 276 0.183 0.023 e Liu et al., 2005a
e e
0.250 0.075
SBR-AGS Batch Synthetic textile 1200 e e 0.217 0.006 0.013 Muda et al., 2011
e e e e
1600 0.412 0.010 0.036
MBSR Batch Dairy 2584 7.42 174 0.228 0.138 1.69 Kaewsuk et al., 2010
±
794
UAASB Batch Industrial Estate 945 0.41 180 0.161 0.039 0.07 Asadi et al., 2014
e
1200
Batch Test Batch Sewage 100 e 343 0.70 0.053 1.13 €lükbaş, 2005
Pala and Bo
e
500
CSTR Continuous Petroleum refinery 172 0.81 111 0.049 0.004 0.04 Mizzouri and Shaaban, 2013
e
1617
Activated Sludge Continuous Sewage e 2 10 0.3 0.06 e Metcalf and Eddy et al., 2003
e e e e
10 60 0.6 0.15
 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11
Fig. 7. Relationship between OLR and synthesis yield coefficient (Y); Control ¼
2011). A further analysis reveals that Kd values gradually increase
with an increase in the period to achieve a steady state (Fig. 8). This
can most probably be attributed to the linear relationship between
Kd and cell age. The result reported by Chen et al. (2008) demon-
strates an increasing trend of Kd values from 0.065 1/d on the first
118 days of the aerobic granule formation period, to 0.069 1/d for
the last 335 days. Moreover, Muda et al. (2011) considered that the
increase in Kd values from 0.0075 to 0.0076 1/d between days
140e280 as constant.
4. Discussion
Although research on aerobic granules has increased exponen-
tially, the mechanism of aerobic granules formation with regards to
intermolecular interactions and the manner in which these forces
govern the stability of aerobic granules has rarely been discussed. In
this study, the mechanism of aerobic granules formation to form
well-defined granules can be described as a two-step process. The
first step involves physical agitation to initiate bacterium-to-
bacterium contact or attachment onto a solid surface. The forces
that might be involved in this step are hydrodynamic shear force,
substrate diffusion, gravity force, Brownian movement and cell
mobility (Liu and Tay, 2002).
The second step involves intermolecular interaction, or so called
physicochemical and microbiological interactions, of various trophic
bacteria groups to keep the aerobic granule in stability. Mittelman
(1985) illustrates the distribution of extracellular polymeric sub-
stance (EPS) around an individual cell wall with the structure of
Fig. 8. Relationship between steady state period and endogenous coefficient (Kd); Control ¼
9
:, Batch1 ¼ C, Batch2 ¼ *, Batch3 ¼ þ, Batch4 ¼ -.
repeat units of exopolysaccharides that carries functional groups
such as hydroxyl (OeH), carboxyl (C]O) and amines (NeH) was
given by Seviour et al. (2012). According to Flemming and Wingender
(2010), the functional groups interact with each other in terms of
hydrogen bonding, electrostatic and ionic attractive forces for the
stability of the EPS matrix. Besides this, the Van der Waals interaction
that exists between any two EPS components also aids to serve for
EPS stability. Consequently, the ionisable groups presented on the
cell aggregate surface will decrease, and subsequently lower the
polar interaction of EPS with water molecules to promote cell hy-
drophobicity (Wang et al., 2006). Thus, in the sense of thermody-
namics, an increase in cell hydrophobicity simultaneously causes a
decrease in the excess Gibss energy of the surface, which in turns
promotes self-aggregation of cells from the liquid phase to form a
new solid phase (Liu et al., 2009; Liu and Tay, 2002).
Sheng et al. (2010) report that the outer region of aerobic
granules comprises dispersible sludge cells that are glued by the
readily extractable EPS. It is expected that cell aggregates will carry
negative charges due to unbound functional groups. This is
consistent with the report by Wang et al. (2014), which indicated
the negative value in the zeta potential of EPS was mainly due to the
ionization of the anionic functional groups such as carboxylic and
phosphate. Since cell aggregates have the same charge, there is a
repulsive force that inhibits the flocculation process. Here, fila-
mentous microorganism help to bridge the individual cell or cell
aggregates together. Besides, in order to promote the flocculation of
cell aggregates for the formation of solid sludge, the forces involved
in First step process are needed.
:, Batch1 ¼ C, Batch2 ¼ *, Batch3 ¼ þ, Batch4 ¼ -.
10 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11
Fig. 9. A conceptual of granule formation and breakage.
Fig. 9 depicts a summary of the aerobic granule formation based
on the discussion above. The physical agitation force (First step
process) helps to initiate individual cell (Step A) contacts, whilst
intermolecular interaction (Second step process) ensures the ag-
gregation of individual cells (Step B). As the cell aggregates carry
negative charges (Step C), bridging between filamentous bacteria,
divalent (Ca2þ, Mg2þ, etc.) or monovalent (Kþ, Naþ, etc.) metal
cations from the particle surface of wastewater with the dissociated
functional groups on the cell aggregates enhance the flocculation
process. In Step D, polymer (polymeric chains of EPS) entanglement
tends to rearrange the position of cell aggregates to become more
compact and strong cell flocs (Sheng et al., 2010). Lin et al. (2013)
provides a clear illustration of the strong gel structure developed
from a crosslink between alginate-like exopolysaccharides and
Ca2þ. During Step E, cell flocs approach each other in the case there
exists an electrostatic attraction between positive and negative
areas of cell flocs. Shear force shapes and densifies cell flocs (Step E)
to become granular sludge, as shown in Step F. The mature granular
sludge subsequently disintegrates after reaching a certain critical
size, and then causes an overall reduction in granular size
(Verawaty et al., 2013).
The selection of reactor type and H/D ratio for applications at the
real site vary depending on the characteristics of the wastewater
itself. For example, Papadia et al. (2011) concluded that the air
supply system has greatly influenced the efficiency of pilot scale
bioreactors treating textile wastewater. The highest performance
was achieved in Bioflotation and Fixed Bed Biofilm Reactors, which
exhibited an H/D ratio of 5 and 3 respectively. The list presented by
Liu et al. (2014) on aerobic granule reactors treating real waste-
water showed that the H/D ratio for pilot scale reactors in both the
Netherlands and China was 10. Meanwhile, the H/D ratio for pilot
scale reactors treating synthetic wastewater in Spain was 8. How-
ever, at full scale application, Gansbaai WWTP, located in South
Africa, demonstrates that the lowest H/D ratio is 0.39. Thus,
although the main criteria that ensures the successful formation of
aerobic granules is the high H/D ratio reactor, in the case of dealing
with real site applications, the low H/D ratio reactor is preferable.
5. Conclusion
The investigation of the kinetics of mature aerobic granules
shows that aerobic granules developed in reactors with a high H/D
ratio have a shorter setup time period and are more active
compared to those in low H/D ratio reactors. Meanwhile, under
variable organic loading rate and biomass floating events, reactors
with a low H/D ratio exhibit an efficient performance compared to
high H/D ratio reactors. At equal effluent performance and OLR, the
production of biomass in low H/D ratio reactors is less than in high
H/D ratio reactors. This comes along with great benefits in terms of
reducing costs involved in the sludge thickening process for con-
ventional activated sludge systems.
Acknowledgments
This work was financially supported by the Department of
Higher Education Malaysia (FP041-2013B) and University of Malaya
research grants (RG160-12SUS and PV038-2012A). Awang N.A ac-
knowledges the Ministry of Education Malaysia and Universiti Sains
Malaysia for their support in her PhD fellowship.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ibiod.2016.04.028.
References
Adav, S.S., Lee, D.-J., Show, K.-Y., Tay, J.-H., 2008. Aerobic granular sludge: recent
advances. Biotechnol. Adv. 26, 411e423. http://dx.doi.org/10.1016/
j.biotechadv.2008.05.002.
APHA, 2005. Standard Methods for the Examination of Water and Wastewater,
twenty-first ed. American Public Health Association, Washington DC. USA.
Asadi, A., Zinatizadeh, A.A., Sumathi, S., 2014. Industrial estate wastewater treat-
ment using single up-flow aerobic/anoxic sludge bed ( UAASB ) Bioreactor : a
kinetic evaluation study. Environ. Prog. Sustain. Energy 0, 1e9 doi:10.1002/ep.
Awang, N.A., Shaaban, M.G., 2015. The impact of reactor Height/Diameter (H/D)
ratio on aerobic granular sludge (AGS) formation in sewage. J. Teknol. (Sci. Eng.)
 N.A. Awang, M.G. Shaaban / International Biodeterioration & Biodegradation 112 (2016) 1e11
77, 95e103. http://dx.doi.org/10.11113/jt.v77.6690.
Chen, Y., Jiang, W., Liang, D.T., Tay, J.H., 2007. Structure and stability of aerobic
granules cultivated under different shear force in sequencing batch reactors.
Appl. Microbiol. Biot. 76, 1199e1208. http://dx.doi.org/10.1007/s00253-007-
1085-7.
Chen, Y., Jiang, W., Liang, D.T., Tay, J.H., 2008. Biodegradation and kinetics of aerobic
granules under high organic loading rates in sequencing batch reactor. Appl.
Microbiol. Biot. 79, 301e308. http://dx.doi.org/10.1007/s00253-008-1421-6.
De Kreuk, M.K., 2006. Aerobic Granular Sludge: Scaling up a New Technology. Ph.D.
thesis. Technical University Delft, The Netherlands.
Flemming, H.-C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8,
623e633. http://dx.doi.org/10.1038/nrmicro2415.
Han, K., Levenspiel, O., 1988. Extended monod kinetics for substrate product and
cell inhibition. Biotechnol. Bioeng. 32, 430e437.
Kaewsuk, J., Thorasampan, W., Thanuttamavong, M., Tae, G., 2010. Kinetic devel-
opment and evaluation of membrane sequencing batch reactor ( MSBR ) with
mixed cultures photosynthetic bacteria for dairy wastewater treatment.
J. Environ. Manag. 91, 1161e1168. http://dx.doi.org/10.1016/
j.jenvman.2010.01.012.
Kantawanichkul, S., 1990. Application of Hollow Fiber Membrane Bioreactor to
Anaerobic Treatment of Oily Wastewater by Photosynthetic Bacteria: Rhodo-
bacter Sphaeroides. Ph.D. thesis. AIT, Bangkok, Thailand.
Kong, Q., Wang, Z.-B., Shu, L., Miao, M.-S., 2015. Characterization of the extracellular
polymeric substances and microbial community of aoerbic granulation sludge
exposed to cefalexin. Int. Biodeterior. Biodegrad. 102, 375e382. http://
dx.doi.org/10.1016/j.ibiod.2015.04.020.
Kong, Y., Liu, Y.-Q., Tay, J.-H., Wong, F.-S., Zhu, J., 2009. Aerobic granulation in
sequencing batch reactors with different reactor height/diameter ratios.
Enzyme Microb. Tech. 45, 379e383. http://dx.doi.org/10.1016/
j.enzmictec.2009.06.014.
Liang, Y., Li, D., Zeng, H., Zhang, C., Zhang, J., 2015. Rapid start-up and microbial
characterixtics of partial nitrification granular sludge treating domestic sewage
at room temperature. Bioresour. Technol. 196, 741e745. http://dx.doi.org/
10.1016/j.biortech.2015.08.003.
Lin, Y.M., Sharma, P.K., van Loosdrecht, M.C.M., 2013. The chemical and mechanical
differences between alginate-like exopolysaccharides isolated from aerobic
flocculent sludge and aerobic granular sludge. Water Res. 47, 57e65. http://
dx.doi.org/10.1016/j.watres.2012.09.017.
Liu, L., Wang, Z., Yao, J., Sun, X., Cai, W., 2005a. Investigation on the formation and
kinetics of glucose-fed aerobic granular sludge. Enzyme Microb. Tech. 36,
712e716. http://dx.doi.org/10.1016/j.enzmictec.2004.12.024.
Liu, L., Wang, Z., Yao, J., Sun, X., Cai, W., 2005b. Investigation on the properties and
kinetics of glucose-fed aerobic granular sludge. Enzyme Microb. Tech. 36,
307e313. http://dx.doi.org/10.1016/j.enzmictec.2004.09.010.
Liu, X.-W., Sheng, G.-P., Yu, H.-Q., 2009. Physicochemical characteristics of microbial
granules. Biotechnol. Adv. 27, 1061e1070. http://dx.doi.org/10.1016/
j.biotechadv.2009.05.020.
Liu, Y., Liu, Z., Wang, F., Chen, Y., Kuschk, P., Wang, X., 2014. Regulation of aerobic
granular sludge reformulation after granular sludge broken: effect of poly
aluminum chloride (PAC). Bioresour. Technol. 158, 201e208. http://dx.doi.org/
10.1016/j.biortech.2014.02.002.
Liu, Y., Tay, J.-H., 2002. The essential role of hydrodynamic shear force in the for-
mation of biofilm and granular sludge. Water Res. 36, 1653e1665.
Liu, Y.-Q., Tay, J.-H., 2007. Cultivation of aerobic granules in a bubble column and an
airlift reactor with divided draft tubes at low aeration rate. Biochem. Eng. J. 34,
1e7. http://dx.doi.org/10.1016/j.bej.2006.11.009.
Liu, Y.-Q., Tay, J.-H., 2012. The competition between flocculent sludge and aerobic
granules during the long-term operation period of granular sludge sequencing
batch reactor. Environ. Technol. 33, 2619e2626. http://dx.doi.org/10.1080/
09593330.2012.673011.
Long, B., Yang, C.-Z., Pu, W.-H., Yang, J.-K., Liu, F.-B., Zhang, L., Zhang, J., Cheng, K.,
2015. Tolerence to oerganic loading rate by aerobic granualr sludge in a cyclic
aerobic granular reactor. Bioresour. Technol. 182, 314e322. http://dx.doi.org/
10.1016/j.biortech.2015.02.029.
Lotito, A.M., Fratino, U., Mancini, A., Bergna, G., Di Iaconi, C., 2012. Effective aerobic
granular sludge treatment of a real dyeing textile wastewater. Int. Biodeterior.
Biodegrad. 69, 62e68. http://dx.doi.org/10.1016/j.ibiod.2012.01.004.
Ma, J., Quan, X., Li, H., 2013. Application of high OLR-fed aerobic granules for the
treatment of low-strength wastewater: performance, granule morphology and
microbial community. J. Environ. Sci. 25, 1549e1556. http://dx.doi.org/10.1016/
S1001-0742(12)60243-5.
Metcalf and Eddy, Tchobanoglous, G., Burton, F.L., Stensel, H.D., 2003. Wastewater
Engineering: Treatment and Reuse, fourth ed. McGraw-Hill, Boston.
Ministry of Housing and Local Government, 1998. Guidelines for Developers:
Sewage Treatment Plant, second ed., vol. IV (Malaysia).
Ministry of Natural Resource and the Environment., 2009. Environmental Quality
(Sewage) Regulations (2009), under the Laws of Malaysia. Malaysia Environ-
mental Quality Act 1974.
11
Mittelman, M., 1985. Biological fouling of purified-water sytems: part 1, bacteria
growth and replication. Microcontaminat 3, 51e55.
Mizzouri, N.S., Shaaban, G., 2013. Kinetic and hydrodynamic assessment of an
aerobic purification system for petroleum refinery wastewater treatment in a
continuous regime. Int. Biodeterior. Biodegrad. 83, 1e9. http://dx.doi.org/
10.1016/j.ibiod.2013.03.026.
Muda, K., Aris, A., Razman, M., Ibrahim, Z., van Loosdrecht, M.C.M., Ahmad, A.,
Zaini, M., 2011. The effect of hydraulic retention time on granular sludge
biomass in treating textile wastewater. Water Res. 45, 4711e4721. http://
dx.doi.org/10.1016/j.watres.2011.05.012.
Ni, B.-J., Xie, W.-M., Liu, S.-G., Yu, H.-Q., Wang, Y.-Z., Wang, G., Dai, X.-L., 2009.
Granulation of activated sludge in a pilot-scale sequencing batch reactor for the
treatment of low-strength municipal wastewater. Water Res. 43, 751e761.
http://dx.doi.org/10.1016/j.watres.2008.11.009.
Okpokwasili, G.C., Nweke, C.O., 2005. Microbial growth and substrate utilization
kinetics. Afr. J. Biotechnol. 5, 305e317.
Pala, A., Bo € 2005. Evaluation of kinetic parameters for biological CNP
€lükba, O.,
removal from a municipal wastewater through batch tests. Process Biochem. 40,
629e635. http://dx.doi.org/10.1016/j.procbio.2004.01.060.
Papadia, S., Rovero, G., Fava, F., Gioia, D.D., 2011. Comparison of different pilot scale
bioreactors for the treatment of a real wastewater from the textile industry. Int.
Biodeterior. Biodegrad. 65, 396e403. http://dx.doi.org/10.1016/
j.ibiod.2011.01.002.
Pronk, M., de Kreuk, M.K., de Bruin, B., Kamminga, P., Kleerebezem, R., van
Loosdrecht, M.C.M., 2015. Full scale performance of the aerobic granular sludge
process for sewage treatment. Water Res. 84, 207e217. http://dx.doi.org/
10.1016/j.watres.2015.07.011.
Seviour, T., Yuan, Z., van Loosdrecht, M.C.M., Lin, Y., 2012. Aerobic sludge granula-
tion: a tale of two polysaccharides? Water Res. 46, 4803e4813. http://
dx.doi.org/10.1016/j.watres.2012.06.018.
Sheng, G.-P., Yu, H.-Q., Li, X.-Y., 2010. Extracellular polymeric substances (EPS) of
microbial aggregates in biological wastewater treatment systems: a review.
Biotechnol. Adv. 28, 882e894. http://dx.doi.org/10.1016/
j.biotechadv.2010.08.001.
Su, B., Cui, X., Zhu, J., 2012. Optimal cultivation and characteristics of aerobic
granules with typical domestic sewage in an alternating anaerobic/aerobic
sequencing batch reactor. Bioresour. Technol. 110, 125e129. http://dx.doi.org/
10.1016/j.biortech.2012.01.127.
Verawaty, M., Tait, S., Pijuan, M., Yuan, Z., Bond, P.L., 2013. Breakage and growth
towards a stable aerobic granule size during the treatment of wastewater.
Water Res. 47, 5338e5349. http://dx.doi.org/10.1016/j.watres.2013.06.012.
Wang, B.-B., Peng, D.-C., Hou, Y.-P., Li, H.-J., Pei, L.-Y., Yu, L.-F., 2014. The important
implications of particulate substrate in determining the physicochemical
characteristics of extracellular polymeric substances (EPS) in activated sludge.
Water Res. 58, 1e8. http://dx.doi.org/10.1016/j.watres.2014.03.060.
Wang, D., Zheng, W., Li, X., Yang, Q., Liao, D., Zeng, G., 2013a. Evaluation of the
feasibility of alcohols serving as external carbon sources for biological phos-
phorus removal induced by the oxic/extended-idle regime. Biotechnol. Bioeng.
110, 827e837.
Wang, D., Chen, Y., Zheng, X., Li, X., Feng, L., 2013b. Short-chain fatty acid production
from different biological phosphorus removal sludges: the influences of PHA
and gram-staining bacteria. Environ. Sci. Technol. 47, 2688e2695. http://
dx.doi.org/10.1021/es304673s.
Wang, S.-G., Liu, X.-W., Gong, W.-X., Gao, B.-Y., Zhang, D.-H., Yu, H.-Q., 2007a.
Aerobic granulation with brewery wastewater in a sequencing batch reactor.
Bioresour. Technol. 98, 2142e2147. http://dx.doi.org/10.1016/
j.biortech.2006.08.018.
Wang, X.H., Zhang, H.M., Yang, F.L., Xia, L.P., Gao, M.M., 2007b. Improved stability
and performance of aerobic granules under stepwise increased selection
pressure. Enzyme Microb. Tech. 41, 205e211. http://dx.doi.org/10.1016/
j.enzmictec.2007.01.005.
Wang, Z., Liu, L., Yao, J., Cai, W., 2006. Effects of extracellular polymeric substances
on aerobic granulation in sequencing batch reactors. Chemosphere 63,
1728e1735. http://dx.doi.org/10.1016/j.chemosphere.2005.09.018.
Xiao, F., Yang, S.F., Li, X.Y., 2008. Physical and hydrodyanamic properties of aerobic
granules produced in sequencing batch reactors. Sep. Purif. Technol. 63,
634e641. http://dx.doi.org/10.1016/j.seppur.2008.07.002.
Yang, S.-F., Tay, J.-H., Liu, Y., 2004. Inhibition of free ammonia to the formation of
aerobic granules. Biochem. Eng. J. 17, 41e48. http://dx.doi.org/10.1016/S1369-
703X(03)00122-0.
Zhao, J., Wang, D., Li, X., Yang, Q., Chen, H., Zhong, Y., Zeng, G., 2015. Free nitrous
acid serving as apretreatment method for alkaline fermentation to enhance
short-chain fatty acid production from waste activated sludge. Water Res. 78,
111e120. http://dx.doi.org/10.1016/j.watres.2015.04.012.
Zhou, D., Niu, S., Xiong, Y., Yang, Y., Dong, S., 2014. Microbial selection pressure is
not a prerequisite for granulation: dynamic granulation and microbial com-
munity study in a complete mixing bioreactor. Bioresour. Technol. 161, 102e108.
http://dx.doi.org/10.1016/j.biortech.2014.03.001.