Letters in Applied Microbiology 2001, 33, 222Ä…226
The role of cellular polysaccharides in the formation
and stability of aerobic granules
J.-H. Tay, Q.-S. Liu and Y. Liu
Environmental Engineering Research Center, School of Civil and Structural Engineering, Nanyang
Technological University, 50 Nanyang Avenue, Singapore 639798
84/2001: received 14 March 2001, revised 11 June 2001 and accepted 21 June 2001
J . - H . T A Y , Q . - S . L I U A N D Y . L I U . 2001.
Aims: This paper attempts to investigate the role of cellular polysaccharides in the formation
and stability of aerobic granules.
Methods and Results: Three column sequential aerobic sludge blanket reactors (R1, R2 and
R3) were operated at a super®cial air upÅ»ow velocity of 0á3 cmsÄ…1, 1á2 cmsÄ…1 and 2á4 cmsÄ…1,
respectively. Aerobic granules appeared at cycle 42 in R2 and R3 with a mean size of 0á37 mm
in R2 and 0á35 mm in R3, however, aerobic granulation was not observed in R1. After the
formation of aerobic granules, the sludge volume index (SVI) decreased to 55 ml gÄ…1 in R2
and 46 ml gÄ…1 in R3. Aerobic granulation was concurrent with a sharp increase of cellular
polysaccharides normalized to cellular proteins, which increased from 5á7 to 13á0 mg per mg
proteins in R2, and 7á5Ä…13á9 mg per mg protein in R3. The content of polysaccharides in aerobic
granules was 2ą3 times higher than that in the bioŻocci cultivated in R1. The disappearance of
aerobic granules in R2 was tightly coupled to a drop in cellular polysaccharides. After the
reappearance of bioŻocci in R2, the content of cellular polysaccharides were found to be restored
to the level observed in R1.
Conclusions: It appears that the production of cellular polysaccharides could be stimulated by
hydrodynamic shear force and contributes to the formation and stability of aerobic granules.
Signi®cance and Impact of the Study: It is expected that this study would provide useful
information for better understanding the mechanisms of aerobic granulation.
charge of bacteria, and thereby bridge two neighbouring
INTRODUCTION
bacterial cells to each other as well as other inert particulate
Extracellular polymers produced by bacteria, mainly con- matters, and settle out as Żoccus aggregates (Shen et al.
sisting of polysaccharides, have been considered to play an 1993; Schmidt and Ahring 1994). Harada et al. (1988) also
important role both in the formation and stability of bio®lms observed that the extracellular polymers excreted by acid-
and anaerobic granules (Schmidt and Ahring 1994; Liu and ogenic bacteria appeared to assist in cell-to-cell attachment
Tay 2001). Polymers can bridge physically or electrostati- and the enhancement of granule strength and structural
cally to form a three-dimensional structure, which favours stability.
attachment of bacterial cells (Ross 1984). In a pilot-scale Recently, attention has turned to developing aerobic
UASB reactor, Quarmby and Forster (1995) reported that granular sludge (Beun et al. 1999; Peng et al. 1999). The
anaerobic granules tended to become weaker as the surface advantages of aerobic granules include strong microbial
negative charge of cells increased. At usual pH value, structure, good settling ability and high biomass retention.
suspended bacteria are negatively charged and electrostatic Although the effects of cellular polysaccharides on bio®lm
repulsion exists between cells. It had been suggested that and anaerobic granulation are well known, the role of
extracellular polymers can change the surface negative cellular polysaccharides in the formation and stability of
aerobic granules has been hardly studied. Therefore, this
research attempts to provide experimental evidence to show
Correspondence to: Dr Yu Liu, Environmental Engineering Research Center,
the importance of cellular polysaccharides in the formation
School of Civil and Structural Engineering, Nanyang Technological University,
and maintenance of aerobic granules.
50 Nanyang Avenue, Singapore 639798 (e-mail: [e-mail]).
ć 2001 The Society for Applied Microbiology
CELLULAR POLYSACCHARIDES IN THE FORMATION AND STABILITY OF AEROBIC GRANULES 223
cellular proteins by Bicinchonic acid kit (Sigma USA)
MATERIALS AND METHODS
according to the manufacturer's instructions.
Column reactors
Three columns reactors (80 cm height and 6á0 cm in diam- RESULTS
eter) with the same geometrical con®guration and a working
The formation of aerobic granules
volume of 2á3 l were used. Reactor one (R1) was supplied with
air at a velocity of 0á5 l minÄ…1, while reactor two (R2) was The seed sludge had a mean Å»occus size of 0á12 mm
supplied with air at a velocity of 2á0 l minÄ…1 and 4á0 l minÄ…1 (Fig. 1a). At cycle 42, small aerobic granules appeared in R2
for reactor 3 (R3), equivalent to a super®cial air upÅ»ow and R3. Figure. 1(c,d) show morphology of aerobic granules
velocity of 0á3 cmsÄ…1, 1á2 cmsÄ…1 and 2á4 cmsÄ…1, respect- at cycle 66. However, no aerobic granulation was observed in
ively. Reactors were operated sequentially as 5 min of feeding, R1, which was operated at a lower super®cial air upÅ»ow
225 min of aeration, 5 min of settling and 5 min of efÅ»uent velocity of 0á3 cmsÄ…1, and only Å»uffy Å»occi with a mean size
withdraw. EfÅ»uent was discharged at the middle port of the around 0á15 mm were grown (Figs 1b and 2). The sludge
column. A substrate loading rate of 6á0 kg chemical oxygen settling ability, in terms of sludge volumetric index (SVI),
demand (COD) mÄ…3 dÄ…1 was applied. The experiments were was not improved in R1 throughout the entire operating
conducted in a temperature-controlled room of 25°C. After period (Fig. 3). As compared to the seed sludge, aerobic
start-up, dissolved oxygen (DO) concentration was monitored granules have a regular round shape and clear-cut. Figures 2
once or twice a week for one whole cycle, and the DO and 3 show variations of sludge size and SVI with the
concentrations in the reactors were greater than 1á5 mg lÄ…1. operation cycles. After the formation of aerobic granules at
cycle 42, aerobic granules have a mean size of 0á37 mm in R2
and 0á35 mm in R3. The seed sludge had a SVI value of
Media
205 ml gÄ…1, along with the formation of aerobic granules it
Three reactors were inoculated with 650 ml of sludge gradually decreased to an average value of 55 and 46 ml gÄ…1
acclimatized for one week by acetate in a batch culture. The in R2 and R3, respectively (Fig. 3). The settling ability of
synthetic wastewater mainly consisted of sodium acetate as sludge was improved signi®cantly after the appearance of
sole carbon source, and other necessary elements, as aerobic granules in R2 and R3.
described previously (Tay and Yan 1996). In the case of R2, it was observed at cycle 90 that aerobic
granules disappeared, which was indicated by a decrease in
granule size and an increase in SVI (Figs 2 and 3). At cycle
Analytical procedures
120, bioŻocci with a SVI value of 180 ml gą1 and a mean size
The concentration of mixed liquor suspended solids of 0á15 mm were re-predominant in R2, while aerobic
(MLSS) and the sludge volume index (SVI) were deter- granules completely disappeared. The situation in R3 was
mined by standard methods (APHA 1995). Granule size different from that of R2, since aerobic granules persisted
was measured by laser particle size analysis system throughout in the entire operation period (Figs 2 and 3).
(Malvern MasterSizer Series 2600), or an image analysis The aerobic granules formed at high air upŻow velocity
system (Image-Pro Plus, V4á0, Media Cybernetics) with an (Fig. 1d) had a much more regular and round outer shape
Olympus SZX9 microscope. Structure of microbial com- than those cultivated in low air velocity (Fig. 1c). In a
munity was observed by using Image Analysis (IA). column reactor, the super®cial air upÅ»ow velocity acts as a
For the determination of cellular polysaccharides, the cells main hydrodynamic shear force. The super®cial air upÅ»ow
from 5 ml of sludge sample were harvested by centri- velocity in R3 was twice as high as that in R2. Beun et al.
fugation. Cells were then re-suspended in 1 M NaOH (1999) reported that a low super®cial air velocity did not lead
solution and heated in oven at 80°C for 30 min. The extract to the formation of stable aerobic granules in the same type
was recovered by centrifugation at 3220 g for 10 min, and of reactor. It is most likely that the instability of aerobic
then was used to determine cellular polysaccharides using granules in R2 would be in part attributed to lower air
the method of Dubois et al. (1956). The protein content of upŻow velocity applied. This is supported by the fact that
sludge sample was determined by resuspending harvested microorganisms could not form granules in R1, which was
sludge an equal volume of sample treatment buffer supplied with air at the lowest upÅ»ow velocity of 0á3 cmsÄ…1.
(0á0625 M Tris HCl buffer, pH 6á8, sodium dodecyl
sulphate (SDS) 2%, glycerol 10%, 2-mercaptoethanol
Cellular polysaccharides
5%), and was heated at 100°C for 10 min (Alexander et al.
1984). The extract was recovered by centrifugation at 38 Figure 4 shows the variation of cellular polysaccharides
000 g for 30 min at 4°C. The extract was used to determine (PS) normalized to cellular proteins (PN) throughout the
ć 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 222ą226
224 J.-H. TAY ET AL.
Fig. 1 Photos of seed sludge (a) and bioŻocci in R1 (b) and aerobic granules at cycle 66 (R2: (c); R3: (d)) by Image Analysis
Fig. 2 Sludge size vs. the operation cycles. *: R1; s: R2; d: R3 Fig. 3 SVI vs. the operation cycles. *: R1; s: R2; d: R3
polysaccharides remains unchanged in R1. In the case of R2
operation cycles. It can be seen that during cycles 36Ä…42
and R3, the aerobic granules formed during the period from
there is sharp increase of cellular polysaccharides from
cycles 36 to 42 (Figs 2 and 3). The appearance of aerobic
5á7 mg per mg proteins to 13á0 in R2 and 7á5Ä…13á9 mg per
granules correlates closely with a signi®cant increase in
mg proteins in R3, however, the content of cellular
ć 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 33, 222ą226
CELLULAR POLYSACCHARIDES IN THE FORMATION AND STABILITY OF AEROBIC GRANULES 225
polymers could change the surface negative charge of
bacteria, and thereby bridge two neighbouring bacterial
cells physically to each other as well as other inert
particulate matter (Shen et al. 1993; Schmidt and Ahring
1994). It appears that the content of cellular polysaccha-
rides is much higher than the content of cellular proteins
in both Żocci and aerobic granules (Fig. 4). Similar
phenomena were also reported in bio®lm systems (Vande-
vivere and Kirchman 1993), implying that cellular proteins
contribute less to the formation, structure and stability of
granules and bio®lms than polysaccharides.
High hydrodynamic shear forces seem to stimulate the
production of cellular polysaccharides (Fig. 4), as has also
Fig. 4 The cellular polysaccharides (PS) normalized to cellular
proteins (PN) vs. the operation cycles. *: R1; s: R2; d: R3 been observed in the case of bio®lms (Trinet et al. 1991;
Ohashi and Harada 1994). It is therefore reasonable to
conclude that the formation and stability of aerobic granules
cellular polysaccharides. The disappearance of aerobic
are dependent on the hydrodynamic shear force-associated
granules at cycle 90 in R2, corresponded to a simultaneous
cellular polysaccharides production. This also provides a
decrease in cellular polysaccharides (Fig. 4). When bioŻocci
plausible explanation for the phenomena observed in R1 and
re-appeared in R2 after cycle 120 (Figs 2 and 3), the content
R2.
of cellular polysaccharides was restored to the level of the
initial seed sludge. These results provide experimental
evidence that cellular polysaccharides would play an
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