Appl Microbiol Biotechnol (2001) 57:227–233
DOI 10.1007/s002530100766
O R I G I N A L PA P E R
J.-H. Tay · Q.-S. Liu · Y. Liu
The effects of shear force on the formation,
structure and metabolism of aerobic granules
Received: 5 March 2001 / Received revision: 6 June 2001 / Accepted: 17 June 2001 / Published online: 25 July 2001
© Springer-Verlag 2001
Abstract The effect of shear force on aerobic granulation was studied in four column-type, sequential aerobic
sludge blanket reactors. Hydrodynamic turbulence caused
by upflow aeration served as the main shear force in the
systems. Results showed that aerobic granulation was
closely associated with the strength of shear force. Compact and regular aerobic granules were formed in the reactors with a superficial upflow air velocity higher than
1.2 cm s–1. However, only typical bioflocs were observed in the reactor with a superficial upflow air velocity of 0.3 cm s–1 during the whole experimental period.
The characteristics of the aerobic granules in terms of
settling ability, specific gravity, hydrophobicity, polysaccharide and protein content and specific oxygen utilization rate (SOUR) were examined. It was found that the
shear force has a positive effect on the production of
polysaccharide, SOUR, hydrophobicity of cell surface
and specific gravity of granules. The hydrophobicity of
granular sludge is much higher than that of bioflocs.
Therefore, it appears that hydrophobicity could induce
and further strengthen cell–cell interaction and might be
the main force for the initiation of granulation. The
shear-stimulated production of polysaccharides favors
the formation of a stable granular structure. This research provides experimental evidence to show that
shear force plays a crucial role in aerobic granulation
and further influences the structure and metabolism of
granules.
Introduction
The feasibility and efficiency of cell immobilizationbased reactors for removing biodegradable organic matter, nitrogen and phosphate from municipal and industriJ.-H. Tay · Q.-S. Liu · Y. Liu (✉)
Environmental Engineering Research Center,
School of Civil and Structural Engineering,
Nanyang Technological University, 50 Nanyang Avenue,
Singapore 639798
e-mail: cyliu@ntu.edu.sg
al wastewater have been shown (Lazarova and Manem
1994; Rusten et al. 1998; Beun et al. 1999). The performance of an immobilized-cells system for wastewater
treatment greatly depends on the active biomass concentration, the overall biodegradation rates, the reactor configuration and the feeding rates of the pollutants and dissolved oxygen. An upflow anaerobic sludge blanket
(UASB) bioreactor using anaerobic granules is one of
the best known self-immobilization processes and has
been extensively applied to anaerobic wastewater treatment. A very comprehensive literature has been documented for anaerobic granular sludge, both in microbiology and in engineering. Also, granulation by methanogens (Lettinga et al. 1984), acidifying bacteria (Vanderhaegen et al. 1992), nitrifying bacteria (De Beer et al.
1993; Van Benthum et al. 1996) and denitrifying bacteria
(Van der Hoek 1988) have been reported. Over the past
few years, research attention had turned towards developing aerobic granular sludge (Beun et al. 1999; Peng
et al. 1999). As compared with conventional activated
sludge flocs, the advantages of aerobic granular sludge
are the regular, dense and strong microbial structure,
good settling ability, high biomass retention and ability
to withstand a high organic loading rate.
In a biological reactor, the shear force resulting from
hydraulics and/or particle–particle collision is a key factor that influences the formation, structure and stability
of the cell-immobilization community, such as biofilm
and anaerobic granules under hydrodynamic conditions.
In biofilm systems, a higher shear force would result in a
stronger biofilm; and a biofilm tends to become a heterogeneous, porous and weaker structure when the shear
force is weak (Chang et al. 1991; Chen et al. 1998;
Kwok et al. 1998). These reports state that shear force
may play an important role in cell-immobilization systems. However, it should be pointed out that little information is currently available on the essential role of
shear force in the formation of aerobic granules. Therefore, this research attempts to study the effect of shear
force on the formation, structure and metabolism of aerobic granules. This work is expected to be useful for a
228
better understanding of the mechanisms responsible for
aerobic granulation.
tract was recovered by centrifugation at 38,000 g for 30 min at
4 °C. The extract was used for protein analysis, using a Bicinchonic acid kit (Sigma, USA) according to the manufacturer’s
instruction.
Materials and methods
Experimental set-up
Four columns (80 cm high, 60 mm in diameter) with a working
volume of 2.3 l were used as sequential aerobic sludge blanket
reactors during the study, each with the same geometrical configuration. In this experiment, reactors 1–4 (R1, R2, R3, R4) were
supplied with an air velocity of 0.5 l min–1, 2.0 l min–1, 4.0 l min–1
and 6.0 l min–1, respectively. This resulted in a superficial upflow
air velocity of 0.3 cm s–1, 1.2 cm s–1, 2.4 cm s–1 and 3.6 cm s–1 for
reactors 1–4, respectively. All reactors were operated sequentially:
5 min of influent filling, 225 min of aeration, 5 min of settling and
5 min of effluent withdrawal. Effluent was discharged at the middle port of the column. A substrate loading rate of 6.0 kg chemical
oxygen demand(COD) m–3 day–1 was applied. The experiments
were conducted in a temperature-controlled room at 25 °C. After
experimental startup, the dissolved oxygen (DO) concentration
was monitored once or twice a week for one whole cycle, for all
four reactors. The lowest DO (1.5 mg l–1) was recorded in R1, and
3.0 mg l–1 in the other three reactors.
Media
Four reactors were started up using 650 ml of sludge acclimatized
for 1 week by acetate substrate in a batch mode. The composition
of synthetic wastewater used for this study mainly consisted of sodium acetate as sole carbon source and other necessary elements.
Details of the composition can be found elsewhere (Tay and Yan
1996).
Analytical procedures
An effluent sample was analyzed for COD and a sludge sample for
mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids, sludge volume index (SVI), specific oxygen utilization rate (SOUR) and specific gravity, following standard methods
(APHA 1995). Granule size was measured by a laser particle size
analysis system (Malvern MasterSizer 2600), or an image analysis
system (Quantimet 500, Leica Cambridge Instruments). Microbial
observation was conducted using either microscopy or image analysis (IA), while the microbial structure of granules was examined
with a scanning electron microscope (SEM; Stereoscan 420, Leica
Cambridge Instruments). The granule samples were gently washed
with phosphate buffer solution and allowed to settle naturally.
Granules then were fixed with 4% paraformaldehyde and left for
4 h. The fixed granules were dehydrated by successive passages
through 40, 60, 80 and 100% ethanol. Then they were dried either
by Freeze Dryer or Critical Point Dryer (model E3000) and finally
observed by SEM. Cell hydrophobicity was determined with the
method described by Rosenberg et al. (1980). Hexadecane
(0.25 ml) was used as the hydrophobic phase. Hydrophobicity was
expressed as the percentage of cells adhering to the hexadecane
after 15 min of partitioning.
The cells in a 5-ml sample were harvested by centrifugation.
The recovered cells were then resuspended in 1 M NaOH solution
and heated in an oven at 80 °C for 30 min. The extract was recovered by centrifugation at 4,000 g for 10 min and was then used to
determine cell polysaccharides, using the method developed by
Dubois et al. (1956). In order to analyze the content of cell proteins, flocs or granules sample were harvested by centrifugation.
The harvested sludge was resuspended in an equal volume of sample treatment buffer (0.0625 M Tris HCl buffer, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol) and
was heated at 100 °C for 10 min (Alexander et al. 1984). The ex-
Results
In the experiments, four reactors (R1–R4) were operated
at different superficial upflow air velocities, ranging
from 0.3 cm s–1 to 3.6 cm s–1. In a column-type reactor,
the superficial upflow velocity of air is a major cause of
hydrodynamic turbulence and further hydraulic shear
force.
Effect of shear force on the formation
of aerobic granules
The seed sludge used in this work had a mean floc size of
0.12 mm. The evolution of sludge morphology during
operation was monitored using an IA technique. After
7 days of operation, tiny granules with a mean diameter
of 0.28–0.37 mm appeared in R2, R3 and R4, which were
operated at high superficial air velocities. However, there
was no significant improvement in sludge morphology in
R1, which was operated at the lowest superficial air velocity. IA photos for all reactors on day 3 and day 11 are
shown in Fig. 1. On day 3, only irregular and loose-structured bioflocs were observed in all four reactors
(Fig. 1A). On day 11, round-shaped granules with a
clear outer shape were formed in R2, R3 and R4, but no
granular sludge was observed in R1 (Fig. 1A–E). It
appears from Fig. 1 that a certain shear force is necessary
for aerobic granulation; and high shear force seems to
favor the formation of more regular and compact granules. The detailed microstructure of the aerobic granules
taken from R4 was further examined using SEM
(Fig. 1F). It can be clearly seen that the granules have a
very compact bacterial structure, in which cells are tightly linked together, and a rod-like species is predominant.
A similar microstructure was also found in R2 and R3.
Effect of shear force on retainable biomass in reactor
After 2 weeks of operation, all four reactors reached a
steady state, indicated by stable granular sludge/bioflocs
concentrations and constant COD removal efficiencies.
Figure 2 shows the relationship between the stable biomass concentration in the reactor and shear force. A stable biomass concentration of 5.4, 6.5 and 6.9 g MLSS l–1
was achieved in R2, R3 and R4, respectively. However,
only 1.4 g MLSS l–1 was obtained in R1, which is within
the range of biomass concentration in a conventional activated sludge process. Together with Fig. 1, Fig. 2 suggests that the formation of compact, dense aerobic granules would lead to a high reactor biomass concentration.
In addition, the COD removal efficiencies in the four reactors were 94–96%.
229
Fig. 1 Image analysis photographs of bioflocs/granules on day 3
(A reactor 1; R1) and day 11 (B R1, C R2, D R3, E R4) Bar
2 mm. F scanning electron micrograph of granule taken from
R4 on day 11.
Effect of shear force on settling ability
The seed sludge used had a SVI value of 205 ml g–1. It
decreased in R2, R3 and R4 after startup. After the formation of granular sludge, the SVI decreased to a stable
value (62, 55 and 46 ml g–1 in R2, R3 and R4 respectively; Fig. 3). However, a SVI value of 170 ml g–1 was obtained during steady state in R1, in which granulation did
Fig. 2 Biomass retained in the steady-state reactor operated at different superficial upflow air velocities
230
Fig. 3 Sludge volume index (SVI) and specific gravity of sludge
versus superficial upflow air velocity. Black circles SVI, white
circles specific gravity
Fig. 4 Comparison of cell surface hydrophobicity before (white
bars) and after (dark bars) granulation at different superficial upflow air velocities
not occur. Figure 3 clearly shows that granulation of aerobic sludge can significantly improve the sludge settling,
as compared with conventional bioflocs. It seems certain
that the shear force-associated granulation is responsible
for the observed sludge settling shown in Fig. 3.
75.9% in R2. These results indicate that the hydrophobicity of cells in R2, R3 and R4 after granulation is around
50% higher than in the period before granulation. However, there is little change in cell surface hydrophobicity
in R1, in which no granulation was observed during the
whole experimental period. Therefore, it appears that the
formation of aerobic granules is coupled to an increase
in the hydrophobicity of the cell surface.
Effect of shear force on specific gravity
The specific gravity of sludge represents the compactness of a microbial community. Figure 3 shows the effect
of shear force on the specific gravities of bioflocs and
granules observed during steady state in R1–R4. As
pointed out earlier, typical bioflocs were predominant in
R1 and granular sludge prevailed in R2–R4. Figure 3
clearly shows that the specific gravity of granular sludge
is much higher than that of bioflocs. It was also found
that the specific gravity increased with the increase in
shear force in a relatively significant way: that is, higher
shear force led to more compact and denser granules. In
fact, it can be seen in Fig. 3 that the data for the specific
gravity and SVI of granules are consistent with each other. Similar phenomena have been widely reported in biofilm reactors: that is, denser and thinner compact biofilms were obtained under high shear conditions (Chang
et al. 1991; Vieira et al. 1993; Kwok et al. 1998).
Effect of shear force on hydrophobicity
In the environmental engineering literature, it has been
recognized that the hydrophobicity of the cell surface
plays an important role in the self-immobilization and
attachment of cells to a surface. The hydrophobicities of
cell surfaces exposed to different shear forces are shown
in Fig. 4. It was found that the hydrophobicity of a cell
surface was somehow improved as the shear force increased. A very significant difference in cell hydrophobicity was observed before and after the formation of granular sludge. In R4, cell surface hydrophobicity increased
from a value of 54.3% during the period with no granulation to 81.2% after granulation. The hydrophobicity increased from 50.6% to 75.1% in R3 and from 53.0% to
Effect of shear force on the ratio of cell polysaccharides
to proteins
So far, it has been well known that polysaccharides can
mediate both cohesion and adhesion of cells and play a
crucial role in maintaining structural integrity in a community of immobilized cells. Figure 5 shows the effect
of superficial upflow air velocity on the ratio of sludgepolysaccharides (PS) to sludge-proteins (PN). It can be
seen that the PS/PN ratio increases significantly with the
increase in shear force, in terms of superficial upflow air
velocity. A similar phenomenon was also observed in a
biofilm system (Vandevivere and Kirchman 1993). It is
worth pointing out that the content of polysaccharides in
granular sludge is at least two-fold higher than that in
flocs. It is also observed that the content of polysaccharides is much higher than the content of proteins in both
flocs and granular sludge. This in turn implies that cell
proteins would contribute less to the structure and stability of a microbial community. Higher shear force seems
to stimulate the production of polysaccharides, as compared with cell proteins. In fact, it has been generally observed in biofilm systems that high shear force can induce the biofilm to secrete more polysaccharides, which
in turn would result in a balanced biofilm structure under
the given hydrodynamic conditions (Trinet et al. 1991;
Ohashi and Harada 1994; Chen et al. 1998).
Effect of shear on SOUR
In this study, microbial activity of microorganisms is
characterized by the SOUR, in terms of milligrams of
231
Fig. 5 Effect of superficial upflow air velocity on the ratio of
sludge-polysaccharides (PS) to sludge-proteins (PN) and specific
oxygen utilization rate (SOUR). Black circles PS/PN ratio, white
circles SOUR
Fig. 6 The relationship between SVI and hydrophobicity of
cells
oxygen consumed by a milligram of cell proteins per
hour. The influence of shear force on the SOUR is
shown in Fig. 5. The SOUR quasi-linearly increased
with the increase in shear force, in terms of the superficial air velocity. It is obvious that the increased shear force
could stimulate the respiration activities of microorganisms in a very significant manner. The growth yield of
microorganisms in R1 was around 0.48 mg MLSS mg–1
COD, 0.35 mg MLSS mg–1 COD for R2 and 0.33 mg
MLSS mg–1 COD for R4. In fact, 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 is utilized in carbon dioxide production,
the fewer cells are produced (Selna and Schroeder 1979).
energy of the surface, which favors solid (cells)–liquid
phase separation, that is, microbial aggregation. Therefore, it is reasonable to consider that a higher hydrophobicity of the cell surface would result in a more
strengthened cell-to-cell interaction and, further, a dense
and stable structure. In fact, Fig. 6 clearly shows that the
SVI of cells almost linearly decreases with the increasing hydrophobicity of cell surfaces. It appears from
Figs. 4 and 6 that hydrophobicity might be the main inducing force for the initiation of sludge granulation. It is,
at least, one of the forces for maintaining the stable microbial structure of granules. In fact, there is strong evidence to show that the hydrophobicity of the cell surface
is an important affinity force in the self-immobilization
and attachment of cells (Marshall and Gruickshank
1973; Pringle and Fletcher 1983). Previous research indicated that the hydrophobicity of microorganisms would
play a crucial role in the formation of anaerobic granules
(Mahoney et al. 1987; Tay et al. 2000). Mahoney et al.
(1987) reported that the non-granular sludge washed out
from UASB reactors was more hydrophilic than the reactor sludge.
It has been generally believed that cell polysaccharides can mediate both cohesion and adhesion of cells
and play a crucial role in maintaining the structural integrity of the biofilm and anaerobic granule matrix
(Fletcher and Floodgate 1973; Christensen 1989;
Schmidt and Ahring 1994). Figure 5 indicates that the
PS/PN ratio increases with the shear force, in terms of
superficial upflow air velocity, up to a relatively stable
value. Also, it is worth noticing that the polysaccharide
content of granules is much higher than that of bioflocs.
In fact, Vandevivere and Kirchman (1993) found that the
content of exopolysaccharides was five-fold greater for
attached cells than for free-living cells. This in turn implies that the polysaccharides would greatly contribute to
the self-immobilization process of cells, e.g. aerobic/
anaerobic granulation. Figure 5 also reveals that the
polysaccharide content of granules is nine-fold higher
than the protein content of granules. It is most likely that
cell proteins contribute less to the structure and stability
of aerobic granules. Higher shear force seems to stimu-
Discussion
In R1 operated in a superficial air velocity of 0.3 cm s–1,
aerobic granules did not form during the whole experimental period; and only bioflocs were observed. However, regular-shaped granules were successfully cultivated in R2, R3 and R4, which were operated at a relatively
high superficial upflow air velocity. Figure 1 clearly
shows that smooth, dense and stable aerobic granules
were formed only under high shear strength. In fact,
Beun et al. (1999) also reported that lower superficial
gas velocity did not favor the formation of stable aerobic
granules in the same type of reactors.
Figure 4 clearly shows that the formation of stable
granules was closely correlated with an increase in the
hydrophobicity of the cell surface. Hydrophobic binding
has a prime importance for cell-to-cell interaction, which
may induce the initial self-immobilization of bacteria
and further keep the bacteria tightly together. This would
serve as the first step towards to microbial granulation.
In fact, it appears from Fig. 4 that the surface hydrophobicity of granules is much higher than that of bioflocs.
In a thermodynamic sense, increasing the hydrophobicity
of cell surfaces causes a decrease in the excess Gibbs
232
late the production of cell polysaccharides. In fact, it has
been generally observed that high shear force can induce
the biofilm to secrete more exopolysaccharides, which in
turn results in a balanced biofilm structure under the given hydrodynamic conditions (Trinet et al. 1991; Ohashi
and Harada 1994; Chen et al. 1998). It has been reported
that colanic acid, an exopolysaccharide of Escherichia
coli K-12, is critical for the formation of the complex
three-dimensional structure and depth of E. coli biofilms
(Danese et al. 2000). It seems reasonable to consider that
cell exopolysaccharides can play a crucial role in building-up and maintaining the architecture of biofilm and
granular sludge. Ohashi and Harada (1994) observed a
proportional relationship between the polysaccharide
content of cells and biofilm density. Therefore, it appears
that the shear-associated production of cell polysaccharides would provide a plausible explanation for the high
gravity of sludge observed at high shear force (Fig. 3).
The metabolic network of cells includes interrelated
catabolic and anabolic reactions. The catabolic activity of
microorganisms is directly correlated with the electron
transport system activity, which can be described by the
SOUR. Figure 5 indicates that the SOUR of cells was
stimulated in a significant way by shear force, in terms of
superficial upflow air velocity. As pointed out earlier, the
shear force may trigger the production of cell polysaccharides. In research on biofilms, the shear force-associated
phenomena are usually attributed to a simple physical effect (Vieira et al. 1993). However, it appears from Fig. 5
that the microbial community may respond to shear force
by metabolic changes and some biological events should
be involved in shear-associated phenomena. The effect of
shear force on the ratio of polysaccharides to oxygen utilization rate (OUR), in terms of milligrams of oxygen utilized per hour, is shown in Fig. 7. It was found that this
ratio increased with the shear force. Such an observation
implies that when the shear force is increased, much more
energy generated by catabolism would be used for the
production of polysaccharides rather than for growth purposes. It is most likely that when the shear force exerted
on granular sludge is high, the granules would have to
regulate the metabolic pathway so as to maintain a balance with the external shear force, through consuming
non-growth-associated energy. In fact, inhibition of the
energy-generating function had been found to prevent the
development of competence for cell aggregation in many
systems (Calleja 1984; O’Toole et al. 2000). Consequently, the catabolic activity of cells would play a role in the
development of granular sludge.
Shear force, in terms of superficial upflow air velocity,
is important in the aerobic granulation process. The results
show that a superficial air velocity higher than 1.2 cm s–1
must be satisfied in order to produce aerobic granules.
Shear force has significant effects on the microbial structure and metabolism of microorganisms. It was found that
high shear stimulates the production of polysaccharides
and improves the hydrophobicity of granular sludge. It appears that the compactness and structure of granular
sludge are highly dependent on cell hydrophobicity and
Fig. 7 The ratio of sludge-polysaccharides to oxygen utilization
rate (OUR) versus superficial upflow air velocity
the polysaccharide content of cells. It is reasonable to
consider that hydrophobicity might act as an inducing
and further maintaining force for aerobic granulation.
The shear-stimulated polysaccharide production may
strengthen the structure of aerobic granules and plays an
important role in building-up and maintaining architecture
of granular sludge. It is expected that this research should
be useful for the production of aerobic granules and the
development of aerobic granular sludge-based bioreactors
for handling high-strength organic wastewater.
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