Journal of Environmental Chemical Engineering 8 (2020) 103681

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Journal of Environmental Chemical Engineering

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Long-term aerobic granular sludge stability through anaerobic slow feeding, T

fixed feast-famine period ratio, and fixed SRT
Oliver Terna Iorhemena,*, Mohamed Sherif Zaghloula, Rania Ahmed Hamzab, Joo Hwa Taya,1
a
Department of Civil Engineering, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada
b
Department of Civil Engineering, Ryerson University, 350 Victoria St. Toronto, ON, M5B 2K3, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: The deterioration of the structural integrity of aerobic granular sludge (AGS) in long-term operation is a major
Aerobic granular sludge (AGS) limitation of the AGS biotechnology. The present study explored AGS long-term stability at semi-pilot-scale using
Extracellular polymeric substances (EPS) the combined strategy of long anaerobic slow feeding, 1:3 fixed ratio of feast-famine period within each cycle,
Feast-famine regime and selective discharge of mature granules. Biomass characteristics, extracellular polymeric substances (EPS),
Granule stability
dissolved oxygen and removal profiles were monitored. Results indicate that the 1:3 ratio of feast-famine period
Selective discharge
Wastewater treatment
controlled the EPS content at a suitable level to allow for granular sludge stability. The system exhibited high
proteins (PN) content of EPS, resulting in high PN/polysaccharides (PS) ratios: 0.01–19 and 2.05–13.15 for
loosely-bound EPS (LB-EPS) and tightly-bound EPS (TB-EPS), respectively. Selective withdrawal of mature
granules from the bottom of the reactor resulted in a good mix of new and old granules in the system. The
strategy allowed for system stability as the reactor was operated for over 240 d without any granule disin-
tegration. High removals of COD (98.3 ± 0.8 %), NH3-N (98.5 %), and TN (89 ± 6 %) were achieved.
Phosphate-P removal was in the range 50–100 %. EPS producers (Thauera, Flavobacterium and Meganema) and
slow growing bacteria (Acinetobacter and Simplicispira) in the system contributed to the AG stability.

1. Introduction However, the major shortcoming impeding the full-scale application

Aerobic granular sludge (AGS) technology has emerged as a novel
biotechnology for the effective treatment of wastewater. The biomass
granules are microorganisms that have been made to agglomerate to-
gether under special conditions in the absence of any biocarriers, i.e.
microbe-to-microbe self-immobilisation with no medium [1]. As such,
the granules are dense microbial consortia packed with different mi-
crobial species and typically contain millions of organisms per gram of
biomass that can collectively biodegrade wastewater pollutants [2].
The AGS technology exhibits numerous advantages over the conven-
tional activated sludge process. These advantages include outstanding
settleability, strong microbial structure, high biomass retention, high
resilience to toxic chemicals, and good ability to handle high organic
and shock loading rates [2,3].
Due to its unique features, the AGS technology is applicable for both
municipal and industrial wastewaters. The technology has been suc-
cessfully applied for the treatment of high-strength organic wastewater,
removal of nitrogen and phosphorus, sulphate and nuclear waste, and
biosorption of heavy metals [4–9].
of the AGS technology is the long-term system operational instability
and granule disintegration problems [3,10]. Deterioration in granule
stability over time impacts the efficiency of wastewater treatment and it
is a major issue affecting the effectiveness of AGS in full-scale appli-
cations [3,11]. Hypotheses proposed that disintegration may be at-
tributed to the growth of filamentous population [12,13] and anaerobic
degradation within the granule core [14,15]. Hence, ensuring long-
term adequate structural integrity of granules is currently one of the
major challenges hindering wider full-scale application of AGS.
Previous studies have demonstrated that anaerobic slow feeding
allows for the selection of slow growing bacteria (phosphate accumu-
lating organisms (PAOs) and/or glycogen accumulating organisms
(GAOs)) that contributed to smooth, dense and stable AGS [16]. AGS
stability was also demonstrated through selection of slow-growing ni-
trifying bacteria [17]. In addition, anaerobic slow feeding promotes the
production of storage polymers under the anaerobic conditions; and,
this controls the excessive aerobic growth on readily available carbon
that causes unstable AGS [18]. Through this feeding regime, the in-
fluent with high carbon content passes through the settled AGS bed,

⁎
Corresponding author.
E-mail address: oliver.iorhemen@ucalgary.ca (O.T. Iorhemen).
1
Deceased.

https://doi.org/10.1016/j.jece.2020.103681
Received 27 September 2019; Received in revised form 7 January 2020; Accepted 9 January 2020
Available online 10 January 2020
2213-3437/ © 2020 Elsevier Ltd. All rights reserved.
O.T. Iorhemen, et al.
enabling the substrate to penetrate the entire granule depth and become
converted to storage polymers by PAOs or GAOs. During the aeration
phase, the rate of bacterial growth on the internally stored carbon
source becomes slow [19]. In turn, there is starvation of fast-growing
heterotrophic microorganisms during the famine period which keeps
their growth in check.
Feast-famine regime of the sequencing batch reactor (SBR) opera-
tion involves the transition from feast period that is characterised by
the presence of organic matter in the bulk liquid to the famine period
when organic matter is absent in the bulk liquid [20]. During the
famine period, bacteria grow on stored compounds [21]. Alternating
the feast and famine conditions is believed to play a key role in AGS
stability as the periodic starvation has a strong effect on cell hydro-
phobicity [22]. Long starvation period was found to be favourable for
AGS stability [23]. Starvation period of the SBR cycle reported in the
literature ranges from 60 to 80 % [24,25].
With the mass transfer issues, big size granules experience anaerobic
processes within the granule core which results in disintegration. The
scheduled withdrawal of mature AGS from the bottom of the reactor is
proposed in this research to allow for a good mix of both old and new
granules. The withdrawal of mature granules, through fixing of the
solids retention time (SRT) would allow for the removal of ageing
granules thereby maintaining the AGS size stability. The fixing of the
SRT as a result of AGS selective discharge will also preserve the mi-
crobial diversity within the system unlike in situations where disin-
tegration occurs and there is biomass washout during decanting. It was
previously indicated that operating AGS reactor under long SRT results
is a major cause of process deterioration [26].
The present study, therefore, investigated the formation and long-
term stability of AGS through the combined strategy of anaerobic slow
feeding, a fixed feast-famine period ratio within each cycle, and fixed
SRT through selective discharge of mature granules at the bottom of the
reactor.
2. Materials and methods
2.1. Experimental set-up
The reactor used for the experiment was made from acrylic
(Plexiglas) glass. It had a height-to-diameter (H/D) ratio of 8 and was
15 cm in diameter. The working volume was 19 L. The SBR mode of
operation was adopted as it is common with AGS systems. The details of
the SBR operation are shown in Table 1. Paintair ceramic diffusers
(AS40) were used in providing fine air bubbles at the bottom of the
reactor at a superficial upflow air velocity of 3 cm/s. The withdrawal of
effluent was done via a port located in the middle of the reactor height,
resulting in a volumetric exchange ratio (VER) of 50 %. Mature gran-
ules were removed from the bottom of the reactor every 3–4 d to
maintain the fixed SRT of 15 d. The schematic diagram of the experi-
mental setup is presented in Fig. 1.
2.2. Seed sludge and media
Settled return activated sludge (RAS) obtained from a wastewater
treatment plant in Calgary was used to start up the reactor. The RAS
had an initial mean particle size of 121 μm. Prior to starting the system,
Table 1
SBR operational conditions.
COD concentration (mg/L) Anaerobic filling time (min) Aeration Time
(min)
1027 ± 47 60 172
716 ± 36 60 172
514 ± 33 47 125
321 ± 33 47 125
Journal of Environmental Chemical Engineering 8 (2020) 103681
the RAS was acclimated to the substrate for 10 d in a batch mode. The
cultivation/ maturation process took 64 d, after which, mature granules
were harvested and used to implement the experimental design. All
experiments were conducted at room temperature (20 ± 2 °C).
Synthetic wastewater was prepared from the following compounds:
sodium acetate, sodium propionate, NH4Cl, KH2PO4, K2HPO4,
CaCl2·2H2O, MgSO4·7H2O, and FeSO4·7H2O. Micronutrients were pre-
pared from: H3BO3, ZnCl2, CuCl2, MnSO4·H2O, (NH4)6·Mo7O24·4H2O,
AlCl3, CoCl2·6H2O, and NiCl2 as detailed elsewhere [27]. The experi-
ment was conducted in four (4) runs: Run 1, Run 2, Run 3, and Run 4.
The details of the chemical oxygen demand (COD) concentrations for
the experimental runs, cycle time, aeration period and the feast-famine
conditions are presented in Table 2. For the feast-famine period ratio, it
was recommended that the feast phase should have a duration no
longer than 20–30 % and famine period higher than 60 % of the cycle
time [20] as long famine period was found to be favourable for AGS
stability [23]. A famine period of 75 % of the aeration period was also
suggested [28]. Based on these, SBR cycle analyses were conducted, as
shown in Fig. SI 2 of the Supplementary Information, and the values
shown in Table 2 below obtained. The COD:N:P ratio was maintained at
100:5:1.
2.3. Analytical methods
Biomass characteristics - mixed liquor suspended solids (MLSS),
mixed liquor volatile suspended solids (MLVSS), and 30-min sludge
volume index (SVI30) - were determined according to standard methods
[29]. SVI5 was measured in a similar way to SVI30 but with the settling
time modified from 30 min to 5 min [30]. COD was determined using
Hach kits following the United States Environmental Protection Agency
reactor digestion method (COD low-range and high-range). The total
nitrogen (TN) was analysed using Shimadzu Total Nitrogen Measuring
Unit (TNM-L). Ammonia nitrogen (NH3-N) was analysed using the
salicylate method TNT Plus 830 (ultra low-range), 831 (low-range), and
832 (high-range). Nitrite, nitrate, and PO43− were analysed using Me-
trohm Compact IC Flex. The readings for nitrite, nitrate, and PO43−
from the IC were then converted to nitrite-N, nitrate-N, and phosphate-
P, respectively. The average particle size was determined by a laser
particle size analysis system (Malvern MasterSizer Series 2000). The
profile of dissolved oxygen (DO) concentration within the mixed liquor
was determined using EcoSense ODO200 DO meter.
2.4. Extraction of extracellular polymeric substances
The extraction of extracellular polymeric substances (EPS) was done
according to the protocol described by Liang et al. [31] with some
modifications. Approximately 10 mL of the mixed liquor was with-
drawn from the reactor during the aeration phase and centrifuged at
4 °C and 2000 g for 15 min [31]. The collected supernatant liquor was
filtered through 0.22 μm polyethersulfone (PES) sterile filter to obtain
the soluble EPS (sEPS). The bottom sediments were re-suspended to
10 mL volume using reverse osmosis (RO) water. 60 μL of 37 % for-
mamide was then added to the suspension. Formamide was chosen to
improve the extraction of tightly bound EPS (TB-EPS) and decrease
contamination from intracellular substances [1]. The suspension was
subsequently put in an orbital incubator (20−30 rpm) at 4 °C for 1 h.
Settling time (min) Decanting time Idle time (min) VER Cycle time HRT (h)
(min) (%) (h)
5 2 1 50 4 8
5 2 1 50 4 8
5 2 1 50 3 6
5 2 1 50 3 6
2
O.T. Iorhemen, et al. Journal of Environmental Chemical Engineering 8 (2020) 103681

Fig. 1. Schematic diagram of the experimental setup.

Table 2 2.6. Microbial community analysis

Details of experimental runs and details of feast-famine conditions.
Runs COD Cycle Aeration time Feast Feast Famine
DNeasy PowerSoil Kit from QIAGEN, Inc. (MD, USA) was used for
concentration time (min) -famine period period the extraction of genomic deoxyribonucleic acid (DNA) from the
(mg/L) (min) period (min) (min) granular sludge. 16S rRNA gene sequencing was done using the
ratio Illumina MiSeq platform with primers 357wF (5′-CCTACGGGNGGCW-
GCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′), which cov-
1 1027 ± 47 240 172 1:3 43 129
2 716 ± 36 240 172 1:3 43 129 ered V3–V4 hypervariable regions. Details of the data analysis are as
3 514 ± 33 180 125 1:3 30 95 reported in the literature [9].
4 321 ± 33 180 125 1:3 30 95

3. Results and discussion

After 1 h, the suspension was then centrifuged at 4 °C and 5000 g for
15 min. The collected supernatant was filtered through 0.22 μm PES
sterile filter to obtain the loosely bound EPS (LB-EPS). The bottom se-
diments were re-suspended again to 10 mL using an extraction buffer
(2 mM Na2HPO4·12H2O, 4 mM NaH2PO4·H2O, 1 mM KCl, 9 mM NaCl,
pH 7). The pH of the resulting suspension was adjusted to about 11
using 1 M NaOH. The suspension was put in an orbital incubator
(20−30 rpm) at 4 °C for 3 h. After extraction, the suspension was cen-
trifuged at 4 °C and 10,000 g for 15 min. The supernatant was collected
and filtered through 0.22 μm PES sterile filter as TB-EPS.
2.5. Analysis of extracellular polymeric substances
The extracted sEPS, LB-EPS, and TB-EPS were analysed for PS and
PN since they are the major components of EPS [32]. The PS content
was determined using the phenol-sulphuric acid method [33] with
glucose as the standard. The PN content was determined using Pe-
terson’s modification of the Lowry method [34].
The three-dimensional excitation and emission matrix (3D-EEM)
fluorescence spectra were measured using a fluorescence spectro-
photometer by collecting a series of emission spectra over a range of
excitation wavelengths. The extracted TB-EPS samples were scanned
over excitation wavelengths from 200 to 600 nm with 5 nm steps and
emission wavelengths from 220 to 600 nm with 5 nm steps in order to
identify the fluorescent compounds present in complex mixture. The
rest of the procedure is as detailed elsewhere [35].
3.1. Granule development and biomass characteristics
A transition from flocculent sludge to dense and granular sludge was
observed starting from day 2 of operation. By day 4, the mean size of
the sludge was 313 μm. The reactor became predominantly granular by
day 11 (i.e. mean size of granules > 200 μm). The granules stabilised
and became mature by day 30. The profiles of biomass mean size, SVI30,
MLSS, MLVSS, and SVI30/SVI5 are presented in Fig. 2. The mean size
increased steadily from 121 μm in RAS to 501 μm on day 11 and con-
tinued to increase thereafter as shown in Fig. 2(a). There was corre-
sponding improvement in sludge settleability as the SVI30 decreased
from 120 mL/g on day 1 to 73 mL/g on day 11. At steady state, the
mean diameter of the granules was 658 ± 92 μm. The system was al-
lowed to run for another 30 d to ensure stability before implementing
the experimental design. During the granule maturation period (days
35–64), biomass sampling was not performed regularly as was the case
during granule formation (days 1–34). The latter period was to allow
for granule stability prior to conducting the planned experiment.
During this latter period, the SVI30/SVI5 ratio kept increasing which
implies the system lost the flocculent biomass and granules dominated
the system.
The mature granules had a clear and regular outline as shown in
Fig. 3(a) and (b). The granules exhibited a compact microbial structure
with spherical outer shape. Fig. 3(c) and (d) show the surface mor-
phology with high concentration of bacteria within the same granule.
There was a dominance of rod-shaped bacteria with some appearance of
filamentous bacteria. The filamentous bacteria acted to bridge other
bacteria species together in the AGS matrix. This is similar to the

3
O.T. Iorhemen, et al.
Fig. 2. Profile of reactor (a) biomass mean size and SVI30; (b) MLSS, MLVSS, and SVI30/ SVI5.
Wiegant's "Spaghetti Theory" of anaerobic granulation where fila-
mentous Methanothix attach on precursors, forming a network via a
branched growth process where other bacteria are entrapped in its
knots [36]. The hydrodynamic shear stress of the upflow liquid and
biogas would subsequently make the 'spaghetti’ structured aggregate
resulting from growth and entrapment to become denser and spheri-
cally shaped. In a similar way, the combination of rod-shaped and fi-
lamentous bacteria in the present study is a precursor for AGS stability.
DNA analyses presented in Section 3.5 show the predominance of rod-
shaped bacteria in all the samples - Thauera sp., Azoarcus sp., Aquimonas
sp., Paracoccus sp., and Flavobacterium sp.
The biomass concentration profile and the settleability of sludge
indicated by SVI5 and SVI30 in the designed experimental phase are
presented in Fig. 4. As it is typical of AGS systems, high biomass con-
centration was maintained in the reactor. There was system upset on
day 112 of operation due to malfunctioning of a valve. This resulted in
washout of some granules. Despite the system upset, the MLSS con-
centration dropped slightly, followed by system recovery and biomass
increase after a few days.
3.2. Effect of EPS content on AGS stability
The extraction of EPS was not done until 15 d had elapsed after the
beginning of each run. This was to obtain results at only steady state
conditions of each run. After 15 d, the EPS was extracted and analysed
at intervals of 5–10 d. The frequency of extraction was to allow for the
monitoring of variations in the course of time during each run.
Fig. 5(a–c) shows the distribution of EPS in the system.
The majority of the EPS was tightly bound to the sludge as shown in
Fig. 5(a) and (b). The TB-EPS PN was comparatively higher than the
corresponding TB-EPS PS. TB-EPS PN showed mean values of
108 ± 61, 83 ± 30, 93 ± 32, and 89 ± 24 mg/g MLVSS for Runs 1,
2, 3, and 4, respectively. This resulted in high PN/PS ratios (Fig. 5c).
The range of PN/PS ratio for LB-EPS and TB-EPS were 0.01–19 and
2.05–13.15, respectively. This is due to the easily degraded PS
4
Journal of Environmental Chemical Engineering 8 (2020) 103681
providing energy for the bacteria rather than PN during the famine
period [37]. The long famine period adopted in the present study may
have contributed to the increase in the PN/PS ratios. It was indicated
that the hydrophobic components of EPS, notably PN, are responsible
for the formation and maintenance of microbial aggregates [38]. High
PN/PS ratios were also reported in the literature for AGS [19].
The feast-famine regime (long famine period of endogenous activity
after a short feast period of abundance of organic matter) allowed for
stressed culture conditions for the production and consumption of EPS
[39]. The EPS produced in the feast phase were later consumed during
the famine period when bacteria spent a longer time under endogenous
respiration conditions [38]. As PS is more easily consumed by bacteria
during the famine phase, more PN is left in the granule matrix to
maintain its structure. Hence, the combination of feast and famine
periods adopted in this study controlled the AGS EPS content at a sui-
table level to allow for granular sludge stability. EPS concentrations in
excess of 200 mg/g VSS have been indicated to block the porosity of the
granules, limiting the transportation of nutrients from the bulk into the
granules, and consequently the death of the bacteria [39]. In this study,
the mean TB-EPS concentrations were 95.6 ± 35.8 mg/g MLVSS and
26.2 ± 13.1 mg/g MLVSS for PN and PS, respectively. These values are
substantially below the threshold indicated in the literature. This im-
plies that the feast famine ratio adopted contributed in maintaining a
healthy porous granular structure that allowed for the transport of
nutrients into the granule layers.
The 3D-EEM spectra of the TB-EPS in the system are shown in Fig. 6
below. All the runs showed one main peak from the 3D-EEM fluores-
cence spectra. The peak for Run 1 was identified at the excitation/
emission wavelengths (Ex/Em) of 350/380 nm. This was similar for
Runs 2, 3, and 4 whose peaks were observed at the Ex/Em wavelengths
of 310/370 nm, 340/380 nm, and 330/370 nm, respectively. A sum-
mary of the 3D-EEM peaks of TB-EPS are presented in Table 3.
The peaks for the runs lie in the region typically identified in the
literature as aromatic protein-like substances [40,41]. This is a clear
indication that protein-like substances were the most abundant in the
O.T. Iorhemen, et al. Journal of Environmental Chemical Engineering 8 (2020) 103681

Fig. 3. (a) Mature granules in the reactor; (b) SEM image of granule; (c) SEM image of granule at ∼5000 magnification; (d) SEM image of granule at ∼4000
magnification.

TB-EPS extracted from the reactor throughout the experimental dura-
tion. This is consistent with the results of EPS analysis above where the
amount of PN was high which resulted in high PN/PS ratios. This also
explains the stability of granules observed in this study.
3.3. Effect of selective discharge of mature granules on AGS stability
Controlled biomass withdrawal from the AGS SBR has been in-
dicated to allow for the discharge of proportional aging granules and
the retention of enough new granules which in turn control the SRT
since long SRTs result in granule deterioration [26]. Selective sludge
discharge has also been reported to promote good settleability, high
pollutant removal efficiency, and microbial diversity [42]. The healthy
mix of mature and new granules in the system will control AGS particle
size, preventing dominance of large size granules that disintegrate with
time due to oxygen diffusion limitations and anaerobic processes within
the granule core [43]. Thus, the selective withdrawal of mature gran-
ules from the bottom of the reactor at regular intervals in this study
allowed for the removal of big granules that would have disintegrated
with time, hence keeping the AGS particle size in check. This eventually

Fig. 4. Biomass concentration and sludge settleability during the experimental period.

5
O.T. Iorhemen, et al.
Fig. 5. (a) EPS-PN profile, (b) EPS-PS profile, (c) Proteins to polysaccharides ratio (PN-PS) of EPS.
resulted in a good mix of new and old granules in the system. The feast-
famine regime and the selective discharge of granules allowed for AGS
stability as the system was run for over 240 d without any disintegra-
tion.
3.4. Reactor performance
3.4.1. Profiles of organic matter
The system exhibited consistent high COD degradation throughout
the duration of the experiment as shown in Fig. 7. The mean removal
efficiency was 98.3 ± 0.8 % for all the experimental runs, i.e. the
different COD concentrations. The high removal efficiency attained in
the present study is consistent with previous findings where organics
removal efficiency was above 96 % for both municipal and industrial
wastewaters [44,3]. Generally, AGS has been proven to efficiently re-
move organics [11,19]. The dense nature of the granular biomass en-
hances the performance of the biological reactor, making it more effi-
cient than the activated sludge process mainly due to the higher MLVSS
concentration and the better sludge settleability which resulted in high
biomass retention.
3.4.1.1. Effect of anaerobic feeding on organic matter removal. The
anaerobic feeding regime adopted in this study allowed for the
uptake of COD during the feeding phase. At the end of the anaerobic
feeding phase, the ratio of the influent COD concentration to the COD
concentration at the onset of aeration was 0.42 for Run 1, 0.28 for Run
2, 0.33 for Run 3, and 0.12 for Run 4. Since the reactor was operated at
6
Journal of Environmental Chemical Engineering 8 (2020) 103681
VER of 50 %, the initial COD concentration in an SBR cycle is
approximately half of that in the influent (assuming a complete
degradation of COD in each cycle). At the end of the anaerobic
feeding phase, COD reduction was observed. This can be attributed to
the carbon uptake by both PAOs and GAOs where they are present [16].
Moreover, the depletion of COD during the feeding phase points to the
presence of PAOs and GAOs.
3.4.1.2. Organic matter degradation within SBR cycle.. The COD
degradation profile during the SBR cycle was determined for each run
as shown in Fig. SI 2 of the supplementary material. The DO
concentration profile within the mixed liquor was also monitored as
an indicator of oxygen uptake during the cycle (Fig. SI 5 of the
supplementary material). A rapid drop of COD concentration was
observed in all the experimental runs during the aerobic phase. After
30 min of aeration, the influent COD concentration decreased by 98.5 %
and 98 % for Runs 1 and 2, respectively. For Runs 3 and 4, the influent
COD was degraded by 97.5 % after 10 min for Run 3 and 97.6 % after
5 min of aeration for Run 4. Afterwards, the COD concentration stayed
almost constant (depleted) for the reminder of the cycle for Runs 1–4,
indicating the occurrence of a long famine phase in the reactor. A
transition from the feast period to the famine period corresponds with
an increase in the DO concentration, approaching saturation as
previously described [20,45]. The DO concentration in the feast
phase was considerably lower than during the famine period as
microorganisms utilised the DO during the feast period to degrade
the organic matter; and, once the COD was depleted in the famine
O.T. Iorhemen, et al. Journal of Environmental Chemical Engineering 8 (2020) 103681

Fig. 6. 3D-EEM of TB-EPS of AGS of different runs (a) Run 1, (b) Run 2, (c) Run 3, (d) Run 4.

Table 3 98 ± 5. Effluent nitrite-N and nitrate-N exhibited quite a range of

3D-EEM peaks of TB-EPS. values, although generally low values as presented in Fig. 8(b). The
Run COD Cycle time HRT Peak (nm)
performance for TN was also consistently high, attaining a mean re-
(mg/L) (h) (h) (Ex/Em) moval efficiency of 89 ± 6 %. The high removal of TN observed in this
study indicates that nitrification and denitrification occurred in the
1 1027 ± 47 4 8 350/380 system. Microbial analyses (Section 3.5) show the presence of various
2 716 ± 36 4 8 310/370
3 514 ± 33 3 6 340/380
bacteria involved in the biological removal of nitrogen in all the runs:
4 321 ± 33 3 6 330/370 Azoarcus, Thauera, Aquimonas, Paracoccus, Pseudomonas, Acidovorax,
and Flavobacterium [46–48].
AGS provides suitable conditions for nitrification (aerobic layer)
phase, the DO concentration increased to reach the saturation levels in and denitrification (anoxic layer). It is widely reported that AGS has a
water. stratified structure that allows for the existence of aerobic, anoxic, and
anaerobic conditions within the same granule [5,10]. The produced
nitrate-N during nitrification was used as electron acceptor in the inner
3.4.2. Profiles of nitrogen removal
anoxic zones of the granule particle for denitrification. Denitrification is
The system showed high NH3-N removal throughout the experi-
normally the limiting step for TN removal since there is the requirement
mental duration as shown in Fig. 8(a). The mean removal efficiency was

Fig. 7. Profile organics degradation.

7
O.T. Iorhemen, et al.
Fig. 8. (a) Profile of NH3-N removal (b) Effluent nitrite-N and nitrate-N (c) Profile of TN removal.
for an anoxic interior in AGS or anoxic condition with adequate carbon
sources [49]. Nitrification is easily achieved in AGS systems due to the
high retention of nitrifying biomass in the granular system. The suc-
cessful denitrification observed in this study demonstrates the presence
and action of an anoxic interior part of the granule towards nitrogen
removal.
With very low nitrite-nitrogen and nitrate-nitrogen in the effluent
(Fig. 8b), the high TN removal achieved is attributed to both simulta-
neous nitrification and denitrification as well as biological assimilation
for growth. Microbial analyses (Section 3.5) show the presence of
known denitrifiers: Azoarcus sp, Thauera sp, Aquimonas sp, Paracoccus
sp, Pseudomona sp, Acidovorax sp, and Flavobacterium sp. In addition, the
high biomass concentration in AGS systems results in increased mi-
crobial consumption of nutrients for growth and cell maintenance [50].
Similar studies have also reported high performance in terms of ni-
trogen removal in AGS systems [11,51,52,10].
3.4.3. Profiles of phosphorus removal
The removal profile of phosphate-P is presented in Fig. 9. Phos-
phorus release during the anaerobic feeding phase was higher in Runs 1
and 2. Higher relative abundance of PAOs was detected in the microbial
analysis during these runs (Section 3.5). High phosphate-P removals:
86 ± 15 % and 92 ± 13 % were attained for Runs 1 and 2, respec-
tively.
There was a system washout on day 112, which affected the re-
moval of phosphate-P. The phosphate-P released during the anaerobic
feeding phase started to decrease after the system upset. As such,
8
Journal of Environmental Chemical Engineering 8 (2020) 103681
phosphate-P uptake in the subsequent aerobic phase also reduced, re-
sulting in a drop in phosphate-P removal efficiency. Runs 3 and 4 which
followed this system upset achieved phosphate-P removal of only
68 ± 15 % and 68 ± 11 %, respectively. The system disturbance
caused a shift in the microbial population with a major drop in PAOs
and the appearance of GAOs which outcompeted with PAOs for the
available carbon (Section 3.5). While GAOs uptake carbon during
anaerobic conditions, they are neither able to release phosphorus
during anaerobic conditions nor uptake phosphorus during aerobic
conditions [4,53].
The removal of phosphate-P in AGS systems is attributed to the
stratified structure of aerobic granule and the SBR mode of operation
with a long anaerobic feeding phase used in this study. The large size of
granule resulted in oxygen diffusion limitation, allowing for a layered
structure with aerobic, anoxic, and anaerobic conditions from the outer
surface to the inner core [5]. This structure created suitable micro-
environments for enhanced biological phosphorus removal (EBPR). In
addition, a previous study found that the anaerobic slow feeding, sub-
sequent aerobic phase, and phosphate availability in the influent allows
for the selection for PAOs [16]. Therefore, the existence of PAOs in the
microbial community analysis coupled with the consequent phosphate-
P removal can be an evidence for EBPR in AGS.
Many studies on AGS have reported efficient phosphorus removal
from wastewater [54,44,10]. Successful EBPR process relies on the
enrichment of PAOs which uptake phosphorus from the bulk liquid
during the aerobic phase. Recent studies have reported another per-
spective to this. The EPS matrix of AGS has been demonstrated to play a
O.T. Iorhemen, et al. Journal of Environmental Chemical Engineering 8 (2020) 103681

Fig. 9. Profile of Phosphate-P removal.

salient role in both biological phosphorus and nitrogen removal pro- Runs 1–4. Additionally, Acidovorax (a facultative bacteria) was found at
cesses [55,56]. The selective discharge of mature dense granules from 1.86, 4.34, 4.26, and 3.65 % in Runs 1–4, respectively. Acidovorax sp
the bottom of the reactor may have also contributed to the removal plays the dual role of COD removal and denitrification [48].
efficiency of phosphorus achieved in this study. It has been previously For denitrification activity, three different denitrifiers were detected
demonstrated that the selective discharge of large and dense granules in the system. Aquimonas sp, belonging to the Rhodanobacteraceae fa-
saturated with phosphorus results in improved phosphorus removal mily and Gammaproteobacteria class, was identified at 8.63, 10.50, 1.47,
[57]. and 2.50 % in Runs 1–4, respectively. Aquimonas sp is aerobic bacteria
and a heterotrophic denitrifier [64]. The two other denitrifiers detected
were Paracoccus and Pseudomonas. Paracoccus sp (belonging to the
3.5. Microbial community analysis
Rhodobacteraceae family and Alphaproteobacteria class) was identified at
18.01, 21.50, 4.88, and 3.50 % in Runs 1–4, respectively. Pseudomonas
The microbial community analysis of the AGS in this study is pre-
sp, belonging to the Pseudomonadaceae family and Gammaproteobacteria
sented in Fig. 10.
class, was detected during all the runs, at 0.53, 0.90, 1.25, and 0.41 %
Thauera sp (belonging to the family Rhodocyclaceae and
for Runs 1–4, respectively. The presence of these three denitrifiers ex-
Gammaproteobacteria class has been shown to be abundant in AGS
plains the removal of nitrate (hence total nitrogen) from the system.
treating high-strength organics wastewater [7]. In this work, the COD
Furthermore, for granular sludge stability and phosphorus removal,
concentration started at 1027 ± 47 mg/L and the relative abundance
Flavobacterium belonging to the family Flavobacteriaceae and class
of Thauera sp was 23.04 %. As the influent COD concentration was
Bacteroidia, was found at 2.91, 2.71, 4.75, and 2.81 %, respectively in
reduced, a declining trend of the relative abundance of Thauera sp was
Runs 1–4. Flavobacterium sp is known to increase in the course of
observed where it decreased to 10.54 %, 1.33 %, and 8.54 % on days
granulation from flocculent sludge to young granules, playing the
84, 168, 197, at COD concentrations of 716 ± 36, 514 ± 33, and
salient role of supporting mature granules through the production of
321 ± 33 mg/L, respectively. In addition to their ability to survive and
EPS, performing denitrification and accumulating phosphorus [7,65].
take part in important functions in both aerobic and anoxic conditions,
The relative abundance of Flavobacterium sp appears to be similar ir-
Thauera sp was indicated to play a critical role in both granule forma-
respective of the COD concentration, pointing to the stable AGS ob-
tion and stability [7,46]. Thauera sp was reported to be an essential
served in this study. Moreover, Meganema sp, belonging to the class
population for the degradation of organics in industrial wastewater
Alphaproteobacteria, was found at 4.46 and 2.70 % in Runs 1 and 2,
treatment plants [58]. The presence of Thauera sp in AGS has been
respectively. Runs 3 and 4 had negligible relative abundance of Mega-
previously reported [59]. Azoarcus sp (Rhodocyclaceae family and Be-
nema sp. Meganema sp is known to store polyphosphates under aerobic
taproteobacteria class) was detected at very low relative abundances
conditions but is unable to assimilate carbon under aerobic conditions
during Runs 1 and 2, at 0.46 % and 0.26 %, respectively. However,
[66]. Acinetobacter of the family Moraxellaceae was detected at relative
there was a considerable increase of Azoarcus sp to 25.02 % and 27.95
abundances of 0.15 and 0.18 % for Runs 1 and 2. This is a known PAO
% during Runs 3 and 4, respectively. As the COD concentration reduced
and its presence alongside Meganema sp in Runs 1 and 2 correspond
to 514 ± 33 and 321 ± 33 mg/L during Runs 3 and 4, the influent TN
with the high phosphate-P removal observed during these Runs (Section
concentration was reduced correspondingly (to maintain the COD:N:P
3.4.3). For contribution to AGS stability, the genus Acinetobacter is also
ratio of 100:5:1) to about 25 and 15 mg/L, respectively. Azoarcus sp and
known for EPS secretion and has high self-aggregation ability and ad-
Thauera sp mostly occur together in wastewater, and are functionally
hesion capability to solid surface [59]. Simplicispira (belonging to the
crucial denitrifiers [60]. Their presence indicated the existence of an-
Gammaproteobacteria class) which takes part in both of phosphorus
oxic conditions within the granule. Other studies reported that the
removal and denitrification was found at 0.57, 2.09, 0.60, and 0.37 %
Betaproteobacteria is also the most abundant class in municipal waste-
in Runs 1–4, respectively.
water treatment plants worldwide, largely responsible for the removal
Moreover, microbial species that compete with PAOs for available
of both organic matter and nutrients [61,62].
carbon but do not contribute to biological phosphorus removal were
Similarly, a variety of microbial species known for carbon de-
also detected. Candidatus Competibacter, belonging to the family
gradation were identified across all the runs. Leadbetterella sp which is
Competibacteraceae and class Gammaproteobacteria, was detected in
known for degrading carbohydrates [63] was identified at 6.34, 3.04,
Runs 3 and 4 at relative abundances of 9.99 and 11.03 %, respectively.
1.78, and 0.48 % in Runs 1–4, respectively. The relative abundance of
Candidatus Competibacter is a GAO that competes with PAO for available
Leadbetterella sp shows a decreasing trend as COD concentration de-
carbon. The presence of GAOs generally indicates the failure of phos-
creased from Run 1 to 4. Similarly, two other COD degraders were
phorus removal performance as they have no contribution towards
detected - Aeromonas sp and Hydrogenophaga sp [48]. Aeromonas sp was
biological phosphorus removal [53]. Runs 3 and 4 during which Can-
identified at 5.47, 3.05, and 0.31 % in Runs 1–3, respectively. Hydro-
didatus Competibacter was detected had reduced phosphorus removal
genophaga sp was found at 1.81, 2.38, 2.06, and 2.13 %, respectively in

9
O.T. Iorhemen, et al.
Fig. 10. Microbial analysis of the system at the genius level.
efficiency. Also identified during Runs 3 and 4 are two more GAOs,
Candidatus Contendobacter and Defluviicoccus at 1.72 % (Run 3) and
6.22 % (Run 4) for Candidatus Contendobacter, and 1.98 % (Run 3) and
2.82 % (Run 4) for Defluviicoccus, respectively. It is to be noted that
Runs 3 and 4 came after the system washout on day 112. The appear-
ance of these GAOs points to a shift in the microbial population re-
sulting from the system upset.
In terms of AG stability, the presence of Thauera, Flavobacterium and
Meganema in all the runs points to the stable reactor operation.
Flavobacterium is known to support granule formation by EPS produc-
tion that bind cells together [47]. Thauera and Meganema are also EPS
producers. Slow growing bacteria, notably PAOs, ammonia-oxidizing
bacteria (AOBs) and nitrite-oxidising bacteria (NOBs), have been in-
dicated to improve AG stability [19,67]. Hence, the presence of Aci-
netobacter and Simplicispira also point to AG stability.
4. Conclusion
The long-term stability of AGS was explored through the combined
strategy of long anaerobic slow feeding, fixed 1:3 ratio of feast-famine
period within each SBR cycle, and selective withdrawal of mature
granules. Four experimental runs were implemented, each lasting about
60 d, with COD concentration ranging from 300 to 1000 mg/L and cycle
time of 3–4 h. There was carbon uptake from the bulk liquid during
anaerobic feeding, allowing for the bacteria to utilise stored carbon
10
Journal of Environmental Chemical Engineering 8 (2020) 103681
during the famine period, thus keeping the growth of heterotrophic
bacteria in check. The 1:3 fixed ratio of feast-famine period within each
SBR cycle controlled the EPS content at a suitable level, allowing for
AGS stability as the system was run for over 240 d without granule
disintegration. The selective withdrawal of mature granules from the
bottom of the reactor resulted in a good mix of new and old granules in
the system. The system achieved 98.3 ± 0.8 %, 98 ± 5 %, 89 ± 6 %
removal of COD, NH3-N, and TN, respectively. Phosphate-P removal
was 88.8 ± 14 % (runs with PAOs) and 68.4 ± 11 % (when PAOs
reduced and GAOs appeared). EPS producers (Thauera, Flavobacterium
and Meganema) and slow growing bacteria (Acinetobacter and
Simplicispira) in the system contributed to the AG stability observed in
this study.
CRediT authorship contribution statement
Oliver Terna Iorhemen: Conceptualization, Data curation, Formal
analysis, Investigation, Writing - original draft, Writing - review &
editing. Mohamed Sherif Zaghloul: Data curation, Formal analysis,
Investigation, Writing - review & editing. Rania Ahmed Hamza: Data
curation, Formal analysis, Investigation, Writing - review & editing. Joo
Hwa Tay: Conceptualization, Funding acquisition, Supervision.
O.T. Iorhemen, et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the Natural Sciences and Engineering
Research Council of Canada. The services of the Centre for Health
Genomics and Informatics in the Cumming School of Medicine,
University of Calgary are also acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jece.2020.103681.
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