Bioresource Technology 169 (2014) 344351

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Microbial communities of aerobic granules: Granulation mechanisms

Yi Lv a, Chunli Wan a, Duu-Jong Lee a,b,c,, Xiang Liu a, Joo-Hwa Tay d
a

Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai 200433, China
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan
c
Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan
d
Department of Civil Engineering, University of Calgary, Calgary, Canada
b

h i g h l i g h t s
 Microbial communities of mature and growing aerobic granules were probed.
 Sliced sample study showed a spherical core with anaerobic Rhodocyclaceae.
 High-throughput sequencing showed that ocs were transited to young granules.
 Microbial community data supported deterministic mechanism for granulation.

a r t i c l e

i n f o

Article history:
Received 10 June 2014
Received in revised form 30 June 2014
Accepted 1 July 2014
Available online 8 July 2014
Keywords:
Slicing
PCRDGGE
Spatial distribution
Microbial community

a b s t r a c t
Aerobic granulation is an advanced biological wastewater treatment technology. This study for the rst
time identied the microbial communities of sliced samples of mature granules by polymerase chain
reaction (PCR) amplication and denaturing gradient gel electrophoresis (DGGE) technique and those
of whole growing granules by high-throughput sequencing technique. The sliced sample study revealed
that mature granules have a spherical core with anaerobic Rhodocyclaceae covered by an outer spherical
shell with both aerobic and anaerobic strains. The growing granule study showed that the occulated
ocs were rst transited to young granules with increased abundances of Flavobacteriaceae, Xanthomonadaceae, Rhodobacteraceae and Microbacteriaceae, then the abundances of anaerobic strains were
increased owing to the formation of anaerobic core. Since the present granules were cultivated from occulated ocs, the microbial community data suggested that granules were formed via a deterministic
rather than via a random aggregationdisintegration mechanism.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Aerobic granulation is an emerging biotechnology for wastewater treatment (Adav et al., 2008a). Biological cells can be aggregated into a compact form without a carrier in sequencing batch
reactor (SBR) so the yielded granules have high biomass content,
excellent settling rate, and supreme resistance to toxicity in feed
wastewaters (Adav et al., 2008b). Owing to the high retention
capability of slow-growing strains, the aerobic granules were
applied in various industrial wastewater treatments that need synergetic growths of different groups of bacteria (Rosman et al.,
2014; Jemaat et al., 2014; Li et al., 2014; Wan et al., 2014a,b).

Corresponding author at: Department of Environmental Science and

Engineering, Fudan University, Shanghai, 200433, China. Tel.: +86 21 65642018;
fax: +86 21 65643597.
E-mail address: djlee@ntu.edu.tw (D.-J. Lee).
http://dx.doi.org/10.1016/j.biortech.2014.07.005
0960-8524/ 2014 Elsevier Ltd. All rights reserved.

Aerobic granules were generally produced by cultivating occulated sludge ocs under starvation and hydrodynamic stress (Lee
et al., 2010). Specic functional strains in the seed sludge would
secret excess extracellular polymeric substances (EPS) for assisting
granulation (Adav et al., 2009). Li et al. (2008) noted that high
organic loading rates reduced microbial diversities in seed sludge
and identied that b- and c-Proteobacteria and Flavobacterium
were the dominating species in their glucose granules. Zhao et al.
(2013) cultivated aerobic granules with piggery wastewaters at
high chemical oxygen demand (COD) and nitrogen (N) loadings.
These authors noted that strains Thauera, Zoogloea and heterotrophic and autotrophic denitriers were present in their granules.
At very high organic loading rates, Adav et al. (2009) noted strains
Zoogloea resiniphila, Acinetobacter sp. clone JT2 and bacterium clone
P1D1-516 retained in their aerobic granules. Winkler et al. (2013)
noted from their nitrifying granules that Nitrosomonas was the
dominant ammonia-oxidizing bacterium (AOB) in seed sludge
while in the formed granules both Nitrosomonas and Nitrosospira

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Y. Lv et al. / Bioresource Technology 169 (2014) 344351

were present in equal amounts. Liu et al. (2010) noted that the occulated ocs and the aerobic granules collected from the same
reactor did not show microbial community difference, suggesting
that granulation posed low microbial selection pressure on the
seed strains. However, Song et al. (2010) noted that the seed sludge
from beer wastewater treatment plant with dominant strains Paracoccus sp., Devosia hwasunensi, Pseudoxanthomonas sp. can be used
to cultivate mature granules. Conversely, the seed sludge from
municipal treatment plant with dominant strains Lactococcus raffinolactis and Pseudomonas sp. needed longer time to cultivate
mature granules. Li et al. (2010) noted that high organic loading
rate favors growth of bacterial granules over lamentous granules.
Therefore, if the function strains were enriched in seed sludge, the
microbial community would experience little change over granulation; otherwise, signicant microbial community change is
expected.
Both granules and occulated ocs co-existed in large-scale
reactor with real wastewaters (Liu et al., 2010). Short settling time
is commonly applied to facilitate washout of occulated sludge
ocs and ne aggregates from the reactor, which is often referred
to hydrodynamic selection pressure for granulation. Weissbrodr
et al. (2012) claimed that washout is a microbial selection process
while Zoogloea was enriched in their dense granules and lamentous Burkholderiales dominated in their loose granules. Verawaty
et al. (2012) added uorescent-labeled crushed granules into
labeled occulated ocs and noted that the ocs would attach onto
the crushed granules to minimize their washout, so shorten the
granulation time. Zhou et al. (2014) labeled the occulated biomass with uorescent microspheres in a continuous-ow bioreactor and noted that bioocs detach, collide and aggregate randomly
in granulation process.
Although the layered structure of mature granules were proposed in literature, few reports revealed the stereological distribution data of dominating strains in mature granules and in growing
granules. The mechanisms for aerobic granulation were proposed
as a sequential process including intra-, inter- and multi-generic
cell-to-cell attachments (Palmer et al., 2001). Liu and Tay (2002)
proposed a four-step mechanistic granulation mechanism as follows: (1) cell-to-cell contact; (2) initial attachment to form aggregates; (3) enhancement in attachment by secreted extracellular
polymeric substances (EPS); (4) hydrodynamic shear force to stabilize the three dimensional structure of the granule. Conversely,
Zhou et al. (2014) negated the mechanistic approach and claimed
that the granulation is a random coagulation process. This study
cultivated aerobic granules in acetate + propionate wastewater
using waste activated sludge as seed sludge. Then the cultivated
granules, slices on mature granules or whole young and mature
granules, were probed on their microbial communities by polymerase chain reaction (PCR) amplication and denaturing gradient
gel electrophoresis (DGGE) technique or by high-throughput
sequencing technique. The mechanisms for granulation were discussed based on the community data.

to 1 min, 5 min efuent withdrawal and 5 min idle time. The initial
dissolved oxygen and pH of aeration phase were 7.52 mg l1 and
7.05, respectively. The composition of the synthetic wastewater
was: peptone 400 mg l1, yeast exact 250 mg l1, NH4Cl 3.74 mM,
KH2PO4 4.85 mM, CaCl2 0.27 mM, MgSO4 0.21 mM, FeSO4
0.13 mM, NaHCO3 0.15 mM. The carbon source in municipal
wastewater was acetate and propionate at molar ration 3:1, giving
an organic loading rate (OLR) of 1.5 kg COD m3d1. The test was
performed at 29 1 C.
2.2. Granules sampling
Two batches of samples were collected. Five mature granules
were collected on 70 d in operation. Using OCT compound to
embed single granule and then placed the embedded granules at
20 C for 30 min (Adav et al., 2010a,b), the granules were sliced
layer by layer with their size and thickness being listed in Table 1.
The volume of each slice was determined by:

h2

pR2  X 2 dx

h1

where h2 denotes the distance between the distal edge of the layer
to equator and h1 denotes the distance between the proximal edge
of the layer to equator. R denotes the radius of sphere and X denotes
the distance from equator of the sphere.
In the startup phase of the reactor, the seed sludge, and the
granules from early stage of size 1.5 mm to later stage of size
6.5 mm were collected. The whole microbial communities of these
collected granules were probed using high-throughput sequencing
technology.
2.3. DNA extraction, PCR, DGGE and high-throughput sequencing
The genomic DNA of granule samples was extracted using
Power SoilTM DNA Isolation Kit (Mobio, USA). The nanodrop ND3300 uorescence spectrophotometer (Thermo, USA) was used to
quantify extracted DNA with picogreen (SIGMA, USA) as the dye.
The extracted DNA was stored at 20 C. Subsequently, the
Table 1
Slicing of collected granules on 70 d in operation.
No.

Diameter (mm)

Layers (B)

Distance from surface (lm)

12

10

12

10

10

0583
5831167
11671750
17502333
23332917
29173500
0700
7001400
14002100
21002800
28003500
0500
5001000
10001500
15002000
20002500
25003000
0800
8001600
16002400
24003200
32004000
0800
8001600
16002400
24003200
32004000

2. Methods
2.1. Granule cultivation
The seed sludge was collected from the reux sludge stream of a
wastewater treatment plant in Shanghai, China.
A 6 cm SBR of total volume of 5 l and working volume of 2.2 l
was used. Air bubbles were supplied through an air sparger at
the bottom of the reactor with a supercial air velocity of
2.95 cm s1. The volume exchange was 0.75. The reactor was operated in successive cycle and each cycle included 5 min feeding,
195 min aerating, gradually decreasing settling time from 30 min

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Y. Lv et al. / Bioresource Technology 169 (2014) 344351

variable V3 region of the bacterial 16S rDNA gene sequence was

amplied by PCR with forward primer 8F and reverse primer
518R. PCR amplication was performed with a BIO-RAD (USA)
using a 50 ll (total volume) mixture containing 1.5U of Taq DNA
polymerase, 5 ll of 10PCR buffer minus Mg2+, 1 ll of 10 mM
dNTP mixture, 1.5 ll of 50 mM MgCl2, 0.5 ll of the forward primer
(10 lM) and 0.5 ll of the reverse primer (10 lM), 1 ll of the DNA
extraction solution (50 mgl1). The PCR temperature program
started with 10 min of activation of the polymerase at 94 C.
Twenty PCR cycles were then conducted, with the rst two cycles
consisting of 1 min denaturation at 94 C, 1 min annealing at 55 C
and 1 min synthesis at 72 C. With the same temperatures for
denaturation and synthesis, the annealing temperature was subsequently decreased by 0.1 C for each cycle since cycle 3 until reaching 52 C. Finally, after 30 PCR cycles, a 10 min extension step at
72 C was performed.
The PCR-amplied DNA products were separated by DGGE on
8% polyacrylamide gels with a linear gradient of 3055% denaturant (100% denaturant 1=4 40% v/v formamide plus 42% w/v urea)
using the DCodeTM Universal Mutation Detection System (Bio-Rad,
USA). Gels were run for 9 h at 140 V in 1TAE buffer maintained
at 60 C. Selected DNA bands from the DGGE gels were aseptically
excised, puried by Axygen DNA extraction kit (USA) and re-amplied
by the same PCR procedure described previously. After cloning and
sequencing, the sequences were analyzed in comparison with the
16S rDNA sequences in the GenBank by BLAST search (National
Centre for Biotechnology Information, US National Library of
9

Rhodocyclaceae
Moraxellaceae
Flavobacteriaceae
Cytophagaceae

8
7

Medicine) for species identication. The relative intensity of each

DGGE band was determined by Gel-pro analyzer (MediaCybernetics,
Rockville, MD, US). From the band intensity data, the gray ratio of
acquired bands can be obtained by dividing the gray value by the
sample volume.
The Roche Emulsion-PCR technology was adopted to prepare
single molecular PCR product. The amplied genomic samples
were taken for high-throughput sequencing by Ion Torrent PGM
(Life Technology, New York, US).
3. Results and discussion
3.1. Image and microbial communities of sliced samples
The collected aerobic granules were smooth on surface appearance and intact in structure (images not shown). The equatorial
slice of granule No. 8 is shown in Fig. S1. The internal core (A)
was white with crowded spots. Moving outward the color turned
yellow, suggesting the presence of excess biomass. The interior of
the granule was compact with layered structure, correlating with
the uorescence results presented elsewhere (Adav et al.,
2010a,b). One essential feature of granules is the compact interior,
inconsistent with numerous granule studies which in fact handled
some forms of aggregates with loose structure.
The DGGE patterns for granules No. 2, 4, 5, 6, 8 were shown in
Fig. S2 with 1 and 10 corresponding to two surface slices and
increased numbers in the gure denoted the inner slices. Based

Microbacteriaceae
Brucellaceae
Rhodobacteraceae

Band 2
Band 6
Band 10
Band 14

Band 3
Band 7
Band 11
Band 15

Band 4
Band 8
Band 12
Band 16

5
Gray Ratio

6
Gray Ratio

Band 1
Band 5
Band 9
Band 13

5
4
3

4
3
2

2
1

1
0

6'

5'

4'

3'

2'

1'

Rhodocyclaceae
Comamonadaceae
Microbacteriaceae
Xanthomonadaceae
Moraxellaceae
Flavobacteriaceae
Rhodobacteraceae
Sphingobacteriaceae

30
25

5'

4'

3'

2'

1'

(b)

(a)

Rhodocyclaceae
Microbacteriaceae
Xanthomonadaceae
Rhodobacteraceae
Moraxellaceae
Sphingobacteriaceae
Flavobacteriaceae
Brucellaceae

8
7

Gray Ratio

15
10

5
4
3
2

0
1

6'

5'

4'

3'

2'

1'

(c)

5'

4'

3'

2'

(d)

8
Rhodocyclaceae
Rhodobacteraceae
Brucellaceae
Moraxellaceae
Flavobacteriaceae
Microbacteriaceae
Sphingobacteriaceae

7
6
Gray Ratio

Gray Ratio

6
20

5
4
3
2
1
0
1

5'

4'

3'

2'

1'

(e)
Fig. 1. Gray ratio (microbial density) of the each layer at family level in the ve granules.

1'

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Y. Lv et al. / Bioresource Technology 169 (2014) 344351

on the DGGE patterns, the gray ratios were the highest at surface
slices and were the lowest at the equatorial slices (Fig. 1). Conversely, the Shannon indices were the highest at equatorial slices
and were the lowest at surface slices (Fig. 2).

The bands 2, 3, 6, 11, 13, 14, 15 in Fig. S2 belong to family Microbacteriaceae, bands 5, 8, 9, 12 belong to family Moraxellaceae, and
band 16, 17, 18 belong to different genera of family Rhodocyclaceae.
Restated, the microbial community of different granules was

2.50
2.45
Shannon index (H')

Shannon index (H')

2.65
2.40
2.35
2.30
2.25
2.20

2.60

2.55

2.15
2.10
1

6'

5'

4'

3'

2'

1'

5'

4'

3'

2'

1'

2.20

2.30

2.15
Shannon index (H')

2.25
Shannon index (H')

(b)

(a)

2.20
2.15
2.10
2.05

2.10
2.05
2.00
1.95
1.90

6'

5'

4'

3'

2'

1'

(c)

5'

4'

3'

2'

1'

(d)

2.65

Shannon index (H')

2.60
2.55
2.50
2.45
2.40
1

5'

4'

3'

2'

1'

(e)
Fig. 2. ShannonWiener diversity index of 16S rRNA gene sequences in the ve granules.

Table 2
Phylogenetic sequence afliation and similarity to the closest relative of amplied 16S rRNA gene sequences excised from DGGE gels of the No. 8 granule.
Band No.

Family

Genus

Similarity (%)

Phylum

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

Sphingobacteriaceae
Microbacteriaceae
Microbacteriaceae
Flavobacteriaceae
Moraxellaceae
Microbacteriaceae
Brucellaceae
Moraxellaceae
Moraxellaceae
Rhodobacteraceae
Microbacteriaceae
Moraxellaceae
Microbacteriaceae
Microbacteriaceae
Microbacteriaceae
Rhodocyclaceae
Rhodocyclaceae
Rhodocyclaceae

Sphingobacterium sp. Ag8

Leucobacter komagatae strain
Leucobacter
Flavobacterium
Acinetobacter
Leucobacter
Pseudochrobactrum
Acinetobacter
Acinetobacter
Rhodobacter sphaeroides strain DB803
Leucobacter komagatae
Acinetobacter
Leucobacter komagatae strain Z3_S_TSA4
Leucobacter
Leucobacter komagatae
Azoarcus indigens strain VB32
Azoarcus indigens strain VB32
Thauera

93
96
98
94
99
100
100
100
100
97
97
100
95
99
97
95
95
100

Bacteroidetes
Actinobacteria
Actinobacteria
Bacteroidetes
Proteobacteria
Actinobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Actinobacteria
Proteobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Proteobacteria
Proteobacteria
Proteobacteria

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Y. Lv et al. / Bioresource Technology 169 (2014) 344351

Fig. 3. Distributions of bacterial strains in L1L7 at level of phylum.

mainly distributed in three phyla: Proteobacteria, Bacteroidetes and

Actinobacteria (Table 2).
3.2. Microbial communities in growing granules
The high-throughput sequencing yielded 7446, 208,512, 10,724,
5229, 4367, 4980, 8515 high quality and effective reads for sample

L1L7, respectively, all with >82% coverage. After quality check, the
read length of majority reads were 450600 with an average length
above 450 and average read number above 500. The number of
operational taxonomic unit (OTU) identied at 97% similarity in
sample L1L7 were respectively 1933, 11,400, 2083, 1231, 1197,
1065 and 1710. The Chao1 indices in all samples were higher than
the numbers of OTU, justifying the adopted 97% similarity for OTU
identication.
In L1L7 communities, the number of bacterial phylum were 9,
13, 11, 11, 10, 10 and 13, respectively, giving a total of 16 phyla
(Fig. 3). All samples were dominated by Proteobacteria, Bacteroidetes and Firmicutes, with Proteobacteria phylum being at the highest
abundance followed by Bacteroidetes and then Firmicutes. Restated,
the most mature L1 (with the largest diameter) was dominated by
Bacteroidetes and Firmicutes; while the seed sludge and young
granules L2 (with the smallest diameter) were dominated by
Proteobacteria.
A total of 92 families were detected in L1L7, with 9 of which
were the dominating families (Fig. 4a). Microbial communities of
L1L7 were distributed at 182 genera of bacteria (Fig. 4b). The heat
map (Fig. 5) suggests that most species in L7 were higher in abundance than the granule samples. Hence, granulation limits the
growth of specic bacterial strains. The clustering in Fig. 5 demonstrates that L6 differed more than did L7 from L1L5. The beta
diversity in Fig. 6 also revealed that the microbial diversity of

Fig. 4. Distributions of bacterial strains in L1L7 at family (a) and genus (b).

Y. Lv et al. / Bioresource Technology 169 (2014) 344351

349

Fig. 5. Richness heat map of microbial community at family level.

L1L5 had high similarity, while L6 was the one departing from all
the other samples including the seed sludge.
3.3. Microbial community and granule formation mechanism
In sliced samples of mature granules, family Microbacteriaceae
was the principal species on the surface slice, which was fastgrowing, obligate aerobic nitrifying bacterium (Kim and Lee,
2011). Other microbes locating at surface slice were family
Sphingobacteriaceae (band 1), family Moraxellaceae genus Acinetobacter (band 8), and family Rhodobacteraceae genus Rhodobacter
(band 10). Family Sphingobacteriaceae is nitrication bacterium
with degradation ability of ammonia and organic matters (Asker
et al., 2008). The genus Acinetobacter has highly hydrophobic cell
membrane and can secret excess EPS, so having high self-aggregation ability and adhesion capability to solid surface (Wan et al.,
2014a,b). The genus Rhodobacter is an anaerobic denitrifying bacterium that is also able to secret EPS for assisting solid attachment.
These predominant species should contribute to the formation of
aerobic granules with their secreted EPS. The family Rhodocyclaceae had a high abundance on the equatorial slice. This observation
suggested that family Rhodocyclaceae was enriched at granule core.
The b-Proteobacteria family Rhodocyclaceae genus Thauera (band 16
and 18) belong to the facultative anaerobic denitrifying bacteria
(Shinoda et al., 2004), which can express denitrication enzyme
in anoxic environment. This strain can produce excess extracellular
polysaccharides and proteins (Jiang, 2011) so should contribute to
granule core strength.
On growing granule studies, correlating with the DGGE results
in Section 3.1, although Rhodocyclaceae belongs to facultative
anaerobic denitrifying bacteria, it was present in both seed sludge
and young/mature granules. At the family level, Flavobacteriaceae,
Xanthomonadaceae, Rhodobacteraceae, Microbacteriaceae were

increased signicantly in abundance from L7 to L6. Growing from

L6 to L5 led to increase in abundance of Rhodobacteraceae. From
L5 to L4, the abundance of Microbacteriaceae was increased. From
L4 to L3, abundance of both Xanthomonadaceae and Microbacteriaceae were increased. Growing from L3 to L2, the abundance of Rhodobacteraceae was increased. From L2 to L1, the abundances of
Flavobacteriaceae and Cytophagaceae were signicantly increased.
Signicant variation in richness diversity occurred from L6 to L5;
while sample L3 and L4 had almost identical microbial community
structures. Thauera was at the least abundance in L6 (young granule) while Flavobacterium was at the most abundant in L1 (mature
granule). Thauera is a facultative anaerobic denitrifying bacterium,
which increased in abundance with granule growth. Also, Rhodobacter with facultative anaerobic denitrifying function emerged in
L6. The abundances of Flavobacteriaceae and Xanthomonadaceae
were high in L6 and L7 which should be supported by the efcient
oxygen transport to surface of large granules.
In the four granulation steps by Liu and Tay ((1) cell-to-cell contact; (2) initial attachment to form aggregates; (3) enhancement by
EPS; (4) hydrodynamic packing), when the activated sludge was
the seed sludge, granulation can start from step (3). Processes to
enhance cell hydrophobicity such as periodic feast-famine conditions in SBR operation can stimulate step (3); high bubbling that
compacts the granular interior can help step (4). Then the spherical-shape granules should have layered structures with different
groups of bacteria of different ecological niches residing at different depths from the surface. Conversely, if the granules were
formed by a random coagulation-detachment mechanism, with
ne biological aggregates attaching and detaching on a large granule matrix in a dynamic manner (Zhao et al., 2014), there should be
no clear layered architecture in the granules. The present observation together with Fig. S1 suggested a concentric spherical structure of the granules: a spherical core with anaerobic strains

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Y. Lv et al. / Bioresource Technology 169 (2014) 344351

Fig. 6. Beta diversity for L1L7. (a) Distance heat map; (b) 2-D PAC analysis; (c) 3-D PAC analysis; (d) phylogenetic tree.

(Rhodocyclaceae) covered by an outer spherical shell with both aerobic strains (Microbacteriaceae, Sphingobacteriaceae, Moraxellaceae)
and anaerobic strains (Rhodocyclaceae). Restated, the microbial
community data herein obtained supported the deterministic
mechanism.
Owing to the compact interior of granules that limited mass
transfer of substances (Tsai et al., 2008), anaerobic strains existed
at the granule core that may lead to deterioration of the granules
in long-term operation (Chen et al., 2007). Adav et al. (2010a,b)
showed that active strains mainly distributed at 200250 lm depth
from their granules surface. Chiu et al. (2006, 2007) noted that dissolved oxygen was completely exhausted by active surface layer so
the interior of granules was at anaerobic condition. However,
Verawaty et al. (2012) and Zhou et al. (2014) noted the occurrence
of attachment and detachment of bioocs from the granule biomass. We believe under high hydrodynamic shear coagulation
and disintegration of ne particles from granules occurred over
the entire granulation process, just the coagulation mechanisms
should dominate in step (1) and step (2) in Liu and Tays scheme.
The layered structure occurs in transition from L6 to L5 should be
attributable to the formation of anaerobic core in the granules,

contributed by the step (3) and step (4) in Liu and Tays scheme.
Conversely, washout is a physical process to screen off ne aggregates, hence the less-occulating strains, from the reactor. Applying
washout to retain large aggregates (and granules) depends on
whether the operators dislike the co-existence of occulated ocs
and granules in their reactors, not as the proposal by Zhao et al.
(2014) that microbial selection pressure is not a prerequisite for
granulation.
4. Conclusions
Microbial communities of aerobic granules were identied from
slices of mature granules and from growing granules. During granulation, the microbial community of seed ocs decreased in diversity, which rst rapidly shifted to a community for young granules
that deviated from seed ocs. Then owing to formation of an intragranular anaerobic regime, a layered structure with anaerobic
Rhodocyclaceae at core covered by an outer spherical shell with
Flavobacteriaceae, Xanthomonadaceae, Rhodobacteraceae and Microbacteriaceae was formed. Granules were formed via a deterministic
rather than via a random aggregationdisintegration mechanism.

Y. Lv et al. / Bioresource Technology 169 (2014) 344351

Acknowledgements
This project is partially supported by NSFC Project 51176037
and by National Science Council.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2014.
07.005.
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