Appl Microbiol Biotechnol (2004) 65: 143 148
DOI 10.1007/s00253-004-1657-8
MINI-REVIEW
. .
Yong-Qiang Liu Yu Liu Joo-Hwa Tay
The effects of extracellular polymeric substances
on the formation and stability of biogranules
Received: 24 March 2004 / Revised: 6 May 2004 / Accepted: 7 May 2004 / Published online: 9 June 2004
# Springer-Verlag 2004
Abstract Biogranulation is a promising biotechnology withstand high organic loading (Schmidt and Ahring
developed for wastewater treatment. Biogranules exhibit a 1996; Tay et al. 2001a). One major drawback of anaerobic
matrix microbial structure, and intensive research has granulation in UASB reactors is the extremely long start-
shown that extracellular polymeric substances (EPS) are a up period, which generally requires 2 8 months for the
major component of the biogranule matrix material in both development of anaerobic granular sludge blankets, and
anaerobic and aerobic granules. This paper aims to review anaerobic granulation technology cannot be applied to
the role of EPS in biogranulation, factors influencing EPS nutrient removal from wastewater. Compared to anaerobic
production, the effect of EPS on cell surface properties of granules, aerobic granules can be developed within
biogranules, and the relationship of EPS to the structural 4 weeks and have the ability to remove nutrients
stability of biogranules. EPS production is substantially efficiently, but their stability is poor (Tay et al. 2001a;
enhanced when the microbial community is subject to Liu et al. 2004a; Yang et al. 2003). Furthermore, anaerobic
stressful culture conditions, and the stimulated EPS granulation is somehow sensitive to the characteristics of
production in the microbial matrix in turn favours the wastewater, e.g. anaerobic granules do not grow success-
formation of anaerobic and aerobic granules. EPS can also fully on some types of wastewater, and sudden disinte-
play an essential role in maintaining the integrity and gration of biogranules without any apparent cause has
stability of spatial structure in mature biogranules. It is been reported (Schmidt and Ahring 1996; Liu et al. 2002;
expected that this paper can provide deep insights into the Punal et al. 2003). Thus, strategies to overcome these
functions of EPS in the biogranulation process. drawbacks and further enhance the stability of biogranules
are highly desired by the wastewater treatment industry.
Extracellular polymeric substances (EPS) are sticky
Introduction materials secreted by cells. There is strong evidence that
EPS are highly involved in adhesion phenomena, forma-
Biogranulation is a process of cell-to-cell aggregation, tion of matrix structure, microbial physiology and
which is, to some extent, different from the formation of improvement of long-term stability of granules (Forster
biofilm because no carrier is needed for biogranulation. 1992; Schmidt and Ahring 1994; Tay et al. 2001b; Qin et
Accumulated information shows that specific operational al. 2004a). This indicates that EPS could play a central
conditions are essential to trigger and enhance cell self- role in the biogranulation process. Extensive research has
aggregation. So far, anaerobic granules are formed mostly been conducted to examine the functions of EPS in
in upflow anaerobic sludge blanket (UASB) reactors, biogranulation. In view of the importance of EPS, this
while only aerobic granules can be developed in paper aims to provide a deeper insight into the functions of
sequencing batch reactors (SBR). Compared to conven- EPS in biogranulation.
tional bioflocs, biogranules show great potential and
capability in wastewater treatment due to their good
settleability, high biomass retention, and the ability to Composition of EPS in biogranules
. .
EPS have been detected in significant amounts in both
Y.-Q. Liu Y. Liu ( ) J.-H. Tay
Division of Environmental and Water Resources Engineering,
aerobic and anaerobic granules, forming a three-dimen-
School of Civil and Environmental Engineering, Nanyang
sional matrix in which bacteria and other particles are
Technological University,
embedded (Grotenhuis et al. 1991; Fang 2000). EPS are
50 Nanyang Avenue,
produced by microorganisms themselves during cultiva-
Singapore, 639798
e-mail: [e-mail] tion, being advantageous in many respects for their
144
survival under various circumstances. From the microbi- of ratios of 1.54 2.23. Indeed, this is consistent with the
ological point of view, EPS can help stabilise membrane finding by Azeredo et al. (1999) that many EPS extraction
structure and may also serve as a protective barrier. EPS methods developed for biofilms were not efficient, and
produced in biogranules contains variable proportions of could somehow promote leakage of intracellular materials.
protein, polysaccharides, nucleic acids, humic-like sub- Although a number of physical and chemical methods, e.g.
stances, lipids, and heteropolymers such as glycoproteins high-speed centrifugation, boiling in acid or alkali, and
(Goodwin and Forster 1985; Horan and Eccles 1986; utilisation of cation exchange resins, are currently
Grotenhuis et al. 1991; Urbain et al. 1993; Jorand et al. available for extracting EPS from biogranules, none has
1995; Frolund et al. 1996). It should be pointed out that yet been adopted as a standard procedure. In addition to
polysaccharide is the only one of these components that is the effect of microbial species, the use of non-standardised
synthesised extracellularly for a specific function, while procedures makes comparison of EPS in terms of content
proteins, lipids, and nucleic acids exist in the extracellular and composition not just unreliable, but impossible.
polymer network due to excretion of intracellular poly-
mers or as a result of cell lysis (Durmaz and Sanin 2001;
Mahmoud et al. 2003). EPS exist in any form of microbial Major factors influencing production of EPS in
aggregate, such as bioflocs, biofilms, and anaerobic and biogranules
aerobic granules. However, the EPS content of biogranules
has been found to be much higher than that in There is no need for microorganisms to secrete excessive
conventional bioflocs and biofilms (Tay et al. 2001a). EPS under normal culture conditions. The enhanced
The environmental engineering literature contains production of EPS observed in biogranules is induced by
contradictory reports on the composition of EPS in some so-called stressful culture conditions (Nichols et al.
biogranules, especially the ratio of carbohydrate to protein. 2004; Qin et al. 2004a). So far, a number of operating
Some researchers have reported protein to be the parameters, including reactor type, substrate composition,
predominant component of EPS in anaerobic granules substrate loading rate, hydraulic retention time, hydrody-
(Fukuzaki et al. 1995), while others found that EPS were namic shear force, settling time in SBR, feast-famine
composed mainly of carbohydrates (Fang et al. 2002). regime in SBR, culture temperature, and so on, are
Current evidence shows that the quantity and the understood to stimulate bacteria to secrete more EPS. The
composition of EPS produced by bacteria depends on a composition of EPS is also related to the characteristics of
number of factors, such as microbial species, growth feed wastewater, e.g. EPS has high protein and DNA
phase, the type of limiting substrate (carbon, nitrogen and levels in protein-grown granules, while high polysaccha-
phosphorous), oxygen limitation, ionic strength, culture ride levels are found in granules grown on other types of
temperature, shear force, and so on (Nielsen et al. 1997; organic substrates (Batstone and Keller 2001). Nitrogen-
Tay et al. 2001b, 2002; Nichols et al. 2004; Qin et al. limiting conditions favour the production of EPS, which in
2004a). This may imply that the composition of extracel- turn accelerates anaerobic granulation (Punal et al. 2000).
lular polymers is variable, and is related to microbial Experimental evidence from research on aerobic gran-
species, the physiological state of the bacteria, and the ulation shows that stressful operating conditions, in terms
operating conditions under which biogranules are devel- of high hydrodynamic shear force, short settling time/
oped. In fact, a shift in bacterial species during hydraulic retention time and periodical feast-famine peri-
biogranulation has been reported (Etchebehere et al. od, significantly stimulate bacteria to produce more
2003; Yi et al. 2003 ); such a microbial shift would affect extracellular polysaccharides than proteins in SBR (Tay
the production and composition of EPS. It seems certain et al. 2001b, 2002; Liu et al. 2003; Qin et al. 2004a,b).
that the diverse EPS compositions reported in the literature EPS production seems to be positively related to the
result, at least partially, from the complexity of mixed specific oxygen utilization rate (SOUR) of aerobic gran-
microbial cultures run under different conditions. Another ules developed in SBR (Tay et al. 2001b; Qin et al. 2004b;
point that needs to be addressed is that EPS includes both Yang et al. 2004). In fact, the catabolic activity of
bound and soluble polymers, the ratio of which may microorganisms is directly correlated with electron trans-
change substantially, even within the same species, under port system activity, which can be roughly described by
various growth conditions. There is evidence indicating the SOUR (Trevors 1984; Lopez et al. 1986); moreover, in
that soluble polymers can be transferred to the supernatant aerobic oxidation processes, the respiratory activity of
after centrifugation, while bound polymers remain tightly cells is coupled to proton translocation activity and a clear
attached to cells and cannot be recovered from the linkage between oxygen reduction and proton transloca-
supernatant (Nielsen et al. 1997). Zhang et al. (1999) tion has been established (Babcock and Wikstrom 1992).
compared different EPS extraction methods for a biofilm This implies that aggregated bacteria can respond to
sample, including regular centrifugation, EDTA extrac- stressful culture conditions by regulating their energy
tion, ultracentrifugation, steam extraction, and regular metabolism. Chan et al. (2004) reported that the purpose
centrifugation with formaldehyde (RCF), and found that of polymer production was to localise iron oxyhydroxide
the RCF method gave the highest yield ratio of mineral precipitation in order to enhance metabolic energy
carbohydrates to proteins, 13.7, for aerobic/sulphate- generation. In general, environmental factors that may
reducing biofilms, with the other methods giving a range influence EPS production and composition can be
145
classified into two categories: (1) changes in environ- interaction if the EPS amount is large (Tsuneda et al.
mental conditions that cause a shift in microbial commu- 2003b).
nity. Subsequently, numbers of EPS-producing microbes Cell surface hydrophobicity has been considered as a
would increase or decrease in the whole microbial triggering force for biogranulation (Liu et al. 2004b).
consortium, which has to adapt to the new environment; Microorganisms with different hydrophobicities have been
(2) the existing microbial community re-regulates the detected in activated sludge (Singh and Vincent 1987;
metabolic pathway of EPS production in response to Jorand et al. 1994), and high cell surface hydrophobicity is
changes in environmental conditions. So far, it has not usually associated with the presence of fibrillar structures
been demonstrated whether the genes for EPS production on the cell surface and specific cell wall proteins (Mcnab
are expressed before or after bacterial granulation, i.e. if et al. 1999; Singleton et al. 2001). In fact, previous
the bacteria initially make EPS and then adhere to each research has shown that the cell wall of bacteria in the
other, or first adhere and then produce EPS. In the former granules is surrounded by an EPS layer (Forster 1992; de
case, the appearance of polymeric materials at the initial Beer et al. 1996; Veiga et al. 1997). This may imply that
site of contact between microbial cells may be due to cell surface hydrophobicity might be related to EPS. Some
migration of polymer molecules already on the cell evidence suggests that proteins and amino acids are the
surface. In the latter case, bacterial adhesion may provide hydrophobic components of the EPS, while polysacchar-
the physiological conditions required for EPS excretion. ides are hydrophilic (Dignac et al. 1998). Jorand et al.
(1998) studied the hydrophobic and hydrophilic properties
of EPS of activated sludge, and found that a significant
Effect of EPS on the characteristics of biogranules proportion of the EPS fraction was hydrophobic. This
implies that hydrophobic EPS would be involved in the
Extracellular polysaccharides are a so-called bioglue, and formation and organisation of microbial aggregates. Since
a high polysaccharide content could facilitate cell-to-cell both hydrophobic and hydrophilic groups are present in
interaction and further strengthen microbial structure EPS, the measured hydrophobicity of EPS indeed reflects
through formation of a polymeric matrix. However, in the average of the hydrophobicity of its all components
recognition of the contribution of extracellular polysac- (Daffochio et al. 1995). Cell surface hydrophobicity and
charides to both anaerobic and aerobic biogranulation, the charge are related to the production, composition and
importance of the EPS properties such as hydrophobicity physical characteristics of EPS (Liao et al. 2001), but no
and charge also need to be accounted for in the specific evidence shows the quantitative contribution of
biogranulation process (Andreadakis 1993; Liao et al. hydrophobic components of EPS to the overall hydro-
2001). Since EPS may accumulate at the cell surface, cell phobicity of biogranules so far. Regarding the importance
surface characteristics such as cell surface hydrophobicity, of surface charge and hydrophobicity, there is evidence
surface charge density, binding site and surface morphol- that a reduction in surface charge is not a requirement for
ogy may be affected. The surface charge has long been the formation of activated sludge, i.e. charge neutralisation
believed to be important in controlling the stability of would not be the main microbial flocculation mechanism
microbial aggregates. Bacteria may carry net negative (Pavoni et al. 1972; Strand et al. 2003), while recent
surface charge when cultivated at physiological pH values research showed that hydrophobic interaction may be
(Rouxhet and Mozes 1990). According to the well-known fundamental in biogranulation (Liu et al. 2004b). In fact,
DLVO theory, when two surfaces have a charge of the study of biofilms has shown that few bacteria are actually
same sign, there is a repulsive force to prevent the in contact with the hydrophilic surface, but are rather in
approach of one cell to another. It has been proposed that contact with the hydrophobic surfaces of other cells
EPS could decrease the negative charge of cell surfaces, (Ghigo 2003).
and thereby bridge two neighbouring cells physically to
each other (Shen et al. 1993; Schmidt and Ahring 1994).
Using a colloid titration technique, granular sludge was EPS-enhanced stability of biogranules
found to be less negatively charged than activated sludge
(Morgan et al. 1990), and Tsuneda et al. (2003a), using a The accumulation of EPS as capsular material and
soft particle electrophoresis technique to investigate the peripheral slime has been correlated with biological
influence of EPS on cell surface electrokinetics, found that adhesion and aggregation processes (Costerton et al.
EPS could increase the softness of the cell surface and 1981; Tay et al. 2001a; Liu et al. 2002). The metabolic
further decrease the negative surface charge density blocking of exopolysaccharide synthesis was found to
surrounding the cell surface, i.e. the EPS layer could prevent microbial aggregation (Cammarota and Sant Anna
hold a lower negative charge compared with that of the 1998; Yang et al. 2004). EPS in granules were
native cell surface. Electrostatic interaction between cells hypothesized to bridge two neighbouring bacterial cells
is closely associated with the amount of EPS produced; physically to each other as well as with other inert
microbial adhesion onto a solid surface can be inhibited by particulate matter, and settle out as aggregates, as shown
electrostatic interaction when the EPS amount is small, schematically in Fig. 1 (Ross 1984; Shen et al. 1993;
while cell adhesion would be enhanced by polymeric Schmidt and Ahring 1994; Tay et al. 2001a).
146
charides in anaerobic granules is almost three times higher
than that in anaerobic bioflocs (de Beer et al. 1996), while
formation of aerobic granules is accompanied by a sharp
increase in cellular polysaccharides relative to cellular
proteins (Tay et al. 2001a). The ratio of extracellular
polysaccharides to proteins by weight in aerobic granules
falls within the range of 2 16 (Tay et al. 2001a,b, 2002;
Jiang et al. 2003; Liu et al. 2003; Qin et al. 2004a,b),
which seems to be higher than that reported for an
anaerobic granulation process. It seems certain that the
characteristics of biogranules are related to the ratio of
polysaccharides to proteins. Previous research has shown
Fig. 1 Schematic representation of extracellular polymeric sub-
stance (EPS)-enhanced biogranulation that anaerobic granules and aerobic bioflocs with a higher
protein/polysaccharide ratio had a lower shear strength and
EPS has been observed in different types of biogranules a poorer settleability (Batstone and Keller 2001; Martinez
by scanning electron microscopy and transmission elec- et al. 2004), while Quarmby and Forster (1995) thought
tron microscopy. In the biogranulation process, EPS that extracellular polysaccharides could contribute greatly
provide an extensive surface area for bacterial binding. to the strength and stability of anaerobic granules. Recent
Furthermore, extracellular polysaccharide matrices sur- research on aerobic granules has provided solid evidence
rounding aggregated bacteria can provide sites available that the specific gravity and mechanical strength of aerobic
for attraction of organic and inorganic materials (Yu et al. granules increased with the increase of ratio of poly-
2001; Sponza 2002). The total concentrations of electro- saccharides to proteins in a very significant way (Tay et al.
static binding sites on EPS were found to be 20- to 30-fold 2001b, 2002).
higher than those reported for bacterial cell surfaces (Liu Evidence shows that the formation of biogranules is a
and Fang 2002). This seems to indicate that EPS of microbial evolution instead of a random aggregation of
neighbouring microbial cells may form a cross-linked suspended microbes (El-Mamouni et al. 1995; Fang 2000;
network by attraction of organic or inorganic materials, as Tay et al. 2001c). It seems a reasonable hypothesis that the
in cationic bridging, and further strengthen the structural spatial distribution of EPS in biogranules should be
integrity of a biogranule (Yu et al. 2001). Microscopic correlated to microbial evolution and distribution during
observation provides visual evidence that EPS with a the formation of biogranules. Investigation into the spatial
filamentous structure is present within and around the distribution of EPS with depth in heterotrophic biofilms
structure of both aerobic and anaerobic granules (Forster showed that EPS production yields tended to decrease
1992; de Beer et al. 1996; Veiga et al. 1997; Tay et al. with biofilm depth (Zhang and Bishop 2001). This is
2001c), while EPS may fill in the intercellular spaces in probably due to the fact that viable biomass loses its
the microcolonies present in biogranules (Macleod et al. ability to produce EPS in the deeper sections of biofilms
1995; Jiang et al. 2002). It is most likely that EPS plays an because of the lower microbial activity resulting from
important role in maintaining the structural and functional lower nutrient availability. In addition, the EPS produced
integrity of granular sludge. So far, no information is by bacteria could be utilised as a secondary substrate in the
available on EPS distribution in the early stages of deeper layers or zones of biofilms and biogranules, where
biogranulation, whereas the spatial distribution of EPS in readily degradable substrates were either not available or
mature granules has been reported. Calcofluor staining and limiting. It appears that the spatial distribution of EPS in
fluorescent microscopy or confocal scanning electron biogranules and biofilms plays an essential role in
microscopy has revealed that approximately 50% of the stabilizing the structure and maintaining the strength of
total amount of EPS is present in a top 40 źm-thick zone microbial aggregates. This is supported by the finding that
on the surface of anaerobic granules, and the rest of the EPS near the edge of granules had a greater effect on shear
EPS is randomly allocated within the deeper parts of the strength than it did in the centre of the granule (Batstone
granules. However, part of the EPS was also found in a and Keller 2001). Finally, it should be stressed that
coating extending outside the granule boundary (de Beer information in the literature on the spatial distribution of
et al. 1996). It should be pointed out that the EPS layer on EPS in biogranules is very limited, and some questions
the surface of biogranules is not found in conventional still remain unanswered, e.g. whether the spatial distribu-
bioflocs. tion of EPS in biogranules is related to operational
Previous research on anaerobic and aerobic granulation conditions or is correlated with microbial distribution and
in different types of bioreactors under a wide spectrum of activity in biogranules. Obviously, there is a strong
operating conditions has shown that the total amount of requirement for further research into such aspects of EPS.
EPS produced is not a decisive factor in the formation and
maintenance of the stability of biogranules. However, the
ratio of polysaccharides to proteins plays a crucial role in
biogranulation (Tay et al. 2001a; Punal et al. 2003). It has
been reported that the content of extracellular polysac-
147
Etchebehere C, Cabezas A, Dabert P, Muxi L (2003) Evolution of
Concluding remarks
the bacterial community during granules formation in deni-
trifying reactors followed by molecular, culture-independent
This review describes the essential role of EPS in the
techniques. Water Sci Technol 48:75 79
formation and maintenance of the structural stability of
Fang HHP (2000) Microbial distribution in UASB granules and its
resulting effects. Water Sci Technol 42:201 208
biogranules. The composition as well as the content of
Fang HHP, Liu H, Zhang T (2002) Characterization of a hydrogen-
EPS in biogranules affects the structure and integrity of the
producing granular sludge. Biotechnol Bioeng 78:44 52
biogranule matrix. Although extensive research has
Forster CF (1992) Anaerobic upflow sludge blanket reactors:
focused on EPS in biogranules, there are still many
aspects of their microbiology and chemistry. J Biotechnol
17:221 232
contradictory research reports on the functional roles of
Frolund B, Palmgren R, Keiding K, Nielsen P (1996) Extraction of
EPS in the biogranulation process. This is partly because
extracellular polymers from activated sludge using a cation
of the very complex composition of EPS, comprising
exchange resin. Water Res 30:1749 1758
proteins, polysaccharides, nucleic acids, lipids, and other
Fukuzaki S, Nishio N, Nagai S (1995) High rate performance and
components. The role of each component of EPS in characterisation of granular methanogenic sludges in upflow
anaerobic sludge blanket reactors fed with various defined
biogranules should be identified. Future studies also need
substrates. J Ferment Bioeng 79:354 359
to examine the effect of the interaction of different EPS
Ghigo JM (2003) Are there biofilm-specific physiological pathways
components, such as polysaccharide-protein, protein-pro-
beyond a reasonable doubt? Res Microbiol 154:1 8
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Goodwin JAS, Forster CF (1985) A further examination into the
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Grotenhuis JTC, Smith M, van Lammeran AAM, Stams AJM,
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Zehnder AJB (1991) Localization and quantification of extra-
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