Research Article: Aerobic Sludge Granulation in A Full-Scale Sequencing Batch Reactor
Research Article: Aerobic Sludge Granulation in A Full-Scale Sequencing Batch Reactor
Research Article: Aerobic Sludge Granulation in A Full-Scale Sequencing Batch Reactor
Research Article
Aerobic Sludge Granulation in a Full-Scale
Sequencing Batch Reactor
Jun Li,1 Li-Bin Ding,1 Ang Cai,1 Guo-Xian Huang,2 and Harald Horn3
1
Department of Municipal Engineering, Zhejiang University of Technology, No. 18 Chao Wang Road, Hangzhou 310014, China
2
Yancang Wastewater Treatment Plant, Haining 314422, China
3
Water Chemistry and Water Technology, Karlsruhe Institute of Technology, Engler-Bunte-Ring 1, 76173 Karlsruhe, Germany
Copyright © 2014 Jun Li et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aerobic granulation of activated sludge was successfully achieved in a full-scale sequencing batch reactor (SBR) with 50,000 m3 d−1
for treating a town’s wastewater. After operation for 337 days, in this full-scale SBR, aerobic granules with an average SVI30
of 47.1 mL g−1 , diameter of 0.5 mm, and settling velocity of 42 m h−1 were obtained. Compared to an anaerobic/oxic plug flow
(A/O) reactor and an oxidation ditch (OD) being operated in this wastewater treatment plant, the sludge from full-scale SBR
has more compact structure and excellent settling ability. Denaturing gradient gel electrophoresis (DGGE) analysis indicated
that Flavobacterium sp., uncultured beta proteobacterium, uncultured Aquabacterium sp., and uncultured Leptothrix sp. were just
dominant in SBR, whereas uncultured bacteroidetes were only found in A/O and OD. Three kinds of sludge had a high content of
protein in extracellular polymeric substances (EPS). X-ray fluorescence (XRF) analysis revealed that metal ions and some inorganics
from raw wastewater precipitated in sludge acted as core to enhance granulation. Raw wastewater characteristics had a positive effect
on the granule formation, but the SBR mode operating with periodic feast-famine, shorter settling time, and no return sludge pump
played a crucial role in aerobic sludge granulation.
necessary factors. A recent study showed that an intensive tank to anaerobic zone. It include an bioreactor consisted of
anaerobic contact of granules and easily degradable organic an anaerobic zone with a size of 25 m length, 25 m width and
carbon at the beginning of each SBR cycle stabilize granular 5 m depth followed by an oxic zone with a size of 55 m length,
growth, phosphorus and nitrogen removal [17]. 25 m width and 5 m depth.
Gansbaai WWTP was reported to be the first full-scale
domestic sewage treatment work in the world using aerobic
2.3. The OD Set up and Operation. In order to meet the
granular sludge technology in an upgrade project [15, 18]. It
treating requirements of the increasing wastewater, the sec-
was designed for 4,000 m3 d−1 of high strength septic influent
ond stage project of the OD process with a treating capacity
consisting of three parallel reactors with a height of 7 m
of 50,000 m3 d−1 came into operation in 2006 (Figure 1(b)).
and diameter of 18 m. Another full-scale SBR in Epe, the
It included a regulation tank, primary sedimentation tank,
Netherlands, was designed for 59,000 person equivalents and
hydrolysis acidification tank, aerobic tank, second sedimen-
treating up to 1,500 m3 h−1 municipal wastewater with a high
tation tank and final sedimentation tank. The aerobic tank
contribution of industrial waste from slaughterhouses [15].
had a size of 90 m length, 35 m width and 3.5 m depth.
Nevertheless, the detailed data of the operational perfor-
The OD was designed and operated in a traditional way.
mance of full-scale applications have not been presented.
The second sedimentation tank was separately built with the
Yancang WWTP was located in Haining, a coastal city
main bioreactor of OD. The returned sludge from second
in Eastern China. Attention has been paid because some
sedimentation tank was pumped to the main bioreactor of
small particles were observed in activated sludge from both
OD.
anaerobic/oxic plug flow process (A/O) and oxidation ditch
process (OD) in this plant since 2008. Particularly, about 60–
79 mL g−1 of SVI indicated that the sludge from these two 2.4. Lab-Scale SBR Set up and Operation. A column type
continuous flow reactors had better settling ability compared lab-scale SBR with an H/D ratio of 2.5, working volume of
with normal activated sludge. 5.0 L and volumetric exchange ratio of 50% was set up in
The major aim of this work was to demonstrate the 2008 (Figure 2(a)). It was reported that Kong had successfully
feasibility of cultivating aerobic granular sludge in an SBR, developed aerobic granules in four SBRs with different H/D
particularly for full-scale application. Successively, a lab-scale ratio of 24, 16, 8 and 4, respectively and a higher reactor
SBR, a pilot-scale SBR and a full-scale SBR were set up H/D ratio such as 20–30 was mostly used in literature to
and used for the treatment of this wastewater through the meet the requirement of the minimal settling velocity for
development of aerobic granular sludge. The characteristics granule formation [19]. So here, we defined that the reactor
of different sludges from A/O, OD and SBR were compared. with an H/D below 4 was considered to be low. The raw
The main factors for aerobic sludge granulation in this full- wastewater was introduced from top of the reactor with a
scale SBR were discussed. volume of 2.5 L per cycle. The lab-scale reactor was aerated
by using a fine bubble aerator and operated in a fill-draw
mode. After inoculation, the SBR was initially operated in
2. Material and Methods successive cycles of 4 h, and each cycle consisted of 1 min
2.1. Wastewater and Inoculating Sludge. Wastewater in Yan- of filling, 120 min of aeration, 60 min of settling, 20 min of
cang WWTP included approximately 30% domestic sewage effluent withdraw and 39 min of idling. After 10 days, the SBR
and 70% industrial wastewater from printing and dyeing, was operated in a cycle of 4 h, which consisted of 1 minute
chemical, textile and beverage. The characteristics of the of filling, 180 min of aeration, 10 min of settling, 20 min of
wastewater were showed as follows: chemical oxygen demand effluent withdraw and 29 min of idling (Table 1). The organic
(CODCr ) of 200–600 mg L−1 , biochemical oxygen demand loading rate (OLR) of the influent was controlled at 3.9–4.5 kg
(BOD) of 50–105 mg L−1 , ammonium nitrogen (NH4 + -N) of COD m−3 d−1 and superficial air velocity was controlled at
28.0–40.0 mg L−1 , total phosphorus (TP) of 2.0–4.0 mg L−1 1.3 cm s−1 . A programmable logic controller (PLC) controlled
and temperature of 18–30∘ C. The average influent BOD/COD the implementation of the pumps, valves and the length of
ratio was only about 0.23 which belonged to bio-refractory every operational batch cycle.
wastewater. The lab-scale, pilot-scale and full-scale SBR were
all introduced with the same raw wastewater and inoculated 2.5. Pilot-Scale SBR System Set up and Operation. A grit
sludge from the second sedimentation tank in OD. chamber, a service tank and two parallel columns constituting
the pilot-scale SBR system was set up in 2009 (Figure 2(b)).
2.2. The A/O Set up and Operation. The A/O process was The two parallel columns had a height of 6 m, an internal
built and came into operation in 2001 treating 10,000 m3 per diameter of 2 m, H/D of 2.5 and a maximum operating flow
day (Figure 1(a)). It included a regulation tank, reaction tank, rate of 120 m3 d−1 . Fine bubble aerators were used in this
primary sedimentation tank, A/O tank, second sedimenta- pilot-scale SBR. The operation was controlled by a PLC.
tion tank, oxidation contact tank and final sedimentation The pilot-scale SBR system would operate in this way: the
tank. The A/O plug flow process in this WWTP was designed raw wastewater was pumped into the grit chamber firstly,
and operated in a traditional way. The second sedimentation and then it flowed into a service tank controlled by electric
tank was separately built with the main bioreactor and the butterfly valve and level controller. When came to the feeding
returned sludge was pumped from second sedimentation period, wastewater was pumped into two parallel columns.
BioMed Research International 3
Figure 1: Photographs of reactors and sludge of A/O, OD and full-scale SBR in Yancang WWTP.
Table 1: A cycle distribution of operation time in lab-scale, pilot-scale, and full-scale SBRs.
The wastewater was introduced from the bottom of the pilot- 2.6. The Full-Scale SBR and Operation. With the expansion
scale reactor. The reactor with a volumetric exchange ratio of service area and urbanization, an A/O with a treating
of 50% was operated in a fill-draw mode. The first stage capacity of 10,000 m3 d−1 constructed in 1999 and an OD with
consisted of 40 min of influent addition, 120 min of aeration, a treating capacity of 50,000 m3 d−1 constructed in 2006 did
60 min of settling and 20 min of effluent withdraw. After 7 not match with the increasing wastewater. A new wastewater
days of operation, it came to the second stage which consisted treatment work with a treating capacity of 50,000 m3 d−1
of 40 min of influent addition, 120 min of aeration, 20 min of was required in this plant. The aims of building the third
settling and 20 min of effluent withdraw (Table 1). stage project in Yancang WWTP were not only to improve
4 BioMed Research International
Figure 2: Photographs of reactors and sludge of lab-scale SBR and pilot-scale SBR in Yancang WWTP.
the treatment ability but also to meet the strict wastewater from top of the full-scale reactor. The full-scale SBR with
discharging standard in China. Consequently, selecting a volumetric exchange ratio of 50%–70% was operated in a
more effective and economic process for treating wastewater fill-draw mode. At the end of 2010, the full-scale SBR was
was an urgent issue. Activated sludge with an SVI of 60– constructed and came into operation. After inoculation, each
79 mL g−1 and some small granules with the size of 30– cycle consisted of 40 min of filling, about 240 min of aeration,
80 𝜇m existing in the A/O and OD process enlightened us 60 min of settling and 30 min of effluent withdraw. After 25
whether it was feasible to cultivate aerobic granular sludge days, the operation cycle consisted of 40 min of filling, about
with SBR process for treating wastewater in Yancang WWTP. 240 min of aeration, 40 min of settling and 30 min of effluent
The A/O plug flow and OD were typical activated sludge withdraw. Six months later, the operation cycle consisted of
process. The only difference was in raw wastewater compared 40 min of filling, about 240 min of aeration, 50 min of settling
with other WWTP. It implied that raw wastewater was the and 30 min of effluent withdraw (Table 1). Accurate aeration
key factor for the formation of these aggregates in typical time was controlled by an intelligent system depending on
A/O plug flow and OD process. If operated in an SBR mode variation of dissolved oxygen.
with certain selecting pressure, aerobic granules would be
easily formed. Hence, a series of lab-scale and pilot-scale 2.7. Analytical Methods. CODCr , NH4 + -N, nitrite (NO2 − -N),
experiments were carried out and proved the feasibility of nitrate (NO3 − -N), sludge volume index with 30 min settling
developing aerobic granules in this treatment plant. After time (SVI30 ), mixed liquor suspended solids (MLSS) and
lab-scale and pilot-scale experiments for successful aerobic mixed liquor volatile suspended solids (MLVSS) were ana-
granulation, the third stage project with a SBR process was lyzed in accordance to the Standard Methods [20]. Biological
built and came into operation in 2010 (Figure 1(c)) and the Oxygen Demand (BOD5 ) was measured using the WTW-
application of aerobic granular sludge was further studied in OxiTop system. The morphology of sludge was observed by
this work. an Olympus CX31 microscope and a digital camera (Canon
The full-scale SBR was divided into four separated tanks EOS 30D). The size of granules was analyzed by an image
for alternative operating. Each tank had an H/D of 0.09, analysis system (Image-Pro Plus 6.0, Media Cybernetics).
a volume of 12,540 m3 with length of 55 m, width of 38 m The element distribution of raw wastewater and granules
and depth of 6 m, respectively. Before raw wastewater flowed was analyzed by X-ray fluorescence (XRF). The toxic organic
into full-scale SBR, it would flow into regulating reservoir, substance in raw wastewater was measured by gas chromatog-
primary sedimentation tank and hydrolysis tank in turn. raphy/mass spectrometry (GC/MS) (SHIMADZU GCMS-
The effluent quality from full-scale SBR was enhanced by QP2010). The extracellular polymeric substances (EPS) of
coagulating sedimentation. The wastewater was introduced granules were extracted using the EDTA extraction method
BioMed Research International 5
Table 3: Similarity coefficients of microbial communities among the Through microscope and digital camera analysis, it could
samples. be found that small granules existed in activated sludge in
both A/O and OD. The A/O operated in a plug flow way while
Lane 1 2 3
in the OD the wastewater was completely mixed. Hence, the
1 1.00 0.72 0.37 substrate concentration in the A/O experienced a change
2 0.72 1.00 0.45 from high to low along the reactor length which matched
3 0.37 0.45 1.00 the feast-famine regime while in the OD the substrate
concentration had almost not changed. Furthermore, the
secondary sedimentation tank with a long settling time (2.5
Table 4: Diversity index (𝐻) and number of the bands in DGGE hours) could not offer enough selecting pressure to wash out
profiles of sludge samples. the poorly settleable sludge. In addition, the aeration tank
Sample 1 2 3
in the A/O and OD process was separated from the second
sedimentation tank. Part of the settled sludge in the second
Number of bands 23 24 21
sedimentation tank was pumped back to the aeration tank
𝐻 2.82 2.89 2.70 for recirculation. In this way, the granule was easily destroyed
by the pump. In conclusion, it was tough to cultivate aerobic
granules in the A/O and OD.
and COD removal rate of 82.2% and 74.0%, respectively.
However, the TN removal rate in full-scale SBR was 59.6% 3.3. DGGE Profile and Bacterial Community Analysis
which was much higher compared with A/O and OD due to
simultaneous nitrification and denitrification effect of aeobic 3.3.1. DGGE Profile and Phylogenetic Analysis. DGGE pro-
granules. TP only decreased from 2.5 mg/L to 1.2 mg/L in full- files (Figure 4) and similarity coefficient analysis (Table 3)
scale SBR since there was no anaerobic phase existed and indicated that under continuous flow (A/O and OD) and SBR
the removal of TP was mainly depend on post-treatment of conditions, the bacterial community composition especially
physical and chemical. Granules in the full-scale SBR showed for bacterial species showed a remarkable difference. Most
the best settling ability and the highest settling velocity among of the bacterial species in activated sludge in A/O and OD
these three processes after granulation (Table 2). It indicates were similar, but a noticeable difference occurred in mature
that aerobic granular sludge in the SBR process is advanta- aerobic granules in the full-scale SBR. It had been previously
geous compared to traditional activated sludge in the A/O reported that the influence of operation conditions and
and OD process. A much higher settling velocity of aerobic reactor format on the community diversity was evidenced by
granules allowed for less settling time in the SBR which in the change in band patterns [29].
turn improved the wastewater treatment efficiency in the Schematic band intensities for DGGE profiles were
SBR. Furthermore, a short settling time favored the growth obtained using BanScan software (Figure 4(b)). There were
of rapidly-settling bioparticles whereas the bioparticles with 32 obvious bands in DGGE profiles with 21–24 bands in
a poor settling ability were washed out [12]. every sample (Figure 4), and there was no obvious difference
BioMed Research International 7
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 11
12 12
13 13
14 14
15 15
16 16
17 17
18 18
19 19
20 20
21 21
22 22
23 23
24 24
25 25
26 26
27 27
28 28
29 29
30 30
31 31
32 32
(a) DGGE profiles of examined samples (b) Schematic diagram of relative band
intensities in DGGE
Figure 4: The DGGE profiles of bacterial communities in three different reactors and all sludge samples were collected on September 8, 2011.
1#: sludge from A/O; 2#: sludge from OD; 3#: sludge from full-scale SBR.
in the number of bands and the diversity index among the than the genus similarity level [30]. It was therefore consid-
samples (Table 4). As measured, similarity coefficients of 1# ered that sequences of these four bands were from Nitro-
and 2# were relatively high (Table 3). Some bands, such as spira, Flavobacterium, Aquabacterium and Thauera genus,
bands 1, 2, 3, 4, 8, 19, 20, 21, 24, 26, 27 and 31 were found in respectively. According to the DGGE profiles, Flavobacterium
all of the samples under three different processes. However, (band 12) dominated in the granules from the full-scale
the relative intensities of the same band in different sample SBR although it was not the dominant group in the initial
profiles were different. Besides, some special bands appeared sludge from the OD used as seed sludge. It was reported
in the DGGE profiles of different samples such as band 14, 16, that Flavobacterium is a significant genera of floc-forming
18 and 20, which only existed in 3# (Figure 4(b)). bacteria [31] producing extracellular polymers to bind cells
Typical bands from the DGGE profiles were separated, re- together. Earlier works also proved that Flavobacterium was
amplified, sequenced and thirteen sequences were obtained. dominant in granules from the full-scale SBR while not
Nucleotide sequences and the abundance of sequenced dominant in seed sludge [32]. It was interesting to note that
DGGE bands were compared (Table 5). Sequence analysis Bacteroidetes (band 13) was dominant in A/O and OD but
showed that bands 10, 12, 16 and 26 had similarity levels of did not exist in the granular sludge in the full-scale SBR.
100%, 99%, 100% and 100%, respectively, which was more Former works had proved that the Bacteroidetes bacterium
8 BioMed Research International
was washed out at short settling times and did not contribute as a bridge between negatively charged groups on the cell
to sludge granulation [33]. In our study, the Bacteroidetes surface which was important in the aggregation progress
bacterium was washed out under SBR operational conditions, [36, 37]. The effect of Ca2+ and Mg2+ enhancing the sludge
but retained under A/O and OD operational conditions due granulation in the SBR was widely recognized [38–40].
to the longer settling time. Nitrospira (band 10) dominated in Some studies indicated that Al and Fe were necessary in
A/O and OD but not in the full-scale SBR after granulation. the development of compact aerobic granules structure with
Thauera (band 26) was present significantly in all three excellent settling properties [41, 42]. Si was also precipitated
reactors and it played an important role in nitrogen removal significantly in granules; it had been reported that Si laid the
[33]. foundations for the aerobic granule structure and supported
the strength of matured granules [41].
3.4. Main Factors for Aerobic Granulation in Full-Scale SBR Large amounts of inorganics composed of Fe, Si, Ca
and P existing in the raw wastewater obviously provided
3.4.1. The Effect of Raw Wastewater on Aerobic Granulation. nuclei to accelerate microbial aggregation. These inorganic
Excellent performance of aerobic granules in the lab-scale solids were found in aerobic granules from this full-scale SBR
SBR, pilot-scale SBR, and particularly full-scale SBR sug- (Figure 6). Huishoff used hydro-anthracite as an additional
gested a necessary discussion about the role of raw wastewater inert support particle accelerating the anaerobic granulation
in aerobic granulation. [43]. Granular activated carbon (GAC) was also added for
Chemical elements in raw wastewater and granules from sludge granulation in the SBR with low-strength wastewater
the full scale SBR were analyzed by XRF (Figure 5). The [44]. Powdered activated carbon (PAC) and GAC were
contents of Na and Cl were 35.43% and 17.59% respectively in added during the start-up of upflow anaerobic sludge blanket
raw wastewater due to the fact that this WWTP is located at (UASB) to accelerate granulation [45]. However, these inor-
the seaside and is treating some industrial wastewater. It was ganics also caused low VSS/SS ratio in sludge.
reported that the presence of salt in the treated effluent did It was believed that the formation and stability of aerobic
not cause a detrimental effect on the operation of the reactor granules were closely related to the sludge EPS [46]. There-
once the aerobic granules were formed [34] or the granular fore, the EPS content of sludge in A/O, OD and full-scale
structure was stable throughout the whole experimental SBR were analyzed respectively (Table 2). The presence of
period when subjected to different salinity [35]. It indicated high PN contents in these three different sludges indicated the
that Fe, Si, Ca and P were precipitated in aerobic granules probable effect of raw wastewater in WWTP, which contained
since the contents of these elements in granules were higher bio-refractory or toxic compounds such as Benzenamine,
than those in raw wastewater. It had been demonstrated that Benzenamine N-methyl- and Isoquinoline (Figure 7). Pre-
the presence of divalent and trivalent mental ions could act vious studies implied that the EPS production, especially
BioMed Research International 9
Si 0.4%
Cl 0.8%
P 0.1%
P 6.4%
Mg 4.1%
Fe 0.5% Si 11.2%
Ca 3.4% Mg 1.1%
Na 3.2% Fe 14.4%
Na 35.5% Ca 7.4%
Fe Si Fe Si
Ca P Ca P
Na Cl Na Cl
Mg Others Mg Others
(a) Raw wastewater (b) Granular sludge in full-scale SBR
Figure 5: Element analysis of raw wastewater and granular sludge in full-scale SBR by X-ray fluorescence (XRF).
×104
3
(1)
(5)
(6)
2
(7)
TIC
(2)
1 (3) (10) (11)
(9)
(4) (8)
0
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0
Retention time (min)
Figure 7: Volatile and semi-volatile organic compounds in raw wastewater by GC/MS analysis: (1) 2-Bromo-2-nitropropane (2) Propanoic
acid, 2-hydroxy-, 2-methylpropyl ester (3) Formic acid, 2-propenyl ester (4) Aniline (5) Anline, N-methyl- (6) Isoquinoline (7) 4-
Aminoheptane (8) 1,2,4-Thiadiazole, 5-amino-3-propyl- (9) Phenol, 3,5-bis(1,1-dimethylethyl)- (10) Pyrimidine-2,4(1H,3H)-dione, 5-amino-
6-nitroso (11) Oxalic acid, isobutyl propyl ester.
DO (mg L−1)
24 60 3
Acknowledgments
45
16 2 This research was supported by the National Natural Science
30 Foundation of China (No. 50878195) and the Project of
8 1 Science and Technology of Zhejiang.
15
0 0
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