Front. Environ. Sci. Eng.
DOI 10.1007/s11783-014-0627-3
RESEARCH ARTICLE
Cultivation of aerobic granular sludge in a conventional,
continuous flow, completely mixed activated sludge system
Xi CHEN1, Linjiang YUAN (✉)1, Wenjuan LU1, Yuyou LI1,2, Pei LIU1, Kun NIE1
1 School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China
2 Department of Environmental Science, Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2013
Abstract Aerobic granules were formed in a conven-
tional, continuous flow, completely mixed activated sludge
system (CMAS). The reactor was inoculated with seed
sludge containing few filaments and fed with synthetic
municipal wastewater. The settling time of the sludge and
the average dissolved oxygen (DO) of the reactor were 2 h
and 4.2 mg$L–1, respectively. The reactor was agitated by a
stirrer, with a speed of 250 r$min–1, to ensure good
mixing．The granular sludge had good settleability, and
the sludge volume index (SVI) was between 50 and 90 mL
$g–1. The laser particle analyzer showed the diameter of the
granules to be between 0.18 and 1.25 mm. A scanning
electron microscope (SEM) investigation revealed the
predominance of sphere-like and rod-like bacteria, and
only few filaments grew in the granules. The microbial
community structure of the granules was also analyzed by
polymerase chain reaction-denaturing gradient gel electro-
phoresis (PCR-DGGE). Sequencing analysis indicated the
dominant species were α, β, and γ-Proteobacteria,
Bacteroidetes, and Firmicutes. The data from the study
suggested that aerobic granules could form, if provided
with sufficient number of filaments and high shear force. It
was also observed that a high height-to-diameter ratio of
the reactor and short settling time were not essential for the
formation of aerobic granular sludge.
Keywords aerobic granular sludge, completely mixed
activated sludge system (CMAS), continuous flow, shear
force, filamentous bacteria, polymerase chain reaction-
denaturing gradient gel electrophoresis (PCR-DGGE)
Received September 11, 2012; accepted August 19, 2013
E-mail: Xi_Chen2011@126.com, yuanlinjiang@xauat.edu.cn
1 Introduction
The activated sludge process is the most widely used
technology for biologic wastewater treatment in the world.
As the sludge in conventional activated systems is
flocculated, these systems have some inherent disadvan-
tages, such as high surplus biomass production, low sludge
age, and continuous solid separation problems. Compared
with flocculated sludge in the conventional activated
sludge system, granular sludge has regular, compact and
strong microbial structure, good settleability, high biomass
retention, and the ability to withstand shock loading.
Previous studies indicated that almost all aerobic granules
were cultivated in either continuously operated reactors,
such as aerobic upflow sludge blanket (AUSB) apparatus
[1], or in discontinuously operated systems, such as
sequencing batch reactors (SBR) [2–7].
However, the actual mechanism of granulation is still the
subject of discussion where factors under consideration are
shear force, settling time, height-to-diameter ratio of the
reactor and the microbial content of the sludge. Mishima et
al. [1], in their study, suggested that mild shear stresses on
the granular sludge, the high dissolved oxygen (DO)
concentration, and the self-tangling growth of filamentous
microorganisms, in the AUSB process, may be responsible
for the formation of granular sludge. In contrast, other
researchers demonstrated that regular-shaped granules
were successfully cultivated with relatively high shear
force, and only fluffy flocs were formed with low shear
force [8]. To enhance the formation of granular sludge, the
settling time of the reactor was always quite short, ranging
from 1 to 20 min. McSwain et al. [9] suggested that a short
settling time was necessary to acquire predominantly
granular sludge. However, Peng et al. [3] cultivated
aerobic granular sludge with quite a long settling time of
2.5 h. Hence, the necessity of a short settling time is
questionable. To date, nearly 100% of aerobic and
2 Front. Environ. Sci. Eng.
anaerobic granules are formed in column-type air or liquid-
lifted upflow reactors, which always have a high height-to-
diameter ratio, compared to completely mixed tank
reactors (CMTR). It is still unclear if the high ratio of
height to diameter of a reactor is indispensable to the
formation of granular sludge. Recently, culture-indepen-
dent molecular techniques have been successfully applied,
to reveal the microbial community structure of activated
sludge in wastewater treatment systems. Zhang et al. [10]
investigated the distribution of ammonia-oxidizing bacter-
ial communities during sludge granulation, in an anaero-
bic-aerobic sequencing batch reactor, using the polymerase
chain reaction-denaturing gradient gel electrophoresis
(PCR-DGGE) method. However, few researchers have
investigated the community structure of granular sludge
cultivated in conventional, continuous flow, completely
mixed activated sludge systems.
In the current study, two conventional, continuous flow,
completely mixed activated sludge systems (CMAS), a
sort of CMTR, were operated. Granular sludge formed
only in the reactor inoculated with sludge containing some
filaments. The study showed, for the first time, the
formation of granular sludge in the CMAS system. Some
preliminary characteristics of the granules were identified,
and the bacterial community structure was analyzed. The
study also discussed the possible mechanism of granule
formation.
2 Materials and methods
2.1 Reactor description
Two conventional, continuous flow, completely mixed
activated sludge systems were operated (Fig. 1). Both
Fig. 1 Schematic diagram of the laboratory-scale CMAS reactor
reactors were made of cylindrical plexiglass, with a
working volume of 18 L, a height-to-diameter ratio of
1.0. The working volume of the clarifier was 4.5 L. Reactor
I was inoculated with sludge from the secondary clarifier of
a local municipal wastewater treatment plant adopting
anaerobic/anoxic/aerobic process in Xi’an, China. The
seed sludge was flocculated and irregular. It contained
some filaments, and had a sludge volume index (SVI)
value of 140 mL$g–1 (Fig. 2(a)). The sludge was fed
with synthetic municipal wastewater. The composition
of the synthetic wastewater is shown in Table 1. A
mechanical mixer was installed in the reactor, and stirred at
250 r$min–1 to ensure good mixing. Air was introduced
from the bottom of the reactors with a flow rate of
568 L$min–1, through two fine bubble aerators. The
average DO concentration was controlled at approximately
4.2 mg$L–1. The reactor was operated at room temperature
(25°C–27°C). The flow rates of the influent and return
sludge were controlled by two peristaltic pumps. The
influent flow rate was 54 L$d–1, resulting in an 8 h
hydraulic retention time (HRT) in the bioreactor, and a
settling time of 2 h. The return activated sludge percentage
of influent was 100%.
The second reactor was operated as control with the
same operation parameters as Reactor I, except the seed
sludge. This reactor was inoculated with sludge containing
no filaments (Figs. 2(b) and 2(c)), and the SVI value of the
sludge was 90 mL$g–1. For Reactor I, because the activated
sludge grew too fast during the cultivated period (day 1 to
day 21), the sludge retention time (SRT) in this reactor was
difficult to maintain around 18 days. The SRT was then
reduced to 9 days from day 22 to keep a relatively constant
organic loading. For the control reactor, the SRT was 18
days during the whole experimental period. The
chemical oxygen demand (COD) of the influent was
 Xi CHEN et al. Cultivation of aerobic granular sludge in a conventional, continuous flow, CMAS 3

Fig. 2 Activated sludge in the reactors designated as: (a) seed sludge of Reactor I, (b) seed sludge of control reactor, (c) Neisser staining
of the seed sludge in the control reactor, (d) and (e) granular sludge in Reactor I, (f) flocculated sludge in the control reactor

about 450 mg$L–1. It should be noted that potato starch wastewater, and represented about 150 mg$L–1 of the
was used to simulate the particulate matter in the municipal COD. Mixed liquor suspended solid (MLSS) in the reactor
4 Front. Environ. Sci. Eng.

Table 1 Composition of the synthetic municipal wastewater  1=2

P
food ingredient and chemical compounds –1
concentration/(mg$L ) GS ¼ , (1)
μV
Na-acetate 79.37
potato starch 122 P is defined by Rushton [12] as follows:
peptone 17.41 1
P ¼ KT n3 D5i ρ, (2)
whole milk powder 96.86 6
yeast 52.24 For turbulent flow (NRe > 10000),
Urea 91.74
D2i nρ
NH4Cl 12.75 NRe ¼ , (3)
μ
K2HPO4$3H2O 37.98
KH2PO4 23.4 where P is power, imparted to the water per unit volume of
MgSO4$7H2O 41.02
the basin (N$m$s–1), µ is absolute viscosity of the water
(0.8737  10–3 N$s$m–2, 26°C), V is the tank volume
CaCl2 5
(0.018 m3), KT is impeller constant for turbulent flow
NaHCO3 25 (0.32) [13], n is rotational speed, 4.17 s–1, Di is impeller
FeSO4$7H2O 10 diameter (0.095 m), ρis density of water (0.9968103 kg
KI 0.06 $m–3, 26°C).
H3BO3 0.3
The shear rate provided by the air diffuser is given as
[14]:
MnSO4$H2O 0.1
 
CoCl2$6H2O 0.49 gρUg 1=2
GA ¼ , (4)
(NH4)6Mo7O24$4H2O 0.11 μ
CuSO4$5H2O 0.70
where g is acceleration due to gravity (9.806 m$s–2), Ug is
ZnSO4$7H2O 0.38 the superficial gas velocity (0.1494 m$s–1).

2.4 Microbial community analysis

was kept within a range of 2800–3500 mg$L–1 during the
experimental period, and the organic loading was main-
tained between 0.38 and 0.48 kg COD$kg–1 MLSS$d–1).
The average concentrations of total nitrogen (TN) and total
phosphorus (TP) were 74 and 10 mg$L–1, respectively.
2.2 Analytical methods
COD, MLSS, mixed liquor volatile suspended solids
(MLVSS), SVI, ammonia, nitrite and nitrate were
measured according to standard methods [11]. The particle
size distribution in both reactors was measured by a laser
particle size analysis system (LS230, COULTER, USA) on
day 61. The microbial composition of the granules in
Reactor I was observed qualitatively, with a scanning
electron microscope (SEM) on day 62. Sludge samples
were taken for microscopic observation, with Gram and
Neisser staining, each week. It is important to note that in
order to get a clear picture, the granules were crushed
before Neisser staining.
2.3 Calculation of shear force
The shear force in the reactor was provided by both stirrer
and air diffuser. The average shear rate GS from the stirrer
is given as follows:
The microbial community structure of the granular sludge in
Reactor I was analyzed by PCR-DGGE. Four milliliter
sludge was collected from the reactor on day 62. Genomic
DNA was extracted using a soil DNA kit (D5625-01, Omega
Bio-Tek Inc., USA). Universal primers, 341F with GC clamp
and 534R, were used to amplify the conserved V3 region of
16S rRNA for most bacteria and Archaea [15]. The
nucleotide sequences of the primers are: GC-341F (5′-
CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGC-
ACGGGGGGCCTACGGGAGGCAGCAG-3′) and 534R
(5′-ATTACCGCGGCTGCTGG-3′). The PCR protocol
was for a 50 μL reaction volume. The PCR conditions
were designed as follows: an initial denaturation at 94°C
for 5 min, then 30 cycles of denaturation at 94°C for 30 s,
annealing at 55°C for 30 s and extension at 72°C for 30 s,
followed by a final extension of 10 min at 72°C. The PCR
product was electrophoresed on a 1.5% agarose gel, to
check for amplicon size and concentration. After electro-
phoresis, 30 μL of the amplified PCR product was loaded
and separated by DGGE on polyacrylamide gel (8%,
37.5:1 acrylamide:bisacrylamide) with a linear 40% to
60% denaturant agent gradient. The gel was run for 13 h at
70 V using 1  TAE buffer maintained at 60°C (Dcode™,
Bio-Rad, USA). The DGGE gel was stained with silver
nitrate solution (1 g$L–1) for 30 min and observed using an
ultra violet (UV) transilluminator (GBOXEF, SYNGENE,
 Xi CHEN et al. Cultivation of aerobic granular sludge in a conventional, continuous flow, CMAS
UK). DNA was recovered by cutting the band of interest of
the gelatin map, then re-amplified, cloned, and finally
sequenced at Sangon Biotech Co., Ltd. (China). 16S rRNA
sequence similarity analysis was performed using the
GenBank server of the National Center for Biotechnology
Information (NCBI) and the BLAST algorithm1).
3 Results and discussion
Formation of granular sludge in a conventional, continuous
flow, completely mixed activated sludge system
Microscopic observation showed that granular sludge
was formed in Reactor I on day 21 (Figs. 2(d) and 2(e)). As
shown in Fig. 2(e), the granular sludge had a relatively
round shape and a clear outer surface. After 70 days of
operation, Reactor I was still working and the granular
sludge in the reactor was stable. For the control reactor, the
SVI value of the sludge was always between 50 and 100
mL$g–1. Although the sludge in the control reactor tended
to coagulate to form large flocs (Fig. 2(f)), no granules
were formed in this reactor.
So far, aerobic granules were always formed in column-
type air or liquid lifted upflow reactors, whose reactor
height to diameter ratios were quite high (eg, 17.0) [16–
18]. Liu et al. [19], suggested that the air of liquid upflow
patterns, in column reactors, can create a relatively
homogenous, circular flow along the reactor height,
which could force microbial aggregates to be regular
shaped granules. For CMTR systems, they believed that
microbial aggregates stochastically moved in a dispersed
manner, and thus could only form flocs of irregular shapes
and sizes. In our study aerobic granular sludge was formed
in a CMAS system, with a height-to-diameter ratio of 1.0.
This phenomenon contradicted the hypothesis proposed by
Liu et al. This suggested that high height-to-diameter ratio
of the reactor may be not essential for the formation of
aerobic granular sludge.
It is generally thought that a short settling time can
create a selective pressure on organisms resulting in two
responses; to be washed out or to bind together and form
easily settling granules [20]. Almost all the reactors in the
previous studies had relatively short settling times, varying
from 1 to 20 min. McSwain et al. [9], suggested that a short
settling time was necessary to select for predominantly
granular sludge. However, Peng et al. [3], cultivated the
aerobic granular sludge in an SBR reactor with a 2.5 h
settling time. In our experiment, the settling time of the
sludge was 2.0 h. Although this settling time was quite
long, aerobic granules did form in Reactor I. This
observation seems to imply that a short settling time may
not be essential for the formation of granules, in some
cases.
1) http://www.ncbi.nlm.nih.gov
5
It is suggested that hydrodynamic shear force would
have a significant influence on the formation of aerobic
granules. Beun et al. [21], found that a low superficial air
velocity of 0.014 or 0.020 m$s–1 did not lead to the
formation of stable aerobic granules. However at a
relatively high superficial air velocity of 0.041 m$s–1
granulation did occur, with the formation of smooth,
dense and stable aerobic granules. These results seem to
indicate that aerobic granulation is a phenomenon
associated very closely with the hydrodynamic conditions
present in the reactors. A similar phenomenon was also
observed by Wu et al. [22]. In the current experiment, the
shear force in the reactor was provided by both the stirrer
and the air diffuser. The superficial gas velocity, Ug,was
0.1494 m$s–1, which was about 4 times the superficial air
velocity in the experiment of Beun et al. [21]. The shear
rate provided by the stirrer (GS) and the air diffuser (GA)
were 18.98 s–1 and 1293 s–1, respectively. This indicated
that the shear force in the reactor was quite strong, which
was beneficial for the formation of granules.
3.1 Bioreactor performance
The MLSS and SVI of seed sludge, containing filamentous
bacteria, was compared to seed sludge, lacking filamentous
bacteria, in 2 conventional, continuous flow, completely
mixed activated sludge system reactors. Fig. 3(a) shows
the change in MLSS and SVI values of the sludge in both
reactors. For Reactor I, after about 21 days, the SVI value
decreased from 140 to 82 mL$g–1 and the MLSS value
increased significantly to 4038 mg$L–1. To adjust the
sludge loading, 4 L sludge was withdrawn from Reactor I
on day 22. The SVI value increased to 110 mL$g–1 on day
26, and then suddenly decreased to 30 mL$g–1 the next day.
In the following days, the SVI value of the sludge was
maintained between 30 and 90 mL$g–1, which falls within
the SVI range of aerobic granules, from 20 to 100 mL$g–1
[2–4].
Fig. 3(b) illustrates the changes in COD and NH3-N in
both reactors. It shows that when Reactor I reached stable
state, the COD removal efficiency was always higher than
89%, and the concentration of NH3-N in the effluent was
lower than 1.0 mg$L–1. The peaks for COD and NH3-N, in
Reactor I, were due to the withdrawal of sludge in this
reactor on day 22. On this day, the MLSS in Reactor I
suddenly decreased from 4200 to 3500 mg$L–1, resulting
in the dramatic increase of sludge loading from 0.32 to
0.39 kg COD$kg–1 SS$d–1. The higher sludge loading rate
led to lots of free bacteria in the effluent and thus increased
the COD of the effluent. When the sludge was withdrawn
from the reactor, many nitrifying bacteria were lost. Since
the nitrifying bacteria usually grow slowly, the nitrification
capacity of Reactor I was weakened, giving rise to the high
NH3-N in the effluent.
6 Front. Environ. Sci. Eng.
Fig. 3 (a) Changes of mixed liquor suspended solids (MLSS) and sludge volume index (SVI) values in the given time period in both
reactors, (b) Changes of chemical oxygen demand (COD) and NH3-N in the given time period in both reactors
3.2 Detection of filamentous bacteria by Gram and Neisser
staining
During the experimental period, growth of filamentous
bacteria was not detected in both reactors by Gram and
Neisser staining (Fig. 4). This was due to the particulate
matter (potato starch) in the influent. Because there was a
lot of starch in the influent, part of the starch granules was
retained within the sludge, and not all the substrate was
consumed outside the sludge [23]. The presence of starch
in the sludge led to the absence of a substrate gradient. The
lack of a substrate gradient failed to provide advantageous
conditions for the development of filamentous bacteria in
the sludge.
3.3 Characteristics of the granules and effect of the
filaments in the seed sludge
Granule size of the sludge is an important parameter for
characterizing granulation. The size distribution of the
granules in Reactor I and the flocculated sludge in the
control reactor was measured by a laser particle size
analysis system on day 61. As shown in Fig. 5(a), granules
of a wide range of diameters coexisted from 0.18 to 1.25
mm.
Fig. 5(b) is the scanning electron micrograph of the
granule surface on day 62, showing the predominance of
rod-like and sphere-like bacteria. Few filaments grew in
the granules. The distribution of microorganisms differed
slightly from the results of Tay et al. [4], which showed
cells tightly linked together with only rod-like species
predominant in the outer surface. There were also many
cavities between the bacteria. It was conjectured that the
substrate transfer from the bulk to the granules and the
reverse intermediate or by-product transfer from inside of
the granules to the bulk, could have been strengthened by
these cavities.
It is interesting to note that although the SEM view
showed few filaments in the granules, microscopic
observation, verified by both Gram and Neisser staining,
showed an absence of filaments growing on the surface of
the granules (Figs. 4(a) and 4(b)). Mishima et al. [1],
proposed that filamentous bacteria can get entangled, and
contribute to the formation of granular sludge. In the
current study granules only formed in Reactor I, which was
inoculated with sludge containing filamentous bacteria. It
was postulated that filaments in the seed sludge may
function in the way suggested by Mishima et al. With
greater shear force, the seed sludge collided continuously
and became entangled with each other through filaments.
Zoogloea grew on flocs and filaments, and also in the
cavities between them. At the same time, the filaments
served as a backbone to strengthen the structure of the floc.
With the help of filaments, and the high shear force, the
flocculated sludge became more and more compact and
smooth, and finally formed into granules. In the control
reactor, no granules were formed because there were no
filaments in the system during the entire experimental
period (Figs. 4(c) and 4(d)).
3.4 Community structure of the granules
The microbial community structure of the granular sludge
on day 62 was analyzed using PCR-DGGE, and each band
of the gel was considered as an operational taxonomy unit
(OTU). The predominant bands were excised from the gel.
DNA in these bands were reamplified, cloned and
sequenced. Fig. 6(a) shows the DGGE profile of the
microbial community from the aerobic granules. Fig. 6(b)
illustrates the evolutionary relationship of clones and their
closest neighbors, using a phylogenetic tree. The bacteria
in the granules can be classified into three categories. The
species of (1), (2), and (3) belonged to Bacteroidetes,
Proteobacteria, and Firmicutes, respectively. The com-
 Xi CHEN et al. Cultivation of aerobic granular sludge in a conventional, continuous flow, CMAS 7

Fig. 4 Gram and Neisser staining of the sludge in both reactors. (a) Gram staining of granules in Reactor I, (b) Neisser staining of
crushed granules in Reactor I, (c) Gram staining of sludge in the control reactor, (d) Neisser staining of sludge in the control reactor

Fig. 5 (a) Distribution of particle size of sludge, measured by a laser particle size analysis system, in both reactors on day 61, (b) Surface
view of granules in Reactor I, at high magnification, by SEM
8 Front. Environ. Sci. Eng.

Fig. 6 (a) Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profile of the microbial community within
the aerobic granules, (b) Evolutionary relationship of clones collected from the DGGE gel and their closest neighbors, shown as a
phylogenetic tree

parative analysis of these partial 16S rRNA sequences,
shown in Table 2, revealed the phylogenetic affiliation of
the 14 sequences retrieved. The dominant bacteria were
closely related to α-Proteobacteria (3 OTUs), γ-Proteo-
bacteria (3 OTUs), Bacteroidetes (3 OTUs), Firmicutes (2
OTUs) and β-Proteobacteria (2 OTU).
Previous studies showed that Proteobacteria (α-, β- and
γ-) were often dominant in the activated sludge of
wastewater treatment plants [24,25]. Our results agreed
well with the previous studies. As shown in Table 2,
Proteobacteria accounted for a large amount of all
bacteria, and most of them belonged to the α- and γ-
subdivision. It is interesting to note that all the γ-
Proteobacteria in the granules were identified as a type
of Pseudomonas species. In Reactor I, they might play an
important role in degrading the organic matter of the
influent. In addition to Proteobacteria, Bacteroidetes were
also dominant in the granular sludge, and this also
coincided well with recent research [26]. Among all the
bacteria, the uncultured α-Proteobacterium (Band 1, α-
Proteobacteria) was detected in soil with high potential for
diesel oil degradation. Uncultured β-Proteobacterium
(Band 2, β-Proteobacteria) was found in soil contaminated
by aliphatic hydrocarbon [27], and Bacteroidetes often
appeared in oil-polluted seawater [28]. These three
aforementioned bacteria might be able to degrade hydro-
carbon hydrolyzed from whole milk powder and possess
the hydrophobic nature that would be beneficial to the
formation of granular sludge [29]. The Lactococcus
raffinolactis (Band 6, Firmicutes) was also found to be
dominant in the granular sludge, which agreed well with
the studies of Song et al. [30]. Song et al., discussed the
effect of seed sludge on the characteristics and microbial
community of aerobic granular sludge. They suggested
that Lactococcus rafinolactis would be the dominant
species when the seed sludge was taken from a municipal
wastewater treatment plant [27]. In our reactor, it is
postulated that Lactococcus rafinolactis may play a key
 Xi CHEN et al. Cultivation of aerobic granular sludge in a conventional, continuous flow, CMAS 9

Table 2 Species identification of selected DGGE bands

submission number
band closest relatives (accession No.) identity/% putative division origin
(from NCBI)
1 KC206008 uncultured α-Proteobacterium 100 α-Proteobacteria adapted soil microflora for oil degradation
(JQ919497.1)
2 KC206009 uncultured β-Proteobacterium 99 β-Proteobacteria aliphatic hydrocarbon-contaminated soil
(AM935413.1)
3 KC2060010 Chryseobacterium sp. bk_48 (HQ538681.1) 99 Bacteroidetes activated sludge
4 KC2060011 uncultured Pseudomonas sp. (HQ658836.1) 99 γ-Proteobacteria municipal wastewater treatment plant
5 KC2060012 uncultured Rhodobacteraceae bacterium 100 α-Proteobacteria full-scale wastewater treatment process
(AF368183.1)
6 KC2060013 Lactococcus raffinolactis (NR_044359.1) 100 Firmicutes activated sludge
7 KC2060014 uncultured Diaphorobacter sp. 100 β-Proteobacteria UASB reactor treating domestic waste-
(JX301649.1) water
8 KC2060015 uncultured Bacteroides sp. (KC110186.1) 100 Bacteroidetes wastewater treatment plant
9 KC2060016 uncultured bacterium (HQ471747.1) 100 activated sludge process
10 KC2060018 uncultured Ethanoligenens sp. 100 Firmicutes sediment from surface-flow wetland
(GQ183418.1)
11 KC2060017 uncultured α- Proteobacterium 95 α-Proteobacteria membrane bioreactor
(JN679095.1)
12 KC2060019 Pseudomonas sp. (JQ595540.1) 100 γ-Proteobacteria wastewater treatment system
13 KC2060021 uncultured Bacteroidetes bacterium 100 Bacteroidetes activated sludge degrading phenol
(DQ322192.2)
14 KC2060020 Pseudomonas sp. (JQ749635.1) 99 γ-Proteobacteria biologic system treating vinyl chloride

role in the fermentation process of glucose hydrolyzed
from potato starch. However, their role in the formation of
granular sludge is not yet clear.
4 Conclusions
The major conclusions of this study are summarized as
follows: our study was the first to show that aerobic
granular sludge was formed in a conventional, continuous
flow, completely mixed activated sludge system, inocu-
lated with sludge containing filamentous bacteria. Fila-
mentous bacteria and high shear force may play a key role
in the formation of granular sludge. An increased height-
to-diameter ratio of the reactor and short settling time were
not essential in the granule formation process. The granular
sludge had good settleability, and the SVI value was
between 50 and 90 mL$g–1. The diameter of the granules
was 0.18–1.25 mm. The SEM view showed sphere-like
and rod-like bacteria were predominant, and only a few
filaments grew in the granules. The dominant species in the
granules were α, β, and γ-Proteobacteria, Bacteroidetes,
and Firmicutes. The uncultured α-Proteobacterium (Band
1, α-Proteobacteria), uncultured β-Proteobacterium (Band
2, β-Proteobacteria), and Bacteroidetes may be able to
degrade hydrocarbon hydrolyzed from whole milk powder,
and possess a hydrophobic nature that would be beneficial
to form granules.
Acknowledgements This research was supported by the National Natural
Science Foundation of China (Grant No. 50878180).
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