Water Research 36 (2002) 1653–1665

Review
The essential role of hydrodynamic shear force in the
formation of biofilm and granular sludge
Yu Liu*, Joo-Hwa Tay
Environmental Engineering Research Centre, School of Civil and Structural Engineering, Nanyang Technological University,
50 Nanyang Avenue, Singapore 639798, Singapore
Received 13 October 2000; received in revised form 12 July 2001; accepted 31 July 2001

Abstract

Biofilm and granular sludge processes are promising biotechnology for wastewater treatment. The formation,
structure and metabolism of immobilized microbial community are associated very closely with hydrodynamic shear
force in reactors. Therefore, this paper attempts to review the essential role of shear force in the formation and
performance of biofilm and granular sludge. More compact, stable and denser biofilms, aerobic and anaerobic granules
form at relatively higher hydrodynamic shear force. It is clearly shown that shear force has significant influences on the
structure, mass transfer, production of exopolysaccharides, metabolic/genetic behaviours of biofilms, aerobic and
anaerobic granules. In an engineering sense, hydrodynamic shear force can be manipulated, as a control parameter,
to enhance microbial granulation process. It can be concluded that the knowledge regarding the effects of
hydrodynamic shear force on biofilms and granules is far from complete and much research is still needed to fully
understand the relevant mechanisms. Some of these future research niches are therefore outlined. r 2002 Elsevier
Science Ltd. All rights reserved.

Keywords: Biofilms; Aerobic granules; Anaerobic granules; Hydrodynamic shear force; Structure; Metabolism; Exopolysaccharides;
Flow pattern

1. Introduction biological treatment processes because a very high

The feasibility and efficiency of reactors based on the
biofilms, aerobic and anaerobic granular sludge for
removing biodegradable organic wastes from municipal
and industrial wastewater have been sufficiently demon-
strated [1–5]. Biofilms and granular sludge are dense,
multispecies, microbial communities. None of the
individual species in these microecosystems is capable
of completely degrading complex wastes. Complete
degradation of industrial wastes involves a complex
suite of interactions between the resident species. Thus,
biofilm and granular sludge reactors are desirable in
*Corresponding author. Tel.: +65-7906913; fax: +65-
7910676.
E-mail address: cyliu@ntu.edu.sg (Y. Liu).
biomass concentration can be reached in the treatment
systems. The high biomass concentration means that
contaminant transformation is rapid, highly concen-
trated or large volumes of waste can be treated in
reactors, which take up a small amount of space. In
granular sludge reactors, the large size and relatively
high density of individual granules have excellent
settleability as compared to conventional activated
sludge flocs [3,6–8]. The good settleability of the
granules would simplify the separation of purified
effluent from the granular sludge.
In biological reactors, detachment force resulting
from gas or liquid flow and particle–particle collision
is a key factor that influences the formation, structure
and stability of biofilms, aerobic and anaerobic granules.
In a biofilm system higher hydrodynamic shear force

0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 3 - 1 3 5 4 ( 0 1 ) 0 0 3 7 9 - 7
1654 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
results in a stronger biofilm, and biofilm tends to
become a heterogeneous, porous and weaker structure
when the shear force is too weak [1,9–12]. On the
other hand, there is evidence that a certain hydrody-
namic shear force is required in the formation
of aerobic and anaerobic granules. Absence of
granulation was observed when the shear force was
very weak [7,8,13–15]. These in turn display
the importance of hydrodynamic shear force in
microbial attachment and self-immobilization process.
However, the mechanisms by which hydrodyna-
mic shear force influences the formation, structure
and metabolism of biofilms and granular sludge
are not yet fully understood. Therefore, this paper
attempts to provide up-to-date information on the
role of hydrodynamic shear force in the formation
and performance of biofilm and granular sludge
in biological systems and further to discuss the
mechanisms by which microorganisms respond to shear
force and produce stronger biofilms and granular
sludge.
2. Microbial immobilization
2.1. Classification of cell immobilization
Cell immobilization technology has been used in
bioengineering and environmental engineering areas for
decades. In general, cell immobilization is known as
microbial aggregation, and can be roughly classified into
three categories:
(1) Biofilm. Microorganisms are immobilized or at-
tached onto a solid surface, such as activated
carbon, basalts, plastics, polymers, ceramics and
others [1,16–18].
(2) Microbial aggregates and granular sludge. So far,
aerobic and anaerobic granules have been success-
fully developed [3,7,8]. Microbial granulation can
be regarded as a self-immobilization community of
bacteria.
(3) Entrapped microorganisms. Microorganisms may be
entrapped in hydrophobic gels of photo-cross-
linked polymers or in other types of gels, such as
polyacrylamide [19,20].
To date, biofilm and granular sludge technologies
have been commonly employed in wastewater treatment
practice. A great number of biofilm reactors perform
organics removal, nitrification and denitrification, while
around 900 anaerobic granular sludge units have been
operated across the world [14]. On the contrary,
application of entrapped microorganisms in gels is very
limited due to its instability and short life cycle in
environmental engineering area [21].
2.2. Cell immobilization process
Biofilms and granular sludge indeed can be regarded
as different forms of cell immobilization. So far, it has
been recognized that the formation of biofilms and
microbial aggregates is a multiple-step process, to which
physicochemical and biological forces make significant
contributions [7,17,21–24]. Based on those previous
studies, it is encouraged to propose that cell immobiliza-
tion can be roughly described as a four-step process as
follows.
Step 1. Physical movement to initiate bacterium-to-
bacterium contact or bacterial attachment onto a solid
surface. The forces involved in this step are:
* hydrodynamic force;
* diffusion force;
* gravity force;
* thermodynamic forces, e.g. Brownian movement;
* cell mobility. Cells can move by means of flagella,
cilia or pseudopods. It has been shown that cell
mobility is important for both initial interaction with
the surface and movement along the surface [25].
Step 2. Initial attractive forces to keep stable bacteria-
solid surface and multicellular contacts. Those attractive
forces are:
* physical forces:
* Van der Waals forces;
* opposite charge attraction;
* thermodynamic forces including free energy of
surface, surface tension;
* hydrophobicity and
* filamentous bacteria that can link or bridge
individual cells together.
It should be emphasized that the hydrophobicity of
bacterial surface plays a crucial role in the initiation of
biofilms, aerobic and anaerobic granules [8,17,26–28].
According to the thermodynamics theory, increasing the
hydrophobicity of cellular surface would cause a
corresponding decrease in the excess Gibbs energy of
the surface, which in turn promotes cell-to-cell interac-
tion and further serves as a driving force for bacteria to
self-aggregate out of liquid phase (hydrophilic phase). In
this step, filamentous bacteria would assist in building
up a three-dimensional structure, which provides a
stable environment for the growth of attached bacteria.
* Chemical forces:
* hydrogen liaison;
* formation of ionic pairs;
* formation of ionic triplet;
* interparticulate bridge and so on.
* Biochemical forces
* cellular surface dehydration;
* cellular membrane fusion.
 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
There is evidence that cellular surface dehydration
and membrane fusion may play an important role in
initiating self-immobilization of anaerobic bacteria [27].
It seems that some environmental conditions would
induce cellular surface dehydration and further mem-
brane fusion [29,30].
Step 3. Microbial forces to make attached bacteria or
aggregated bacteria mature:
* production of extracellular polymer, such as exopo-
lysaccharides etc;
* growth of cellular cluster;
* metabolic change and genetic competence induced by
environment, which facilitate and further strengthen
the cell–cell interaction, and result in the high density
of adhering cells.
Step 4. Steady state three-dimensional structure of
microbial aggregate shaped by hydrodynamic shear
forces. The microbial aggregates would be finally shaped
by hydrodynamic shear force to form a certain
structured community. The outer shape and size of
microbial aggregates are finally determined by the
interactive strength/pattern between aggregates and
hydrodynamic shear force, microbial species and sub-
strate loading rate and so on.
It can be seen that for cells in a culture to aggregate or
attach to a solid surface, a number of conditions have to
be fulfilled. Among the factors involved in the formation
of biofilm and granular sludge, shear force has been
sufficiently demonstrated to play a major part
[7,12,13,24,31]. Therefore, in the limit of this review
the focus will be specifically put on the essential role of
hydrodynamic shear force in the formation of biofilms,
aerobic and anaerobic granules.
3. Effect of shear force on biofilms
3.1. Biofilm structures
In a biofilm system, hydrodynamic shear force may
result from liquid/air flow or particle-to-particle attri-
tion. Shear force has been considered as one of the most
decisive factors in the formation of biofilms under
hydrodynamic conditions. Steady state structures of
biofilms are highly dependent on the shear force, which
leads to the equilibrium biofilm thickness and density.
There is evidence that a higher shear force results in a
thinner and denser biofilm [1,12,18,32–34]. Fig. 1 shows
the effects of shear stress on biofilm density and
thickness observed in steady state fluidized bed reactor
[9]. It can be seen in Fig. 1 that biofilm density quasi-
linearly increases with the increase of shear stress, while
biofilm thickness exhibits a decreasing trend. Similar
phenomena were also reported by Ohashi and Harada
1655
80 40
Biofilm density (mg/cm )
3
Biofilm thickness (µ m)
60 30
40 20
20 10
0 0
6.5 7 7.5 8 8.5 9
2
Shear stress (dyne/cm )
Fig. 1. Effects of shear stress on biofilm density and thickness
in steady state fluidized bed reactor (data from Chang et al. [9]).
(K): density; (J): thickness.
[32] and Kwok et al. [1]. In addition, biofilm density was
found to correlate very closely with the self-immobiliza-
tion strength of fixed bacteria, which was determined by
the shear force imposed to the biofilms [10,32]. There-
fore, it appears that a certain shear force in the biofilm
system is necessary in order to produce a compact and
stable biofilm structure, i.e. higher shear force favours
the formation of a more smooth and denser biofilm.
Obviously, hydrodynamic-conditions-associated biofilm
structure in turn would influence the efficiency of
substrate diffusion and ecological selection in biofilms.
In an engineering sense, sloughing phenomena occur
more frequently with thicker biofilms, which results in
instabilities with respect to effluent quality, thus a
compact and stable biofilm is desirable. For achieving
such a purpose, hydrodynamic shear force in the reactor
seems to be an effective tool for manipulation of biofilm
structures.
3.2. Mass transfer
In fixed-film culture, hydrodynamic conditions are
usually considered in relation to mass transfer of
substrate, and substrate flux in biofilms is increased
with increasing hydrodynamic turbulence [33,35–38].
Although advanced tools for dynamic biofilm modelling
are available [39–41], the sensitivity of population
dynamics in biofilms to dynamic detachment mechan-
isms has not been thoroughly investigated even though
in wastewater treatment processes dynamic detachment
conditions are the general case.
To date, the diffusion-reaction theory of biofilm has
been most often employed in biofilm modelling as well
as in determination of mass transfer coefficient of
substance in biofilms. As pointed out earlier, a higher
shear force would lead to a more compact and denser
biofilm. It can be understood that the three-dimensional
structure of biofilms would have significant physical
1656 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
impacts on diffusion behaviours of substance in biofilms,
i.e. the diffusivity of a substance in biofilms would be
inversely related to biofilm density. However, the shear-
force-associated biofilm structure has not been seriously
taken into account in the current diffusion-reaction
theory. Although a large amount of diffusion coefficients
data determined by using the diffusion-reaction theory
are available in the literature, those data have poor
comparability [42–46]. Probably, this in part can be
explained by the shear-force-induced changes of biofilm
structures.
A biofilm with high density of bacteria can show a
surface reaction, which is not subject to the diffusion-
reaction theory [47–49]. Williamson and McCarty [50]
applied the diffusion coefficients determined by using
only particulate nitrifying biomass, filtered onto a
membrane, to verify the kinetic model of biofilm. It is
evident that filtered biomass does not have the same
three-dimensional structure as a grown biofilm under
hydrodynamic conditions; thus diffusion behaviours of
substrate in such artificial biofilms would not be
representative. Hence, it seems necessary to prudently
review once again the validity of the existing kinetic
models for biofilms, in which biofilm structure is a
neglected parameter.
Recent research has challenged the classical diffusion-
reaction theory. In their study on combined effects of
substrate strength and flow velocity on effective diffu-
sivity in open channel flow biofilm reactor, Beyenal and
Lewandowski [51] demonstrated that effective diffusiv-
ities increased with increase in glucose concentration,
but decreased with increase in flow velocity. High
effective diffusivities at high glucose concentrations
exhibit lower biofilm densities; on the other hand,
reduced effective diffusivities at high flow velocities
show higher biofilm densities [52]. Morgenroth and
Wilderer [53] further showed that not only the strength,
but also the mode of detachment should be taken into
account in mathematical modelling and laboratory
experiments. It should be realized that hydrodynamic
conditions indeed have a dual effect on the behaviours of
mass transfer in biofilms, i.e. high turbulence would
facilitate substrate diffusion in biofilms; however, shear-
force-enhanced biofilm density in turn reduces the
diffusivity of substrate in biofilms. The observed
diffusivity of substrate would be a net result of these
two phenomena. Therefore, the shear-force-related
structure of biofilm must be incorporated in future
modelling and optimal design of biofilm reactors
operated under different hydrodynamic conditions.
3.3. Exopolysaccharides production
Another important shear-force-associated phenomen-
on in biofilm culture is known as the overproduction of
extra-cellular polymers (ECPs), mainly consisting of
6.5
PS/PN (g/g)
5.5
4.5
4 8 12 16 20
Superficial air upflow velocity (m/h)
Fig. 2. Effects of superficial air upflow velocity on the ratio of
biofilm-PS to biofilm-PN in steady state three-phase fluidized-
bed reactor (data from Lertpocasombut [59]). PS: polysacchar-
ides; PN: proteins.
exopolysaccharides [10,32,54,55]. The exopolysacchar-
ides can mediate both cohesion and adhesion of cells,
and play a crucial role in maintaining structural integrity
of the biofilm matrix [56–58]. Fig. 2 shows the effect of
superficial air upflow velocity (a major hydrodynamic
shear force) on the ratio of biofilm-polysaccharides to
biofilm-proteins in steady state three-phase fluidized bed
reactor [59]. It can be seen that the content of biofilm-
polysaccharides is at least 4.5-fold higher than that of
biofilm-proteins, and higher superficial air upflow
velocity seems to stimulate the production of biofilm-
polysaccharides. It has been generally observed that
high shear force can induce the biofilms to secrete more
exopolysaccharides, which in turn would result in a
balanced biofilm structure under the given hydrody-
namic shear force [10,32,55]. In fact, during the early
stages of biofilm formation, a high exopolymer produc-
tion was observed, which would promote the initial cell
adhesion on the support surfaces [57,59,60].
Danese et al. [61] used a genetic approach to examine
the potential role of colanic acid, an exopolysaccharide
of Escherichia coli K-12 in biofilm formation, and they
found that colanic acid production is not required
for surface attachment. Rather, colanic acid is critical
for the formation of the complex three-dimensional
structure and depth of E. coli biofilms. Ohashi and
Harada [32] reported a linear relationship between
the content of exopolysaccharides in biofilm and biofilm
density. These clearly indicate that exopolysaccharides
in biofilms play a key role in building up and
maintaining the architecture of biofilms. It has been
hypothesized that a physical phenomenon rather than a
biological effect is responsible for the observed relation
between shear force and biofilm structure [18], but
the shear-force-triggered exopolysaccharides production
exhibits metabolic changes of biofilms, and a biological
 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
event must be involved. However, the mechanisms
by which hydrodynamic shear force stimulates the
production of polysaccharides are not yet clear in a
biological sense.
3.4. Energy metabolism
Similar to suspended microbial cultures, metabolic
network of bacterial film basically includes interrelated
catabolic and anabolic reactions. However, the relation-
ship between hydrodynamic shear force and energy
metabolism of biofilms has hardly been studied so far.
Lertpocasombut [59] investigated the kinetic behaviours
of biofilms developed in a three-phase fluidized-bed
reactor coupled to a quadrupole mass spectrometer, and
determined the ratio of specific dissolved oxygen (DO)
utilization rate (rDO ) to total organic carbon (TOC)
removal rate (rTOC ) at different superficial air upflow
velocities. The relationship between the superficial air
upflow velocity and rDO =rTOC ratio is thus presented in
Fig. 3. This figure indicates that much more DO was
consumed in oxidation of per unit TOC at higher
superficial air upflow velocity, i.e. much more dissolved
organic carbon would be converted to carbon dioxide
rather than for biosynthesis. Vanderborght and Gilliard
[98] also reported that up to 89% of the organic carbon
was oxidized to carbon dioxide in the same type of
reactor. The biochemical reactions associated with
bacterial metabolism result in an approximately linear
relationship between oxygen utilization and carbon
dioxide production, i.e. oxygen utilization and cell
production oppose each other, and the more oxygen
utilized in carbon dioxide production, the fewer cells
produced [62].
In aerobic oxidation process, the respiratory activity
of cells can be indirectly determined by the measurement
1 5
rDO/rTOC (mol/mol)
0.8
0.6
0.4
5 10 15
Superficial air upflow velocity (m/h)
Fig. 3. Effect of superficial air upflow velocity on the rDO =rTOC
ratio in steady state three-phase fluidized-bed reactor (data
from Lertpocasombut [59]).
1657
of the proton translocation activity, and the
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium
chloride (INT) can be used as artificial proton acceptor
to quantify the catabolic activity of biofilms. Fig. 4
shows that high hydrodynamic shear stress on
the biofilm leads to an increase of the specific INT–
dehydrogenase activity of biofilms. Similar results were
also obtained in a three-phase fluidized bed reactor [59].
These seem to suggest that biofilms might respond to
hydrodynamic shear force by regulating metabolic
pathways that determine the substrate flux flowing
between catabolism and anabolism.
The catabolic activity of microorganisms is directly
correlated with the electron transport system activity
[63,64]. In aerobic oxidation process the respiratory
activity of cells couples to the proton translocation
activity and a clear linkage of oxygen reduction to
proton translocation has been established [65].
The magnitude of catabolic activity would be propor-
tionally related to the activity of proton translocation
across cell membrane. Recent research on anaerobic
granulation showed that the proton-translocation-
induced dehydration of cell surface could facilitate
and strengthen the cell–cell interaction, and further
led to the high density of microbial community [27,29].
The proton-translocation–dehydration of cells has
been proposed to be a major mechanism responsible
for anaerobic granulation [27]. On the other hand,
it has been demonstrated that inhibition of energy-
generating function would prevent the development of
competence for cell aggregation in many systems [22,66].
It should be pointed out that the effects of hydro-
dynamic shear force on the energy metabolism of
biofilms are not yet completely understood, and relevant
information is very limited in the environmental
engineering literature.
INT-dehydrogenase activity (Abs./mg )
4
3
2
1
20 0
0.4 0.8 1.2 1.6
Tip velocity (m/s)
Fig. 4. Effect of shear force on the INT-dehydrogenase activity
of biofilm (data from Liu and Tay [97]).
1658 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
3.5. Molecular biology
In the past few years, much research progress has been
made in understanding the formation and development
of bacterial biofilms by using genetic and molecular
approaches. Researchers have begun to identify the
genetic components required for the formation of single-
species biofilms [54,61,66,67]. Danese et al. [61] exam-
ined the role of the outer membrane protein, antigen 43
in the formation of E. coli biofilm, and their results
showed that E. coli can use both common and specific
gene sets for the development of biofilms under various
growth conditions. O’Toole et al. [66] further reported
that the catabolite repression control protein that plays
a role in the regulation of carbon metabolism is
necessary for biofilm formation in Pseudomonas aerugi-
nosa. When Acinetobacter calcoaceticus BD413 cells in
monoculture biofilms were exposed to free DNA
solution, Hendrickx et al. [68] observed that the cells
lying on top of the biofilm that were subject to high
shear force displayed the highest transformation fre-
quencies in terms of the ratio of cells transformed to
recipient cells. In addition, Castellanos et al. [69]
reported changes in cell surface hydrophobic protein
induced by environmental conditions, which leads to the
primary phase of attachment of cells to surfaces. These
seem to indicate that transformation might be related to
culture conditions, and would play a major role in the
dissemination of genetic information in biofilms.
Microorganisms must often cope with stressful
environmental conditions, and they have developed
sophisticated cooperative behaviours and intricate com-
munication capabilities. By utilizing those capabilities,
microbial community may develop complex spatio-
temporal patterns in response to adverse growth
conditions [70]. Davies et al. [67] demonstrated that a
cell-to-cell signal is involved in the development of
Pseudomonas aeruginosa biofilms. It is known that
bacterial cell-to-cell communication relies upon the
interaction of a small diffusible signal molecule with a
sensor or transcriptional activator to couple gene
expression with cell population density [71]. As Watnick
and Kolter [72] pointed out, the bacteria in a multi-
species biofilm are not randomly distributed but rather
organized to best meet the needs of each. Similarly, in
the UASB granules, different groups of bacteria carry
out sequential metabolic processes [73]. In order to have
high metabolic efficiency, the granule-associated cells
would present in an organized structure, and signalling
mechanisms to organize the syntrophic species can be
predicted [74]. Quorum sensing is found to modulate
both intra- and inter-species cell-to-cell communication,
and plays a central role in enabling bacteria to construct
complex community structures [75]. It has been pro-
posed that biofilm formation can proceed through
multiple, convergent signalling pathways, which are
regulated by various environmental signals [76]. In order
to be more resistant to the stressful environmental
conditions, such as hydrodynamic shear force, heat,
cold, starvation, pH and so on, biofilm-associated cells
would have to adapt their behaviours and pattern of
gene expression as a function of the environmental
situation. In this aspect, biofilm-specific exopolysacchar-
ides and quorum-sensing specific effects may be the
reason for such environment-induced resistance. Sus-
pended cells indeed can respond to a variety of
environmental stresses by altering their metabolism
and genetic expression [77–79]. However, little informa-
tion is currently available for possible shear-force-
induced genetic/molecular changes of multiple species
biofilms, and further investigation is needed.
4. Effect of hydrodynamic shear force on aerobic
granulation
Recently, research efforts had been turned towards
developing aerobic granules in upflow sequencing batch
reactors (USBR) [7,8,24,80–82]. As compared to con-
ventional bioflocs, the advantages of aerobic granules
can be seen as good settling ability, high retainable
biomass concentration and ability to withstand high
organic loading rate. Some characteristics of aerobic
granules cultivated in USBR are summarized in Table 1.
4.1. The formation and structure of aerobic granules
Aerobic granulation can be regarded as a self-
immobilization process. Similar to the formation of
biofilms, hydrodynamic shear force would have a
significant influence on the formation and structure of
aerobic granules. Tay et al. [8] reported that when the
USBR operated at a low superficial air upflow velocity
0.008 m/s, no granules were observed in the system, but
only fluffy flocs (Fig. 5a). On the contrary, regular-shape
granules were successfully developed in the USBR
operated at a high superficial air velocity of 0.025 m/s
(Fig. 5b). Beun et al. [7] also observed that a low
superficial air velocity did not lead to the formation of
stable aerobic granules in USBR; however, at a
relatively high superficial air velocity, granulation
occurred and because of the high shear strength smooth,
dense and stable aerobic granules formed. In their study
on aerobic granulation in oxygen aerobic upflow sludge
bed reactor, Shin et al. [84] concluded that the
granulation was governed by the physical stress on
granular sludge. These results seem to indicate that
aerobic granulation would be a phenomenon associated
very closely with the hydrodynamic conditions present
in reactors.
The specific gravity of sludge and sludge volume index
(SVI) represent the compactness of a microbial structure.
 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665 1659

Table 1
Some characteristics of aerobic granules

SVI (ml/g) Settling velocity Diameter SOUR Specific gravity Reactor type References
(m/h) (mm) (mg O2/g h) (g/ml)

40.8–143 2.0–8.0 AUSBa reactor [83]

86.4 0.5–2.5 AUSBa reactor [84]
30–40 2.35 96.3 SBR [81]
80–100 0.3–0.5 SBR [82]
20–45 0.5–1.6 76.2 1.0068–1.0072 SBR [85]
1.9–4.6 SBR [7]
>16.2 around 1.0 SBR [86]
50–85 30–35 1.1–2.4 55.9–69.4 SBR [8]
72 3.0 1.040–1.054 SBR [80]
a
Aerobic upflow sludge blanket.

Fig. 5. (a) Bioflocs cultivated at a superficial air upflow velocity of 0.008 m/s; and (b) granules formed at a superficial air upflow
velocity of 0.025 m/s in USBR (from Tay et al. [8]).

Tay et al. [24] studied the effects of hydrodynamic shear 200 1.010
force on the structure of aerobic granules developed in

Specific gravity (kg/l)

USBR. In USBR, superficial air upflow velocity may act 150
1.008

as a major hydrodynamic shear force exerted on

SVI (ml/g)

1.006
bacterial community. It was found that the specific
100
gravity of aerobic granules increased with the increase of
1.004
hydrodynamic shear force, while the SVI decreased from
180 to 40 ml/g (Fig. 6). It must be pointed out that at a 50
1.002
superficial air upflow velocity of 0.3 cm/s, aerobic
granulation did not occur, and only conventional 0 1.000
bioflocs similar to Fig. 5a were developed. As compared 0.0 1.0 2.0 3.0 4.0
to those bioflocs, aerobic granulation significantly Superficial air upflow velocity (cm/s)
improves the settleability of sludge. Figs. 1 and 6 clearly
indicate that high hydrodynamic shear force would Fig. 6. Effects of superficial air upflow velocity on the specific
favour the formation of more compact and denser gravity and SVI of aerobic granules developed in USBR [24].
(K): SVI; (J): specific gravity.
aerobic biofilms and granules. In fact, higher granule
density associated with a lower SVI can ensure a more
efficient biosolid–liquid separation, which is important 4.2. Cellular polysaccharides
in wastewater treatment systems for the successful
operation of the process and production of high-quality The shear-force-associated production of cellular
effluent polysaccharides has been discussed earlier for biofilm
1660 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
16
PS/PN (mg/mg)
12
8
4
0 16
0.0 1.0 2.0 3.0
Superficial air upflow velocity (cm/s)
Fig. 7. Effects of superficial air upflow velocity on the PS/PN
ratio and SOUR of aerobic granules [24]. (K): PS/PN; (J):
SOUR.
system. Recent studies by Tay et al. [24] showed that
superficial air upflow velocity in USBR had a profound
influence on the production of cellular polysaccharides
(Fig. 7). It can be seen that the ratio of sludge-
polysaccharides (PS) to sludge-proteins (PN) increases
significantly with the increase of hydrodynamic shear
force. A similar phenomenon was also observed in a
biofilm system (Fig. 2). It is worth pointing out that the
content of polysaccharides is much higher than the
content of proteins in granular sludge. Vandevivere and
Kirchman [87] also found that the content of exopoly-
saccharides normalized to proteins was 5-fold greater
for attached cells than for free-living cells. These in turn
imply that the polysaccharides would highly contribute
to the attachment and self-immobilization processes of
bacteria, and the contribution of cellular proteins to the
structure and stability of granule-associated bacteria
would be less important. In fact, it was shown that the
disappearance of aerobic granules in USBR was tightly
coupled to a drop of cellular polysaccharides [88]. It
seems that high hydrodynamic shear force could induce
both aerobic granules and biofilms to secrete more
cellular polysaccharides, which would lead to a balanced
microbial structure of aerobic biofilms and granules
under the given hydrodynamic conditions, i.e. the role of
cellular polysaccharides is crucial in maintaining the
stability of aerobic biofilms and granules.
4.3. Microbial activity
In the environmental engineering field, microbial
activity may be characterized by the specific oxygen
utilization rate (SOUR) in terms of mg oxygen
consumed by mg cell proteins per hour. The influence
of hydrodynamic shear force on the SOUR of aerobic
granules is shown in Fig. 7 [24]. The SOUR of aerobic
granules quasi-linearly increased with the increase of
hydrodynamic shear force in terms of superficial air
upflow velocity. It is obvious that the increased
26 hydrodynamic shear force somehow could stimulate
the respiration activity of granules. Figs. 3, 4 and 7
SOUR (mgO2/mg h)
24 suggest that aerobic biofilms and granules can metabo-
lically respond to hydrodynamic shear force, and those
22
metabolic changes in turn should favour the formation
20 of a strong and stable microbial community against the
stressful hydrodynamic conditions.
18
5. Effect of hydrodynamic shear force on anaerobic
4.0
granulation
5.1. General consideration
In the past twenty years, the feasibility and efficiency
of upflow anaerobic sludge blanket (UASB) technology
for removing high-strength biodegradable organic
wastes from industrial wastewater has been sufficiently
demonstrated [3,5,13]. To date, more than 900 UASB
units are operating all over the world [14]. Granular
sludge is the main prominent characteristic of UASB
reactors as compared to other anaerobic technologies,
and the performance of the UASB systems is strongly
dependent upon granulation process with a particular
organic waste. In the UASB reactor, anaerobic bacteria
form granules through self-immobilization of cells.
Anaerobic granules have advantages of keeping high
biomass hold-up, microbial activity and good settle-
ability. It should be pointed out that as compared to
aerobic biofilm and granular sludge processes, little is
known of the role of hydrodynamic shear force in the
formation of anaerobic granules.
5.2. Formation of anaerobic granules in UASB
In UASB reactors, hydrodynamic shear force is
mainly due to liquid upflow velocity and gas production.
Anaerobic granulation proceeds well at relatively high
liquid upflow velocity, while absence of granulation at
weak shear force has been reported [13–15,89]. Alphe-
naar et al. [13] observed that the granulation process in
UASB reactor was favoured by the combination of high
liquid upflow velocity and short hydraulic retention time
(HRT). In fact, the liquid upflow velocity and HRT are
interrelated. So far, the effects of liquid upflow velocity
on anaerobic granulation have been explained by the
selection pressure theory [90]. The selection pressure
may result from any environmental condition, e.g.
temperature, HRT, upflow velocity, organic loading
rate, pH, reactor configuration and seed sludge and so
on. A low liquid upflow velocity may allow dispersed
bacterial growth and would be less favourable for
microbial granulation. A relatively high liquid upflow
velocity could cause washout of dispersed bacteria and
those bacteria competents for aggregation would be kept
in the reactor.
 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
5.3. Rapid production of anaerobic granules
It appears from the above discussion that the liquid
upflow velocity has a pronounced effect on the granula-
tion process in UASB reactors. Thus, research attempts
have been made to develop a strategy for expediting
granulation process by controlling hydrodynamic shear
force in UASB reactors. Noyola and Moreno [6]
conducted a series of experiments to investigate the
effect of liquid upflow velocity on the formation of
anaerobic granules in UASB reactors. Experiments
showed that flocculant anaerobic sludge could be
converted to a relatively active anaerobic granular
sludge by enhancing agglomeration with only hydro-
dynamic stress in a very short time, less than 8 h. It can
be seen in Fig. 8 that settleability of those anaerobic
granules in terms of SVI and sludge settling velocity
were significantly improved as the liquid upflow velocity
increased, and the increased settleability of granules in
turn reduced washout rate of sludge from 46% to 2%.
These results suggest that the formation of anaerobic
granules could be enhanced through a purely physical
aggregation resulting from the hydrodynamic stress
applied to the anaerobic flocculant sludge by increasing
the liquid upflow velocity.
One major problem encountered with UASB reactor
is the long startup period required for the development
of anaerobic granules since anaerobes are slow-growing
bacteria. In cases where the inoculation is done with
municipal digester sludge, it usually takes 2–8 months
before the process can be put in operation. In view of the
long startup period, enhanced granule formation as
demonstrated by Noyola and Moreno [6] is highly
desirable in order to reduce space-time requirements of
various bioreactors leading to cheaper treatment of
high-strength wastes. The improvements can also lead to
better treatment efficiency with greater capacity to
handle large volumes of wastewater with more compact
55
50
SVI (ml/g)
45
40
35
30
0 20 40 60
Liquid upflow velocity (m/h)
Fig. 8. Effect of liquid upflow velocity on the formation of
anaerobic granules in UASB reactors (data from Noyola and
Moreno [6]). (K): SVI; (J): settling velocity of sludge.
1661
design. It is therefore possible to economize on the
capital investment and subsequent cost of operation.
Use of granular sludge as the seed material in operating
UASB reactors has the advantage of being able to
achieve high organics removal within a short startup
period. However, the availability of granular seed sludge
is limited and the expenses for purchase and transporta-
tion of the inoculum are extremely high. Consequently,
technology for enhanced and fast production of
anaerobic granules is strongly needed and would be
beneficial to wastewater treatment industry.
5.4. Formation of anaerobic granules in other types
of reactors
Anaerobic granular growth has been sufficiently
described for UASB systems but very limited informa-
tion is available for other types of bioreactors.
Vanderhaegen et al. [91] developed anaerobic granules
in stirred reactors, but they found that granular sludge
disappeared within 3 weeks when the reactors were
incubated statically instead of being shaken. The study
of Vanderhaegen et al. [91] in fact provides experimental
evidence showing the importance of hydrodynamic
shear force in maintaining the integrity of granular
sludge. It should be pointed out that flow patterns in
UASB and stirred reactor are different. The experiments
by Vanderhaegen et al. [91] challenge the general belief
that upflow pattern is necessary in order to develop
granular methanogenic sludge. In addition, Pereboom
and Vereijken [92] cultivated methanogenic granules in
full-scale internal circulation reactors (ICR), in which
the average shear rate was approximately two-fold
higher than in UASB reactors.
6. Effect of flow pattern on granulation
So far, nearly 100% of aerobic and anaerobic granules
are produced in column-type air or liquid upflow
reactors, and a high ratio of reactor height to diameter
18 seems to favour the formation of granular sludge. As
Settling velocity of sludge
discussed earlier, the feasibility and efficiency of other
types of bioreactors, such as completely mixed tank
12
reactor (CMTR) in development of aerobic and
(m/h)
anaerobic granular sludge have not been sufficiently
demonstrated in full-scale operation practice. The air or
6
liquid upflow velocity in column reactors has been most
commonly described by its strength [7,13,24,90]. How-
0
ever, the contribution of flow pattern in reactor to
granulation process has hardly been considered in the
environmental engineering literature.
In a hydrodynamic sense, column-type upflow reactor
and CMTR have very different hydrodynamic beha-
viours in terms of interactive patterns between flow and
microbial aggregates. The air or liquid upflow pattern in
1662 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
(a) Circular movement of an aggregate
Fig. 9. Flow patterns in upflow column reactor and CMTR.
column reactors can create a relatively homogenous
circular flow along the reactor height, and microbial
aggregates are constantly subject to such a circular
hydraulic attrition (see Fig. 9a). According to the
thermodynamics, the circular flow could force microbial
aggregates to be shaped as regular granules that have a
minimum surface free energy, provided those aggregates
could be kept in the reactors under given dynamic
conditions. Thermodynamically, such a phenomenon is
very similar to the formation of benthic round-shape
boulders in a natural flowing river system. It is obvious
that in a column-type upflow reactor a higher ratio of
reactor height to diameter can ensure a longer circular
flowing trajectory, which in turn creates a more effective
hydraulic attrition to microbial aggregates. However, in
CMTR microbial aggregates stochastically move with
dispersed flow in all directions. Thus, microbial aggre-
gates are subject to varying localized hydrodynamic
shear force, flowing trajectory and random collision
(Fig. 9b). Under such circumstances, only flocs of
irregular shape and size instead of regular granules
occasionally form, and this is exactly like what happens
in a conventional activated sludge aeration tank, which
is a typical CMTR. The operation practice of conven-
tional activated sludge process supports the above
analysis because microbial granulation has hardly been
reported in CMTR in the past one hundred years of
operation practice.
It seems certain that not only the strength of
hydrodynamic shear force, but also the interactive
pattern between flow and microbial aggregates have
effects on the formation of granular sludge. In this
aspect, the column-type upflow reactor with high ratio
of reactor height to diameter can provide an optimal
interactive pattern between flow and microbial aggre-
gates for granulation. This may be a major reason why
almost 100% of granular sludge only forms in column-
type upflow reactors. In an engineering sense, the
desirable interactive pattern between flow and aggre-
gates might be achieved by controlling reactor config-
urations and operation strategy. Consequently, a better
understanding of the role of flow pattern in granulation
(b) Stochastic movement of an aggregate
process would lead to development of novel types of
granular sludge reactor.
7. Is granulation a general phenomenon?
Granulation by different species such as methanogens,
acidifying bacteria, nitrifying bacteria, denitrifying
bacteria and aerobic activated sludge [8,82,91,93–95]
has been reported. Calleja [22] proposed that aggrega-
tion is a fundamental condition, function, and structure
in biology, and cell aggregation can be defined as the
gathering together of cells to form a fairly stable,
contiguous, multicellular association under physiologi-
cal conditions. So far, various researchers have described
the resulting structures of microbial aggregation with
different terminologies, e.g. film, floc, pellet, pellicle,
cluster, slim, granule and so on. As discussed earlier,
almost all aerobic and anaerobic granules are developed
in air or liquid upflow bioreactors; on the other hand,
microbial granulation seems to never be reported in the
conventional activated sludge process. It appears from
the earlier discussion that microbial granulation is not
strictly restricted to some specific species, e.g. methano-
gens, and would be a phenomenon associated with the
operation conditions and reactor configurations. How-
ever, the contribution of microbial and genetic factors to
granulation should also be taken into account.
The information for microbial aggregation may reside
in the genetic makeup of the microorganisms involved
[22,54,69,96]. This could be the major reason why within
a species, strains differ in their capacity for aggregation,
and some species are more competent for aggregation,
but some are less under the same operation conditions.
As Calleja [22] pointed out, ‘‘the deposition of structural
and regulatory genes may determine whether the
aggregation function of cells is constitutive or induci-
ble’’. If the capacity for aggregation is constitutive, i.e.
whatever the cell is in regard to its cell cycle or its life
cycle, aggregation will be present, provided the environ-
mental conditions allow it to occur. On the contrary, if it
is inducible, then it will be present only when the cells
 Y. Liu, J.-H. Tay / Water Research 36 (2002) 1653–1665
are physiologically competent. So far, there is no
evidence to show that microbial granulation is consti-
tutive or inducible in the environmental engineering
literature. As discussed earlier, the formation of
granular sludge is dependent on the reactor configura-
tion, operation mode and cell metabolism other than
species. Thus, a prudent hypothesis is that granulation is
governed by both microbial constitutive and inducible
capabilities, such as species, physical and physiological
conditions of a culture. It must be realized that gross
engineering events in relation to microbial granulation
can be easily identified; however, the events at molecular
or genetic level still require a more profound under-
standing of the mechanisms of granulation.
8. Summary
This paper shows that hydrodynamic shear force
plays a crucial role in the formation of biofilm, aerobic
and anaerobic granules. It appears that shear force
influences not only the structures, but also metabolic
and kinetic behaviours of biofilms and granular sludge.
However, future research is required to provide quanti-
tative understanding of the following aspects:
* Mechanisms by which hydrodynamic shear force
influences the formation, structures and metabolic
behaviours of biofilm and granular sludge in
molecular and genetic levels of cells must be well
understood.
* It is necessary to know how to incorporate the shear-
force-associated biofilm structure and metabolism
into kinetic models of biofilm.
* Almost 100% of aerobic and anaerobic granular
sludge reactors operate in a gas or liquid upflow
mode. There is very limited information on microbial
granulation in other types of bioreactors. The role of
interactive pattern between shear force and microbial
aggregates should be described in future research.
* In biofilm and granular sludge processes shear force
and growth force represented by sludge loading rate
are two key factors that would govern the structure
and performance of steady state immobilized micro-
bial community. Previous research showed that high
shear force promoted the formation of high-density
biofilm and granules; on the contrary, the strengths
of biofilm and granules were lowered as the sludge
loading rate was increased [12,92]. Thus, the com-
bined effects of hydrodynamic shear force and
growth force on the formation of biofilm and
granular sludge need to be investigated, and the
optimal combination between shear and growth
forces for the formation of stable biofilm and
granular sludge should be outlined.
* Further research is also needed to develop an
operation strategy for expediting granulation process.
1663
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