Biochemical Engineering Journal 58–59 (2011) 69–78

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Biochemical Engineering Journal

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Evolution of bioaggregate strength during aerobic granular sludge formation

Junfeng Wan a,b,c , Irene Mozo a,b,c , Ahlem Filali a,b,c , Alain Liné a,b,c , Yolaine Bessière a,b,c ,
Mathieu Spérandio a,b,c,∗
a
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
b
INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
c
CNRS, UMR5504, F-31400 Toulouse, France

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

Article history: This work investigated the modification of aggregate properties during the formation of granular sludge
Received 7 January 2011 in a sequencing batch airlift reactor (SBAR). The cohesion of biological aggregates was quantified by
Received in revised form 23 July 2011 subjecting sludge samples to two different controlled shear stresses in a stirred reactor. For reference
Accepted 25 August 2011
sludge (without granules), flocs broke and reformed easily, indicating that floc size was controlled by
Available online 31 August 2011
the turbulence micro-scale (Kolmogorov scale, here from 17 ␮m to 62 ␮m). In contrast, granules showed
high strength which enabled them to resist turbulence and their size was no longer imposed by the
Keywords:
Kolmogorov micro-scale. Different steps were observed during the granulation process: a first increase
Aerobic granular sludge
Aggregate strength
of aggregate cohesion associated with a decrease in sludge volume index (SVI), a growth of aggregates
Shear with detachment of fragile particles from the surface and, finally, an increase in the sizes of small and
Cohesion large granules to reach a pseudo-stable size distribution. Results suggest that small particles could have
formed the seeds for new granules, as they were maintained in the bioreactor. Here, granular sludge was
formed in an SBAR with a conventional settling time (30 min), i.e. without particle washout, and with
a low superficial air velocity (SAV = 0.6 cm s−1 ): it is thus demonstrated that high SAV and low settling
time are not necessary to produce granules, but probably only accelerate the accumulation of granules.
It is shown that the increase of cohesion is the initial phenomenon explaining the granule formation
concomitantly with bacterial aggregates densification. It seems important, in the future, to investigate the
reasons for this cohesion increase, which is possibly explained either by bacterial bounding interactions
or the excretion of extracellular polymeric substances (EPS).
© 2011 Elsevier B.V. All rights reserved.

1. Introduction and extracellular polymeric substances [7,8]. However, if mech-

Since the phenomenon of aerobic granular sludge was first
reported in the 1990s [1,2], research on the subject has flourished
and this new technology has shown its significant perspectives
in wastewater treatment [3–5]. Compared with conventional
activated sludge, aerobic granular sludge presents a variety of
attractive properties such as more compact structure, higher spe-
cific activity and better settling properties as required to improve
solid–liquid separation. In most of the studies, granular sludge
was formed in a sequencing batch reactor [6]. Aerobic granules
were obtained by consecutively inoculating with conventional acti-
vated sludge and applying operating conditions which favoured
the development and selection of granules. Because of its com-
pact structure, this particular aggregate is expected to present
similarities with biofilm, which is mainly composed of bacteria
∗ Corresponding author. Tel.: +33 561559785; fax: +33 561559760.
E-mail address: mathieu.sperandio@insa-toulouse.fr (M. Spérandio).
anisms involved in the initial formation of granules have been
the subject of many researches, relationships between microbial
aggregation mechanisms, reactor operational conditions and the
resulting physical properties of granules still need to be clarified.
So far, the literature proposes biological and process conditions
leading to granular sludge formation. Concerning the biological
selection pressure, stability of granules is found to be improved
when selecting slow growing bacteria. This can be done either when
increasing the substrate N/COD ratio [9] to favour nitrifying bacte-
ria or by alternating feast/famine and anaerobic/aerobic periods
[10] to promote selection of phosphate or glycogen accumulating
organisms.
Concerning the process conditions, the literature [11–14]
underlines that aeration rate is an important factor, influenc-
ing granular sludge formation through three main, simultaneous
mechanisms: (i) oxygen transfer, which modifies the dissolved oxy-
gen in the bulk and hence within the granule, (ii) mixing, which
influences mass transfer of substrates in the liquid boundary layer
and (iii) hydrodynamic shear stress, which controls erosion and

1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2011.08.015
70 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78
fragmentation. A high superficial gas velocity (SGV) has been seen
as a major factor enhancing the formation of aerobic granular
sludge but its roles (shear stress, mixing and oxygen transfer) are
still controversial due to the interdependency of these processes
[12]. Investigations have focused more on biological aspects and
aerobic granulation has not been thoroughly studied from the fluid
mechanics point of view [15]. So far, little information concerning
the influence of mechanical stresses on granule size and shape has
been reported. The results of Zima-Kulisiewicz et al. [15] suggest
that the shear stress (tangential force) and elongation stress (nor-
mal force) together with stresses induced by fluctuating velocities
are the main forces influencing granule building. Although the ori-
gin and significance of mechanical stresses in the bioreactor have
been clarified, only few approaches has been developed to esti-
mate whether bioaggregate strength and size are altered by these
stresses, whereas a strong relationship has been reported between
the size of physicochemical flocs and the Kolmogorov microscale
[16]. Particle strength (or cohesion) has rarely been measured on
aerobic granular sludge but it is reasonable to think that this prop-
erty plays a role in granule formation and stability as underlined on
Anammox granules where an increase of input specific power was
shown to affect activity and biomass retention [17]. Because it gen-
erally controls surface detachment (erosion) and particle breakage,
aggregate strength should receive more attention.
Detachment of particles is a crucial phenomenon for aerobic
granular sludge, as it limits the growth rate and controls the stabil-
ity of granules (as well as the specific activity of supported bacteria).
Detachment rate also controls the microbial competition between
fast-growing heterotrophic bacteria and slow-growing autotrophic
bacteria as has been shown numerically and experimentally in the
case of biofilms [18,19]. This phenomenon is of importance as both
kinds of bacteria are necessary for simultaneous carbon and nitro-
gen removal. The significance of detachment is strongly related to
the cohesion of granules. Cohesion is a physical property of the
aggregate defining how it can resist shear and elongation force,
which induce erosion and/or break-up. It is well known that the
strength of aggregates depends on the strength and number of the
bonds holding the aggregates together [20]. Aggregate breakage is
likely to occur if the stress applied to the surface of the aggregate
can counterbalance the bonding strength [21], which means that
the more compact the structure of aggregates is, the more difficult
they are to break. Thus, cohesion is directly related to structure
and is, therefore, highly dependent upon the aggregate formation
process.
Techniques for measuring granule strengths can be derived from
those developed for measuring floc strength. Floc strength has been
largely studied [22,23] using various methods. Most of the methods
used have been impeller-based techniques [24–29]. Floc strength
may be estimated through floc size analysis by examining its break-
up consecutively by exposing to known stresses [30]. The method
consists of subjecting flocs to single or increasing levels of shear
within a vessel and comparing the ratio of the floc size before and
after breakage, thus quantifying the energy input necessary for floc
breakage [31]. Other techniques, such as ultrasonic methods, have
also been adapted to test aggregate strength but are more suitable
for application to mineral flocs than to bioaggregates due to the
effect of ultrasound on bacterial components [32]. Similar studies
have recently focused on characterising granules from anaerobic
processes in the upflow anaerobic sludge blanket (UASB) system.
For example, the resistance to compression forces has been mea-
sured recently [33]. In a previous work [34], anaerobic granules
were subjected to strong mechanical stirring (200 rpm) for 5 min.
The granular strength of anaerobic granules was deduced from the
ratio of solids in the supernatant to the total weight of the gran-
ular sludge. This ratio gives an indicative index of the granules’
resistance when subjected to abrasion and shear, but gives no infor-
mation on the modification of the particle sizes. This method has
been combined with image analysis to compare two different types
of aerobic granules developed in aerobic sequencing batch reactors
(SBRs) [35]: fungal granules from one SBR were larger and weaker,
with a loosely packed, fluffy structure whereas the bacterial gran-
ules from another SBR were smaller and stronger, with a compact
structure and higher decantation feasibility.
Little information is available to understand how the operating
conditions can affect the particle strength in an aerobic granular
sludge process. A study carried out by Di Iaconi et al. [36] investi-
gated the influence of hydrodynamic shear forces on the properties
of granular biomass developed in a sequencing batch aerobic biofil-
ter reactor. They found that granular biomass density increased
quasi-linearly with the increase of shear forces in the reactor and
that EPS content and composition were not affected by hydro-
dynamic shear forces. More recently, an energy-dissipation-based
model has been established by Ren et al. [37] to quantify the shear
stresses in an aerobic granular sludge SBR and predict the rate
of particle detachment. Simulations indicate that shear is induced
mainly by gas bubbles or granule collisions and only slightly by the
fluid. The possible modification of particle strength has not been
integrated in this model. Because the detachment is quantified by
absorbance measurement, no information is provided on the size of
the detached particles and remaining granules. In conclusion, little
is known at present about the evolution of cohesion during granular
sludge formation, and yet this should be helpful in understanding
the mechanism of granule formation.
Consequently, the goal of this study is to characterise the evolu-
tion of aggregate cohesion during the formation of aerobic granular
sludge, and the behaviour of granules under given hydrodynamic
conditions. To determine aggregate strength, biological aggregates
were subjected to a calibrated stress in a stirred reactor and the par-
ticle size distribution was measured. Flocculated biomass was first
characterised and considered as a reference point. Then granular
sludge was developed in a statically filled sequencing batch air-
lift reactor (SBAR) by applying substrate feast and famine periods
coupled with alternating anoxic/aerobic conditions. The analysis
focused on the progressive conversion from a floc-like structure
to granule-like aggregates, and non-significant hydraulic particle
selection was applied during this period (using a 30 min settling
phase). Finally, at the end of the study, granules were collected
after sieving and their specific behaviour was characterised in the
stirred reactor.
2. Materials and methods
2.1. Sequencing batch airlift reactor (SBAR)
The reactor employed was an airlift column with a working vol-
ume of 17 L (internal diameter = 15 cm, total height = 105 cm, H/D
ratio = 7). A plate baffle (length: width = 83:15 cm) placed vertically
in the middle of the reactor divided the column into two zones:
riser and downcomer. Air was introduced through a fine bubble
aerator at the bottom of the reactor, and induced the circulation
of air, liquid and solid. The system is schematically presented in
Fig. 1a.
The column was inoculated with 10 L of conventional acti-
vated sludge taken from the aeration tank of a local municipal
wastewater treatment plant at a mixed liquor suspended solid
(MLSS) concentration of 4.5 g L−1 and a sludge volume index after
30 min (SVI30 ) of 172 mL g−1 . The reactor was fed with a syn-
thetic wastewater having a mixed organic carbon source: glucose,
ethanol, sodium propionate and sodium acetate, each contribut-
ing 25% of the total COD (total = 750–1000 mgCOD L−1 ). The other
components of the wastewater (mg L−1 ) were as follows: NH4 Cl
 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78
Fig. 1. Schematic diagram of the SBAR (a) and cohesion test vessel (b).
(143–191), NaNO3 (610), NaHCO3 (100), KH2 PO4 (100), CaCl2 (30),
MgSO4 ·7H2 O (4) and other necessary elements were similar to the
trace solution described elsewhere [38]. The organic and ammo-
nium loading rates were simultaneously increased during the study
with a constant COD/N ratio of 20 (Table 1) by increasing the influ-
ent concentration or decreasing the cycle duration. The system was
first operated by cycles of 6 h including influent filling (30 min), pre-
anoxic phase (30 min), aeration (240 min), settling (30 min) and
effluent withdrawal (30 min). Reduction of the cycle to 4 h was per-
formed by a proportional reduction of the filling, anoxic and aerobic
phases. A relatively low particle selection pressure was imposed
compared to those used in previous studies on granular sludge
[3,11,14]. The process was initially operated with a high aeration
rate (superficial air velocity SAV = 2.23 cm s−1 ) and a purely aerobic
reaction phase. From day 80, the SAV was reduced to 0.63 cm s−1 ,
and the pre-anoxic phase was introduced from day-120 (by means
of nitrogen gas sparging at the same gas velocity).
2.2. Analytical methods
Sludge samples taken from the column were characterised in
terms of suspended solids (SS) and volatile suspended solids (VSS)
using standard methods [39]. Sludge volume index (SVI) was mea-
sured in a 1000 mL graduated cylinder with the sludge sample taken
from the mixed liquor at the end of the aerobic phase. SVI5 and SVI30
were measured after 5 and 30 min, respectively, so as to compare
aggregates properties with the one proposed for granular sludge
in literature. The particle size distribution (PSD) of aggregates was
analysed using a laser diffraction technique (Mastersizer, Malvern
2000, UK) with a particle size ranging from 0.02 ␮m to 2000 ␮m.
Table 1
Strategy for organic and ammonium loading rates.
Time Duration of OLR (kg COD d−1 m−3 ) ALR (kg N-NH4 d−1 m−3 )
cycle (h)
d70–120 6 1.412 0.071
d120–175 6 1.882 0.094
d175–256 4 2.822 0.141
71
In this study, the optical index of the dispersant was set to 1.330,
which corresponded to the value for the water. As biological aggre-
gates are mainly composed of organic matter, the optical index was
set to 1.596 as indicated in the work of Lambert et al. [40]. Qualita-
tive observations were performed by using either a microscope or
a stereoscopic microscope depending on the size of the aggregates.
2.3. Cohesion tests
Cohesion tests were performed in a stirred vessel, equipped with
an impeller (5 cm in diameter), consisting of a 145 mm × 245 mm
(inner diameter × height) cylinder providing a testing volume of
2.5 L (Fig. 1b). The shear level corresponding to the operating con-
ditions induced during this experiment can be approximated by the
average velocity gradient value (G) for isotropic and homogeneous
turbulence as follows:

P
G= (1)
V
where G is the velocity gradient (s−1 ), P is the power input (W),  is
the dynamic viscosity (Pa s) and V is the suspension volume (m3 ).
The power input (P) was calculated as the energy dissipated by
the impeller:
P = Np N 3 d5 (2)
where Np is the power number given by the manufacturer (4 in
our case: turbine, 4 flat blades),  is the mass density of the fluid
(kg m−3 ), N is the rotation speed (s−1 ) and d is the impeller diameter
(m).
The mechanical stirring system provided an average velocity
gradient of up to 3400 s−1 . By considering the energy dissipation
(ε in m2 s−3 ) in terms of a gradient (G) dependent expression:
ε =  · G2 (3)
where  is the kinematic viscosity (m2 s−1 ), the Kolomogorov
microscale ( in ␮m), corresponding to the size of the smallest
72 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78
Fig. 2. Hydrodynamic test conditions in the vessel and the measurement time: (a) theoretical hydrodynamic characterisation of the system (vessel + impeller); (b) hydrody-
namic test conditions during the three phases: low mixing intensity (phases I and II) and high mixing intensity (phase II) and symbols used for different times.
eddies during mixing (linked to the shear level in the vessel), could
be determined:
 1/4
3
=
ε was close to the Kolmogorov microscale (62 and 17 ␮m, respec-
Based on the previous definitions, a theoretical characterisation
of the cohesion test system was performed in terms of average
velocity gradient and Kolmogorov microscale resulting from a rota-
tion speed as presented in Fig. 2a. As shown in Fig. 2b, the cohesion
test consisted of a three-phase procedure: the vessel was first oper-
ated at a low mixing intensity (300 rpm/250 s−1 ) for 45 min (phase
I); then the shear intensity was increased (1700 rpm/3400 s−1 ) for
45 min (phase II) and, finally, decreased to the original condition
for 45 min (phase III). Those operating conditions lead to turbu-
lent flow regime with impeller’s Reynolds numbers of 12 500 and
70 000, respectively. During the entire period, PSD was measured
5 min before the end of each phase (Fig. 2b). The Kolmogorov
microscale corresponding to the applied G values was 62 ␮m for
a G value of 250 s−1 (300 rpm) during phases I and III, and 17 ␮m
for 3400 s−1 (1700 rpm) during phase II. For each test, the sludge
was sampled at the end of the aerobic phase of the operational
cycle of the SBAR and systematically diluted to 1 g L−1 by using the
ultra centrifuged supernatant (10 000 × g, 15 min) in order to avoid
any modification of the environment, such as the ionic strength,
which is known to have a significant effect on flocculation pro-
cesses.
3. Results
3.1. Characterisation of floc behaviour (reference) during the
cohesion test
As a starting point, the cohesion test was performed on a floc-
culated sludge (Fig. 3a) developed in a purely aerobic SBAR fed
by the same synthetic influent as for the anoxic/aerobic SBAR.
Sludge was sampled from the reactor and placed in the stirred
reactor before various stirring rates were imposed. As can be
observed in Fig. 3b, the particle size distribution of this floccu-
lated biomass was clearly influenced by the hydrodynamics: an
increase in impeller rotation speed resulted in a significant reduc-
tion in median diameter from 80 to 18 ␮m. After reduction of
the velocity gradient, an increase in the median diameter was
observed (Fig. 3c) indicating a reflocculation of the particles and
resulting in full recovery of the initial particle size. Furthermore,
it can be seen that the suspension seemed to be calibrated by
the turbulence scale as the average diameter (70 and 16 ␮m)
(4)
tively). Similarly, it has been demonstrated that chemically induced
flocculation is controlled by hydrodynamic turbulence in stirred
coagulation reactors [41]. The present results suggest that the
approach proposed for physicochemical flocs is still valid for bio-
logical flocs developed in the laboratory with soluble synthetic
substrates.
3.2. Evolution of aggregate properties during granular sludge
formation in SBAR
3.2.1. Suspended solid concentration, SVI and particle size
As shown in Fig. 4, the profiles of MLSS and SVI versus time
varied inversely during the study. Before day 120, MLSS first
increased from 2.5 to 4.5 g L−1 whereas SVI30 decreased from 150
to 100 mL g−1 . Then the pre-anoxic mixing phase was introduced
in the cycle after the static filling phase, from day-120 (by sup-
plying nitrogen gas instead of air at the same gas flow rate). Later
again, a significant decrease of SVI30 from 100 to 45 mL g−1 and
an increase of MLSS up to around 9.5 g L−1 were observed. These
data are thoroughly described in Wan et al. [42]. From day-175, the
organic, ammonium and nitrate loading rates were increased, as
the cycle time was reduced from 6 to 4 h. After a temporary drop
for a short period, MLSS recovered the same level as before, and
larger and larger particles (>200 ␮m) were observed in the airlift
column. MLSS continued to increase during the following months
and stabilised around 15 g L−1 after 350 days. Simultaneously, SVI
decreased to 30 mL g−1 indicating typical properties of granular
sludge.
Aggregates’ PSD in the column was measured on different days
(Fig. 5). The median floc size (d0.5 ) was around 100 ␮m on day-71
(similar to the reference sludge) and decreased slightly during the
first phase. On day-212, bimodal peaks were observed, indicating
that two populations of aggregates were present in the reactor. On
day-260, the size of both these types of aggregates had increased
significantly and the median size of the biggest population (gran-
ules) was around 900 ␮m.
 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78 73

Fig. 4. Evolutions of MLSS, MLVSS and SVI30 .

tion curves and the associated microscopic observations are shown

in Fig. 6. Additionally, Kolmogorov microscales (17 and 62 ␮m) cor-
responding to the two rotation speeds used are presented together
with PSD (Fig. 6). In this experiment, the particle size distribution
should be interpreted considering that two types of bio-particles
co-exist in the reactor.
On day-162 (Fig. 6a), microscopic observation indicated the
appearance of homogeneous ovoid aggregates with co-existence
of smaller flocs and grazing protozoa. During the cohesion test,
the PSD clearly showed that the size of the aggregates remained
almost constant whatever the shear rate applied. The curves were
centred on a diameter of 80 ␮m with a marked tail on the left
which showing the existence of smaller particles (diameter around
10 ␮m). Thus there was no apparent aggregate break-up under the
different hydrodynamic conditions imposed in the stirred reac-
tor.
Hence, compared to the reference sludge, the resistance of
aggregates to mechanical stress was definitely much higher. This
means that strong aggregates were formed in the SBAR due to the
new growth conditions imposed. The size of the aggregates was no

day-71 day-161 day-212 day-260

10

6
% Vol

4
Fig. 3. Example of the cohesion test on a flocculated biomass from a purely aerobic
SBAR: (a) microscopic observation before cohesion test; (b) evolution of size distri- 3
bution during the shear test (cf. Fig. 1b for the colour legend). Kolmogorov microscale
2
for high ( ) and low ( ) rotation speed; (c) evolution of median diameter (d0.5 ) in
volume during the cohesion test.
1
3.2.2. Bioaggregate cohesion
0
During aerobic granular sludge formation, aggregate cohesion 1 10 100 1000
was assessed on different days in the stirred vessel. The charac- d ( m)
terisations were performed on samples collected on day-162 (i),
day-212 (ii) and day-260 (iii) (arrows in Fig. 4). The size distribu- Fig. 5. Evolution of PSD in the SBAR.
74 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78

Fig. 6. Variation of cohesion of aggregates during the formation of aerobic granules (cf. Fig. 1b for the colour legend of size distribution) and corresponding microscopic

observation (bar: 200 ␮m). Kolmogorov microscale for high ( ) and low ( ) rotation speed.

longer calibrated by the turbulence and stabilised well above the
Kolmogorov microscale (17 and 62 ␮m). This result demonstrates
that the initial step of granulation corresponds to an increase in
aggregate cohesion and it should be pointed out that the greatest
decrease of SVI was observed during this initial phase (from 150
to 50 mL g−1 ). At the same time, observations indicated that aggre-
gates became progressively more compact than the initial flocs (and
would probably show higher density).
On day-212 (Fig. 6b) the results suggested that two types
of particles co-existed and presented different behaviours with
respect to hydrodynamic strain. After sampling, the initial parti-
cle size distribution was clearly bimodal with two populations, one
around 50 ␮m (dmax1 ) and one around 210 ␮m (dmax2 ). As shown
by microscopic observation at this stage, large, dense aggregates
(100–600 ␮m) clearly co-existed with small flocculated particles
(10–100 ␮m). Fig. 7a shows the evolution of dmax1 (small aggre-
gates) and dmax2 (large aggregates) during the test. On the one
hand, the size dmax1 is controlled by the turbulence change, i.e.
the Kolmogorov microscale. Like the fragile flocs from the refer-
ence sludge (cf. Section 3.1), small aggregates seem to be broken
at high velocity gradient and re-flocculated at low velocity gradi-
ent, reaching the same size as at the beginning. On the other hand,
the value dmax2 is not significantly affected by modification of the
velocity gradient. These results indicate a second phase in the gran-
ulation process: during this phase, granules grow rapidly but small
fragile particles are still observed. This may be due either to the dis-
persed growth of flocs or to the detachment of fragile particles due
to surface erosion of granules. This phenomenon was specifically
 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78 75

Fig. 7. Evolution of dmax1 and dmax2 for the two respective peaks of the bimodal distribution during the cohesion test: (a) day-212; (b) day-260.

observed after an increase of organic loading rate (OLR) from 1.9 to
2.8 kg COD m−3 d−1 .
In the sample collected on day-260 (Fig. 6c), matured gran-
ules were observed (showing a dense internal core). The PSD again
shows a bimodal distribution. The new values of dmax1 and dmax2
indicate that both the small and large particles grew in size in
the SBR, respectively, to 150 ␮m and 900 ␮m. At this stage, dur-
ing the cohesion test performed in the stirred vessel, both dmax1
and dmax2 remained constant, independently of the velocity gradi-
ent, as shown in Fig. 7b. This means that neither the smaller nor the
larger aggregate population seems to be influenced by the hydro-
dynamic stress induced during the test. Moreover, the size of both
types of particles is much higher than the Kolmogorov microscale
imposed in the vessel (17–62 ␮m). Therefore, the two types of
bioaggregates were very cohesive. Actually, two hypotheses can
be proposed: small particles (still fragile on day-212) were either
lost or progressively became bigger from day-212 to day-260. The
latter phenomenon was probably encouraged in the SBAR because
little particle selection (no washout) was imposed. In our study,
as the settling time was 30 min, it seems reasonable to think that
it was sufficiently long to retain small particles in the bioreactor
and to make their growth possible with respect to their cohesion
properties. Due to this phenomenon, two types of aggregates still
coexisted in the reactor at this stage.
Consequently, these results illustrate a third phase of granule
formation: when the cohesion of all the particles became sufficient
to make them resistant to the hydrodynamic and mechanical strain,
particles could all grow above the turbulence micro-scale. Granules
progressively reached a size ranging from 500 to 1000 ␮m, which
is a common value for aerobic granules [5,43].
ticle volume and thus were not significantly observed in the volume
distribution. When focusing on the evolution of size distribution by
number (Fig. 9b), we note the effect of the increase of velocity gra-
dient: the distribution, originally monomodal (centred on 300 ␮m),
becomes bimodal when granules are subjected to a high rotation
speed (G = 3400 s−1 ). A new population of small particles centred
on a diameter (based on number) of about 50 ␮m appeared and,
even when the low rotation speed (G = 250 s−1 ) was applied again,
no reflocculation was observed. Here, it appears clearly that small
particles are detached from the surface of the granules and form
a new population of particles. It should be pointed out that these
new particles are strong enough to resist the micro turbulence as
they are not calibrated at the Kolmogorov scale. This behaviour is
clearly different from that observed for flocs (cf. Section 3.1). It leads
us to think that those detached particles probably contain dense
microcolonies from the surface of the granules and can constitute
interesting seeds for new granule development. However, more
work will be necessary to confirm this hypothesis and to determine
the microbial diversity in detached particles.
4. Discussion
4.1. Initial formation of granular sludge
Until now, few works have focused on the initial period of
granule formation. Usually, granules have been selected as rapidly

3.3. Final granular sludge

To characterise the granular sludge at steady state accurately,

after 500 days of operation, granules were collected and small par-
ticles were removed by sieving at 400 ␮m. Granules had a distinct
ovoid shape (Fig. 8) and a size from 400 ␮m to 1000 ␮m. The SVI5
and SVI30 of these granules were similarly measured at 20 mL g−1 ,
which is comparable to other studies with pure granule suspen-
sions [5].
As presented in Fig. 9, a cohesion test was performed on granule
suspension. The PSD by volume (Fig. 9a) brings out the high cohe-
sion of granules, the size of which remained almost constant during
the steps of high and low rotation speed. However, a population of
small particles (less than 90 ␮m) appeared at the end of phase II
(). These small particles contributed only slightly to the total par- Fig. 8. Final appearance of isolated granules after 500 days (bar = 400 ␮m).
76 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78
a Phase I Phase II Phase III
14
12
10
Vol (%)
8
6
4 6
2
0 0
1 10 100 1000
d (µm)
Fig. 9. Behaviour of isolated granules when subjected to the cohesion test: (a) PSD by volume; (b) PSD by number.
as possible by a short settling phase and authors have mainly
investigated the mature granules, looking for the best possi-
ble performance. Beun et al. [14] proposed a schematic diagram
illustrating different phases: pellet formation, growth with devel-
opment of filaments, detachment due to shear, growth of bacteria
embedded in polymers, lysis and growth of dense colonies. Our
study provides complementary information and demonstrates
that, whereas initial floc size is clearly calibrated by the Kolmogorov
microscale, aggregates become stronger and progressively resist
the hydrodynamic stress. From the results, successive steps in the
granulation process can be highlighted: (1) an initial increase of
aggregate cohesion associated with a rapid decrease of SVI, (2) a
growth of aggregates with temporary detachment of small fragile
particles, and (3) a growth of all the particles to much larger than
the turbulence micro-scale.
Granular sludge was previously defined as “a particle which can
settle rapidly and which does not coagulate under reduced shear
stress” [43]. In this definition high settling velocity is pointed out
(due to high density and size) but also the fact that granules are
stable particles which cannot agglomerate (unlike flocs). Here we
can introduce a new, complementary definition: granules are par-
ticles whose cohesion is sufficiently high to resist turbulence and
shear stress fluctuations. This high cohesion allows granule size to
increase to much higher than the turbulence microscale (whereas
floc size is controlled by the Kolmogorov scale).
Here, granular sludge was formed in an SBAR with a conven-
tional settling time, i.e. without particle washout. We suggest that
the modification of particle properties, i.e. higher strength and
higher density, which are linked to modifications of microbial
growth, initially explains the apparition of granules. In contrast, a
reduction in settling time, if applied, would probably only acceler-
ate the accumulation of granules. These data suggest that cohesion
is a property which should be considered when characterising gran-
ules and investigating aerobic granulation mechanisms. More work
is definitely necessary to understand the origin of this high cohe-
sion of granules. In this study, granules were obtained by imposing
an anoxic feast period and an aerobic famine period while the aer-
ation rate was relatively moderate. Therefore, it should be pointed
out that the initial increase of cohesion and decrease of SVI did not
follow an increase of shear stress in the reactor as often reported
in the literature but was linked to metabolic selection as proposed
by few authors [9,10,42]. However, the results obtained during the
cohesion test tend to confirm that an increase in hydrodynamic
b Phase I Phase II Phase III
20
18
16
14
Number (%)
12
10
8
4
2
1 10 100 1000
d (µm)
constraints in the SBAR could play a role in the erosion of aggregate
during granule growth.
It is still difficult to give the fundamental reason why cohe-
sion improved. One simple hypothesis is that this phenomena
results from densification, dense colonies having more bonding
interactions and thus being much difficult to break: Adav et al.
[44] demonstrated in pure culture that a high autoaggregation
index was a prerequisite for granule formation. It was assumed
in our work that nitrate diffusion inside aggregates (where oxygen
becomes limiting) could encourage aggregate densification but also
that anoxic carbon storage could improve the conversion of readily
biodegradable substrate into slowly biodegradable internal com-
pounds (see Wan et al. [42]) favouring aggregate stability by the
way of slow growth as already suggested [10]. Furthermore Liu et
al. [45] underlined that formation of granules is associated with a
sharp increase of hydrophobicity. Zhang et al. [46] also observed
an increase of surface charge and hydrophobicity during the for-
mation of aerobic granular sludge together with an increase of
protein/polysaccharide ratio: the increase in protein content was
found to decrease the surface negative charge of bacteria cells,
thus reducing the electrostatic repulsions and favouring bridging
between two neighbouring cells. As it is suggested in literature
that EPS production can explain the difference between the prop-
erties of flocs and granules [45,47,48], more works are currently
planned to evaluate the relation between cohesion of granules and
EPS properties. At present, it is still difficult to give a physico-
chemical explanation of the high cohesion of granules even though
experiments based on specific enzyme attacks tend to show that
either ␤-polysaccharides [49] or ␣-polysaccharides [48] could be
responsible for this characteristic.
4.2. Stabilisation of granular sludge
In a granular sludge system, aggregates can present heteroge-
neous retention times as smaller particles are preferentially lost
after settling, especially if short settling times are imposed. The
steady state is assumed to be reached when external data are sta-
bilised (size, MLSS concentration and activity). This was obtained
in our work after 360 days. It seems reasonable to think that the
diameter of aerobic granules stabilises when the growth rate is
counterbalanced by the detachment rate. Ren et al. [37] found gas
bubbles and particle collisions to be the main reason for the detach-
ment. Here, the cohesion test demonstrated that a high shear rate
 J. Wan et al. / Biochemical Engineering Journal 58–59 (2011) 69–78
generated the release of particles of about 50 ␮m. the dissipated
energy in the SBAR is expected to be lower (around 100 s−1 ) than
that obtained in the stirred vessel. However, Fig. 2a underlines that
similar result could have been obtained with a lower rotation speed
(kolmogorov microscale is around 23 ␮m for 1200 rpm). Further-
more, the solid concentration in the airlift reactor is twelve times
higher than the one used in the cohesion test. Thus the particle colli-
sions due to the high MLSS concentration could locally induce such
detachment, whose resulting small but cohesive particles possibly
constitute the seeds for the formation of new granules.
In long-term experiments, it could be expected that old gran-
ules would show lower cohesion because of deterioration of the
internal structure. Adav et al. [50] indeed observed that the cen-
tres of granules became porous, showing an empty internal heart
after long-term starvation. This was attributed to the fact that the
internal part of the aggregate (exopolymers) could be partly biode-
graded, i.e. used as a secondary substrate in such famine conditions.
Recently, Lemaire et al. [51] also observed large channels and holes,
which probably negatively affect granule cohesion, but these chan-
nels were partly filled by EPS.
In our work, no loss of cohesion was observed after 500 days
of operation. Basically, no significant rupture of granules occurred
during the test for a range of G between 250 s−1 and 3400 s−1 . The
occurrence of these mechanisms is probably linked to the sludge
retention time in the sequencing batch reactor and the duration
of the experiments. In our case, biomass was wasted with effluent
withdrawal (the upper fraction of sludge volume exceeding 50% of
the reactor). Calculations showed that the mean total SRT reached
40 days (it is possible that bigger granules were maintained in the
system longer because of their settling capacity). With these condi-
tions, granules size may be small enough to avoid too much transfer
resistance. Growth is then sufficiently possible in the core of the
aggregate to avoid (or slow down) a loss of integrity and a decrease
of aggregate strength. This is crucial information for possible indus-
trial applications of aerobic granular sludge as the stability of the
granule structure should be guaranteed in order to maintain high
performance.
5. Conclusion
The aggregate strength of flocculated sludge and granular sludge
were assessed in a stirred reactor, by varying the rotation speed.
Special attention was paid to the initial period of granular sludge
formation in a sequencing batch airlift reactor and some important
insights can be underlined:
• It is confirmed that high SAV and low settling time are not pre-
requisites to produce granules if proper metabolic selection (here
by alternating anoxic feast/aerobic famine), is applied.
• At the initial phase of granular sludge formation, an increase
of aggregate cohesion is first observed, whereas SVI decreases
rapidly; then aggregates resist high shear imposed in the stirred
cell.
• Then growth of aggregates is progressively observed. Small
particles are temporarily detached, indicating the role of hydro-
dynamics in particle erosion.
• As no aggregate selection was performed, bimodal particle size
distribution was observed, the small population of detached par-
ticles probably constituting the seeds for new granules. Granules
showed a high cohesion even after 500 days of operation.
• Tests performed on mature granules (diameter greater than
400 ␮m) indicate that high energy dissipation (ε ≈ 10 m2 s−3 )
leads to surface detachment of particles of about 50 ␮m, these
small particles also showing high cohesion.
77
These results show that granules can be defined as microbial
aggregates with a higher cohesion than flocs, which makes it pos-
sible for them to grow to a size much greater than the turbulence
micro-scale. This indicates that a key point in granulation is the
initial increase in aggregate cohesion, which was observed in the
present work after anoxic–aerobic alternance was imposed. Further
work will be necessary to understand the reasons for this cohe-
sion increase, in relation with the biological (formation of colonies)
and chemical (EPS quantity and properties) modifications occurring
inside aggregates.
Acknowledgements
This work was supported by the UT-INSA programme. It is part
of the Ph.D. research of Junfeng WAN. The authors also thank Aldo
Moro and Evrard Mengelle for their technical contributions to this
work.
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