Biotechnology Advances 28 (2010) 882–894

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Extracellular polymeric substances (EPS) of microbial aggregates in biological

wastewater treatment systems: A review
Guo-Ping Sheng a, Han-Qing Yu a,⁎, Xiao-Yan Li b
a
School of Chemistry, University of Science and Technology of China, Hefei, 230026, China
b
Department of Civil Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong

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

Article history: A review concerning the definition, extraction, characterization, production and functions of extracellular
Received 8 September 2008 polymeric substances (EPS) of microbial aggregates in biological wastewater treatment reactors is given in
Received in revised form 27 July 2010 this paper. EPS are a complex high-molecular-weight mixture of polymers excreted by microorganisms,
Accepted 31 July 2010
produced from cell lysis and adsorbed organic matter from wastewater. They are a major component in
Available online 10 August 2010
microbial aggregates for keeping them together in a three-dimensional matrix. Their characteristics (e.g.,
adsorption abilities, biodegradability and hydrophilicity/hydrophobicity) and the contents of the main
Keywords:
Extracellular polymeric substances (EPS)
components (e.g., carbohydrates, proteins, humic substances and nucleic acids) in EPS are found to crucially
Extraction affect the properties of microbial aggregates, such as mass transfer, surface characteristics, adsorption ability,
Microbial aggregates stability, the formation of microbial aggregates etc. However, as EPS are very complex, the knowledge
Sludge regarding EPS is far from complete and much work is still required to fully understand their precise roles in
Stability the biological treatment process.
Surface characteristics © 2010 Elsevier Inc. All rights reserved.
Wastewater treatment

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883
1.1. Definition of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883
1.2. Composition of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883
1.3. Spatial distribution of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883
2. EPS extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
2.1. Evaluation of EPS extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
2.2. Selection of an appropriate extraction method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
3. Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 885
3.1. Conventional chemical colorimetric analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 885
3.2. Innovative methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 886
4. Characteristics of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
4.1. Adsorption characteristics of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
4.2. Biodegradability of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
4.3. Hydrophilicity/hydrophobicity of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
5. Factors influencing EPS production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
5.1. Substrate type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
5.2. Nutrient content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
5.3. Growth phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
5.4. External conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
6. Relationship between EPS and functions of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
6.1. Mass transfer in microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
6.2. Surface charge of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888

⁎ Corresponding author. Fax: + 86 551 3601592.

E-mail address: hqyu@ustc.edu.cn (H.-Q. Yu).

0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.08.001
 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894 883

6.3. Flocculation ability of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889

6.4. Settleability of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889
6.5. Dewatering ability of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889
6.6. Stability of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890
6.7. Adhesion ability of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890
6.8. Formation of microbial aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890
7. Future work needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891

1. Introduction soluble EPS and those forming microbial pellets being bound EPS.
However, their origins are not well known yet. Although the
In biological wastewater treatment systems, most of the micro- interaction between soluble EPS and cells is very weak, previous
organisms are present in the form of microbial aggregates, such as study showed that soluble EPS also have a crucial effect on the
sludge flocs, biofilms, and granules. The presence of extracellular microbial activity and surface characteristics of sludge (Sheng and Yu,
polymeric substances (EPS), a complex high-molecular-weight 2007). However, the study on the soluble EPS is limited, and the EPS
mixture of polymers, in pure cultures, activated sludge, granular mentioned in literature and this review without being specified are
sludge, and biofilms, has been confirmed and observed using various bound EPS. The structure of bound EPS is generally depicted by a two-
electron microscopy techniques. EPS have a significant influence on layer model (Fig. 1) (Nielsen and Jahn, 1999). The inner layer consists
the physicochemical properties of microbial aggregates, including of tightly bound EPS (TB-EPS), which has a certain shape and is bound
structure, surface charge, flocculation, settling properties, dewatering tightly and stably with the cell surface. The outer layer, which consists
properties, and adsorption ability. EPS bind with cells through of loosely bound EPS (LB-EPS), is a loose and dispersible slime layer
complex interactions to form a vast net-like structure with plenty of without an obvious edge. The content of the LB-EPS in microbial
water that protects cells against dewatering (Wingender et al., 1999) aggregates is always less than that of the TB-EPS, and thus may have
and the harm of toxic substances (Sutherland, 2001a). Part of EPS can some influence on the characteristics of microbial aggregates (Li and
serve as carbon or energy sources in conditions of nutrient shortage Yang, 2007; Sheng et al., 2006a).
(Sutherland, 2001a; Zhang and Bishop, 2003). They also accelerate the
formation of microbial aggregates through binding cells closely (Liu
1.2. Composition of EPS
et al., 2004). Thus, the in-depth study of EPS is a matter of great
interest not only in terms of improving our comprehension of
Carbohydrates and proteins are usually found to be the major
biological wastewater treatment, but also improving the efficiency
components of EPS. Humic substances may also be a key component
of such treatment through the optimization of operational para-
of the EPS in sludge in biological wastewater treatment reactors,
meters. This review outlines some important discoveries with regard
accounting for approximately 20% of the total amount (Frolund et al.,
to the EPS extraction, spatial distribution of EPS in microbial
1995, 1996). In addition, lipids, nucleic acids, uronic acids and some
aggregates, analytical techniques, EPS characteristics and also the
inorganic components have also been found in EPS from various
effect of EPS compositions on the functions of microbial aggregates.
matrixes (Frolund et al., 1996; Dignac et al., 1998; D'Abzac et al.,
2010a,b). Their fractions in EPS depended strongly upon the
1.1. Definition of EPS extraction methods and the sludge origins.
The content and compositions of the EPS extracted from various
EPS are present both outside of cells and in the interior of microbial microbial aggregates are reported to be heterogeneous (Wingender
aggregates. Wingender et al. (1999) used the abbreviation “EPS” as a et al., 1999). The variation in the compositions of the extracted EPS is
more general and comprehensive term to represent different classes attributed to many factors, such as culture, growth phase, process
of macromolecules such as polysaccharides, proteins, nucleic acids, parameter, bioreactor type, extraction method, and analytical tool
lipids and other polymeric compounds presented in the interior of used (Nielsen and Jahn, 1999).
various microbial aggregates. Nielsen and Jahn (1999) proposed that
all polymers outside the cell wall, which are not directly anchored to
the outer membrane or murein-protein layer, should be EPS. This 1.3. Spatial distribution of EPS
broad definition of EPS makes the results from the EPS studies
unpredictable and controversial. Actually, the EPS are mainly the The spatial distribution of EPS is reported to be heterogeneous, and
high-molecular-weight secretions from microorganisms, and the can be observed using confocal laser scanning microscopy (CLSM) or
products of cellular lysis and hydrolysis of macromolecules. In fluorescent microscopy after EPS stained by fluorescent dyes or
addition, some organic matters from wastewater can also be adsorbed
to the EPS matrix (Nielsen and Jahn, 1999; Liu and Fang, 2003). The
EPS related with the microbial metabolism may make more sense in
microbial aggregates related studies, and can affect various physico-
chemical characteristics of microbial aggregates.
The forms of EPS that exist outside of cells can be subdivided into
bound EPS (sheaths, capsular polymers, condensed gels, loosely
bound polymers, and attached organic materials) and soluble EPS
(soluble macromolecules, colloids, and slimes) (Nielsen and Jahn,
1999; Laspidou and Rittmann, 2002). Bound EPS are closely bound
with cells, while soluble EPS are weakly bound with cells or dissolved
into the solution. Generally, these two types of EPS can be separated
by centrifugation, with those remaining in the supernatant being Fig. 1. Sketch of EPS structure (adapted from Nielsen and Jahn, 1999).
884 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894
lectins. EPS was concentrated in a sludge floc center (de Beer et al.,
1996) and that some polysaccharides were present around the
network of filamentous fungi (McSwain et al., 2005). EPS are a
major structural component of biofilms, and EPS were distributed in
layers through the biofilm depth and their yield varied along the
biofilm depth (Zhang et al., 1998; Zhang and Bishop, 2001). For
anaerobic granular sludge, most of the EPS were distributed in the
outer layer, and the remainder was distributed through the rest of the
granules (de Beer et al., 1996; Zhang and Fang, 2004). Wang et al.
(2005) reported that the EPS content in the inner layer of aerobic
granular sludge was about four times greater than that in outer layer.
The distributions of various EPS components were also heteroge-
neous. McSwain et al. (2005) observed that the cells and carbohy-
drates were present in the outer layer of aerobic granular sludge,
whereas most of the proteins were found in the inner layer. Chen et al.
(2007) found that in acetate-fed aerobic granules, protein and β-D-
glucopyranose polysaccharides formed the core, whereas, the cells
and α-D-glucopyranose polysaccharides accumulated in the granule
outer layers. In the phenol-fed aerobic granules, proteins formed the
core, and the cells and α- and β-D-glucopyranose polysaccharides
were accumulated at an outer filamentous layer. These results
indicate that the EPS distribution depends on the microbial aggregate
types, structures and origins.
2. EPS extraction
2.1. Evaluation of EPS extraction methods
An ideal EPS extraction method should be effective, cause min-
imal cell lysis, and not disrupt the EPS structure (Frolund et al.,
1996). The extraction efficiency can be defined as the total amount
of EPS extracted from the total organic matter, or the total amount
of EPS extracted from the total EPS pool, for a given cell sample
(Nielsen and Jahn, 1999). It should be noted that the EPS extraction
efficiency differs significantly according to the extraction method
used.
Cell lysis might occur when EPS are extracted. However, the extent
of cell lysis during extraction is difficult to evaluate. Some studies have
taken the protein or nucleic acid content of EPS as an indicator of cell
lysis (Brown and Lester, 1980), but it is recognized that the EPS matrix
usually contains a large amount of proteins and a little nucleic acids,
and thus neither macromolecule is an accurate indicator. However, as
the nucleic acid content in EPS is usually low, a high level of nucleic
acids after EPS extraction indicates severe cell lysis. Adenosine
triphosphate and intracellular enzymes, such as glucose-6-phosphate
dehydrogenase, have been used as intracellular markers (Frolund
et al., 1996). The cell count coupled with microscopy methods, and the
live/dead cells count or staining methods has also been used to
evaluate cell lysis. This is actually based on cell wall integrity, and
hence the cell wall interruption means intracellular content release.
However, this can only be used to evaluate whether cells are
destroyed, rather than to assess whether intracellular material has
leaked out. Sheng et al. (2005a) proposed a UV–visible spectrometry
method to evaluate cell lysis through measuring the release of
intracellular compounds, and applied it to compare EPS extraction
methods for photosynthetic bacteria.
The disruption of macromolecules can take place during EPS
extraction, with boiling and alkaline treatments being reported to
cause particularly severe disruption. NaOH has been found to cause a
change in the polymer composition at pH N 9, and at high pH values,
the disulfide bindings in glycoproteins are broken and uronic acids are
degraded. The changes in the macromolecular composition of the
extracted EPS can also be evaluated using gel permeation chroma-
tography or high-pressure size exclusion chromatography (Frolund
et al., 1996).
2.2. Selection of an appropriate extraction method
A typical EPS extraction procedure is as follows (Nielsen and Jahn,
1999): (1) pretreatment, including sampling, storing, washing, and
homogenizing. This step allows the microbial cells to be dispersed. For
EPS extraction from biofilm and microbial granules, homogenizing is
always needed. (2) EPS extraction from the samples after the
pretreatment. (3) Purification for further analysis. In these steps, the
second step is the important one. EPS components should be
extracted using an appropriate extraction procedure. For soluble
EPS, centrifugation is always used, whereas for bound EPS many
different extraction methods have been developed, and new methods
are still coming to light (Nielsen and Jahn, 1999). These extraction
methods can be classified as physical methods, chemical methods, and
a combination of physical and chemical methods, as listed in Table 1.
The physical extraction methods usually utilize the external forces,
which are created by ultrasonic, centrifugation, or heating, to
encourage the EPS to detach from cells and dissolve in solution. The
chemical extraction methods involve in adding chemical compounds
to disrupt the binding interactions between the EPS and the cells in
order to accelerate the dissolution of the EPS. In general, the
extraction efficiencies of the physical extraction methods are lower
than those of the chemical extraction methods. Furthermore, different
methods have various extraction efficiencies for these components,
which results in various compositions in the EPS (Liu and Fang, 2002a;
Sheng et al., 2005a; D'Abzac et al., 2010b).
To study the compositions and functions of the LB-EPS and TB-EPS in
a sludge sample, the two fractions of the bound EPS may be extracted
separately. As the LB-EPS bound with cell loosely, a mild method (e.g.,
high-rate shear, heating at low temperatures, or high speed centrifu-
gation) should be chosen to avoid the inclusion of the TB-EPS.
Subsequently, a harsh method (e.g., heating at high temperatures,
sonication or chemical extraction methods) should be applied for the
TB-EPS extraction. Li and Yang (2007) modified a heating extraction
method which included a mild step and a harsh step for extracting the
LB-EPS and TB-EPS from activated sludge subsequently. The cell lysis
was not significant after such extraction process. The high speed
centrifugation and ultrasonication were also used to extract the LB-EPS
and TB-EPS from sludge (Ramesh et al., 2007).
As has been highlighted, a number of methods have been
developed and applied to extract EPS from pure or undefined cultures.
The extraction efficiencies of chemical methods, such as cation
exchange resin (CER) (Frolund et al., 1996), EDTA (Sheng et al.,
2005a), and the HCHO/NaOH methods (Liu and Fang, 2002a), are
always higher than those of physical methods, but the application of
chemicals brings about certain problems either in the extraction
process itself or in the subsequent EPS analysis. Alkali treatment can
cause severe cell lysis and the disruption of macromolecules. The
EDTA method has a high extraction efficiency and causes a low degree
of cell lysis. However, the residual EDTA would contaminate the EPS
extraction (Comte et al., 2006b), and then interfere with protein
determination in the Lowry method. The dialysis is always needed for
EDTA extraction method. The dose of HCHO in the HCHO/NaOH
extraction method changes the EPS characteristics and causes
substantial interference in the determination of carbohydrates. EPS
extraction using an enzyme for hydrolysis is a mild and effective
method, but the extraction efficiency is not very high for activated
sludge (Sesay et al., 2006). Because of its high efficiency and low cell
lysis, the CER method has become the most widely accepted EPS
extraction method, largely because the resin can be removed readily,
which means that pollution by chemical reagents is avoided and
subsequent analysis is easier.
It should be mentioned that none of these methods can extract all
the EPS entirely from microbial aggregates. The extraction strategies
for the extractable EPS fractions in activated sludge using various
cation-associated extraction methods were evaluated. Their results
 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894 885

Table 1
Methods for the extraction of EPS from various microbial aggregates.

Method System Mechanism Reference

Physical methods Sonication UASB granule The EPS part from the matrix under the impulsive Quarmby and Forster (1995)
Activated sludge pressure that is created by the sonication. Dignac et al. (1998)
Sonication/centrifugation Activated sludge The EPS dissolve to solution under the impulsive Jorand et al. (1995)
pressure created by the sonication and centrifugal force.
High speed centrifugation Anaerobic sludge The EPS detach from cell surface and dissolve to Fang and Jia (1996)
solution under the centrifugal force.
Heating Activated sludge The molecular movement is enhanced that accelerates Li and Yang (2007)
the EPS dissolution.
Chemical methods Alkaline treatment R. acidophila Alkaline treatment by the addition of NaOH causes Sheng et al. (2005a)
Activated sludge the groups, such as the carboxylic groups, to be ionized, Brown and Lester (1980)
resulting in a strong repulsion between the EPS and
the cells, and thus makes the EPS dissolve in water.
Sulfide Activated sludge Addition of sulfide to sludge could remove Fe by Park and Novak (2007)
formation of FeS, resulting in a significant disintegration
of the sludge floc structure.
EDTA Anaerobic sludge Divalent cations are very important for the crosslinking Fang and Jia (1996)
R. acidophila of charged compounds in the EPS matrix, and thus the Sheng et al. (2005a)
Marinobacter sp. removal of these cations using EDTA causes the EPS Bhaskar and Bhosle (2006)
matrix to fall apart.
CER Biofilms CER removes the divalent cations, thus causing the Jahn and Nielsen (1995)
Activated sludge EPS to fall apart. Frolund et al. (1996)
NaCl P. aeruginosa Cation exchange is promoted by using a high May and Chakrabarty (1994)
concentration of NaCl.
Acidic treatment R. acidophila Improves the repulsive force and disrupts the Sheng et al. (2005a)
interaction between EPS and cells, causing the EPS to
fall away from the cell surface.
NH4OH/EDTA Activated sludge This method combines the pH adjustment and ion Sato and Ose (1984)
exchange methods to improve the extraction efficiency.
Using a strong alkali such as NH4OH reduces cell lysis.
Ethanol extraction Activated sludge Denatures the EPS and reduces the binding force Forster and Clarke (1983)
between EPS and cells.
HCHO/NaOH Activated sludge The addition of HCHO reduces the cell lysis that is Liu and Fang (2002a)
caused by the addition of NaOH.
Glutaraldehyde Activated sludge As glutaraldehyde has the ability to fix cells and Azeredo et al. (1998)
denaturize EPS, it can also be used to extract EPS.
Crown ether Biofilms and activated sludge Crown ether is used to combine divalent metals and Wuertz et al. (2001)
disrupt the binding interaction between EPS and cells.
Enzymatic extraction Activated sludge The carbohydrate and protein-hydrolyzing enzymes Sesay et al. (2006)
were used to disrupt the structure of sludge and
dissolve the EPS.

show that the CER method is highly selective for the Ca- and Mg-
bound EPS, whereas that the sulfide extraction method is selective for
the Fe-bound EPS. On the other hand, the alkaline extraction is found
to be less specific than these two methods, but it is more effective to
extract the Al-bound EPS (Park and Novak, 2007). As no universal
extraction method is available for the quantitative extraction of EPS
from microbial aggregates, a method must be selected and optimized
for each case, taking into consideration the sample characteristics.
Several EPS extraction methods must be compared and the most
appropriate method should be chosen carefully. A combined extrac-
tion was necessary to target different EPS fractions and that repeated
extractions were always needed to obtain a high extraction efficiency.
The cell lysis should be evaluated carefully for the combined and
repeated extractions.
3. Analytical methods
3.1. Conventional chemical colorimetric analyses
The composition of the EPS matrix in biofilms and activated sludge
is reported to be very complex, containing proteins, carbohydrates,
nucleic acids, lipids, humic substances etc. Conventional chemical
colorimetric analyses can be used to quantify their contents in EPS
(Raunkjær et al., 1994). Generally, the carbohydrate content is
measured using the anthrone method or the phenol–sulfuric acid
method. A comparison between the two methods for carbohydrates
determination in EPS showed that the two methods yielded similar
results, but that the coefficient of variation for the anthrone method
was lower than that for the phenol–sulfuric method (Frolund et al.,
1996). The protein content is measured using the Lowry method, the
Bradford method, or the total N-content method. The Lowry method
has a higher recovery than the Bradford method (Frolund et al., 1996).
The total N-content method is more accurate, but the procedures are
complex. Thus, the Lowry method is frequently applied for protein
analyses in EPS characterization. As the phenolic functional groups of
humic acids also react with the Lowry reagent, the appropriate
correction is always needed.
Humic substances are very complex, and there are fewer
appropriate methods for measuring their content in EPS. Frolund
et al. (1995) proposed a modified Lowry method to determine the
humic substance content by correcting the protein interference. The
uronic acid content can be measured using the m-hydroxydiphenyl
sulfuric acid method (Blumenkrantz and Asboe-Hansen, 1973). The
DNA or nucleic acid content is measured using the DAPI fluorescence
method (Frolund et al., 1996), the UV absorbance method (Sheng
et al., 2005a), or the diphenylamine method (Liu and Fang, 2002a).
The UV absorbance method is easy to perform, but is readily interfered
by proteins. The DAPI method for DNA estimation works well, but its
procedures are complex. Therefore, the diphenylamine method could
be used more conveniently and widely.
886 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894

3.2. Innovative methods
The complex compositions make it difficult to analyze the
conformation, structure, distribution and functions of EPS. However,
progress in analytical chemistry has led to the development of new
instruments and techniques for the characterization of EPS, as listed in
Table 2, which has generated a large amount of information about the
structural and functional properties of EPS as well as their environ-
mental behavior.
The EPS presented in microbial aggregates with amorphous-phase
surrounding cells were directly observed using electronic microscopic
technology (Li and Ganczarczyk, 1990). Using the conventional
scanning electron microscopy (SEM) and transmission electron
microscopy (TEM), the microbial aggregates should be fixed and
dewatered first, which would change the original conformation of
EPS. The environmental scanning electron microscopy (ESEM) (Beech
et al., 1996), atomic force microscopy (AFM) (van der Aa and Dufrene,
2002; Li and Logan, 2004) and CLSM (Zhang and Fang, 2001) could be
used to observe the fully hydrated samples to obtain the original
shapes and structures of EPS. After staining by various fluorescence
probes, the spatial distributions of carbohydrates, proteins and nucleic
acids in EPS can also be obtained by CLSM (Staudt et al., 2004).
The chromatography, mass spectrometry and their combination
could be used to qualitatively and quantitatively analyze the EPS
compositions (Dignac et al., 1998). The spectroscopy, including X-ray
photoelectron spectroscopy (XPS) (Dufrene and Rouxhet, 1996;
Omoike and Chorover, 2004; Ortega-Morales et al., 2007), Fourier
transform infrared spectroscopy (FTIR) (Allen et al., 2004; Omoike
and Chorover, 2004; Sheng et al., 2006b), 3-dimensional excitation–
emission matrix fluorescence spectroscopy (3D-EEM) (Esparza-Soto
and Westerhoff, 2001; Sheng and Yu, 2006a), and nuclear magnetic
resonance (NMR) (Manca et al., 1996; Lattner et al., 2003) can be used
to elucidate the functional groups and element compositions in EPS or
microbial aggregates. EPS contain large quantities of aromatic
structures and unsaturated fatty chains with various types of
functional groups, which have fluorescence characteristics. As a
rapid, selective and sensitive technique, 3D-EEM fluorescence
spectroscopy is useful for studying the physicochemical properties
of EPS, as fluorescence characteristics are greatly related to their
structure and functional groups in molecules.
Due to the high sensitivity, good selectivity, and non-destruction
of samples, these spectroscopy techniques could also be used to
characterize the adsorption pollutants to EPS from the changes of
their functional groups in EPS (Manca et al., 1996; Omoike and

Table 2
New analytical techniques used in EPS research.

Analytical techniques Descriptions References

Gas chromatography (GC)
High-performance liquid
chromatography (HPLC)
Gas chromatography–mass
spectrometry (GC–MS)
Scanning electron microscopy (SEM) Observation of the microstructure and EPS of microbial
Transmission electron
microscopy (TEM)
Environmental scanning electron
microscopy (ESEM)
Atomic force microscopy (AFM)
Confocal laser scanning
microscopy (CLSM)
Quartz crystal microbalance (QCM) Be used to study the kinetics of EPS adsorption or cell
X-ray photoelectron spectroscopy
(XPS)
Fourier transform infrared
spectroscopy (FTIR)
Raman spectroscopy
3-Dimensional excitation–emission A sensitive and selective method that needs only
matrix fluorescence
spectroscopy (3D-EEM)
Nuclear magnetic resonance (NMR) Can be used to study the molecular structure of
High-performance size exclusion
chromatography (HPSEC)
Time-of-flight secondary-ion mass
spectrometry (TOF-SIMS)
Qualitative and quantitative analyses of the monosaccharides Dignac et al. (1998) and Ortega-Morales et al. (2007)
and amino acids of EPS after hydrolysis.
Li and Ganczarczyk (1990), Macleod et al. (1995) and Bura et al. (1998)
aggregates after fixation and dewatering. Slices may be
needed for the observation of the interior structure of
microbial aggregates. Combining with energy dispersive
X-ray spectrometer (EDX), the element composition and
distribution of EPS can be obtained.
Technique for observing fully hydrated samples to Beech et al. (1996) and Surman et al. (1996)
obtain the original shapes and structure of EPS.
Be used to observe the surface shape and structure of Beech et al. (1996), van der Aa and Dufrene (2002), and Li and Logan
EPS in the bacteria cells. It can also be used to evaluate (2004)
the adhesion force between cells and solid surface.
Be used to observe the structure of fully hydrated samples. Zhang and Fang (2001), Kawaguchi and Decho (2002), and Staudt et al.
The spatial distribution of EPS also can be obtained using (2004)
various fluorescence probes. The total EPS content can be
evaluated from the CLSM figures through the use of
conversion factors.
Kwon et al. (2006), Long et al. (2009), and Zhu et al. (2009)
adhesion onto material surface in situ and in real-time.
Be used to study the surface functional groups of EPS, Dufrene and Rouxhet (1996), Omoike and Chorover (2004), and
the interactions between EPS and metals, and the role Ortega-Morales et al. (2007)
of EPS in microbial adhesion to substrates.
Be used to study the functional groups of microbial Allen et al. (2004), Omoike and Chorover (2004), and Sheng et al.
aggregates and their associated EPS. The attenuated total (2006b)
reflection technique (FTIR/ATR) can be used to observe
the microbial adhesion in solutions in situ.
Be used to study the chemical structure of EPS in situ. Wagner et al. (2009)
Esparza-Soto and Westerhoff (2001) and Sheng and Yu (2006a)
very small samples and does not destroy the structure
of the samples. Has recently been used to characterize
EPS of various origins.
Manca et al. (1996) and Lattner et al. (2003)
carbohydrates and proteins and the interaction
between EPS and metals.
Can be used to characterize the molecular weight Frolund et al. (1996), Gorner et al. (2003), and Garnier et al. (2005)
distribution of EPS and to evaluate extraction methods.
HPSEC combined with other techniques (e.g., FTIR)
can be used to characterize EPS with different
molecular weight fractions.
Be used to characterize the chemical compositions of EPS Pradier et al. (2005)
and the interaction of EPS and metals.
 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894
Chorover, 2004; Sheng and Yu, 2006a). For example, from the
fluorescence quenching degree of EPS or UV–Vis spectral changes
before and after adsorption, the binding strength of pollutants onto
EPS could be evaluated (Sheng et al., 2008; Pan et al., 2010).
4. Characteristics of EPS
4.1. Adsorption characteristics of EPS
The EPS in microbial aggregates have many sites for the adsorption
of metals and organic matters, such aromatics, aliphatics in proteins,
and hydrophobic regions in carbohydrates (Flemming and Leis, 2002).
This reveals the potential roles of EPS in heavy metal sorption to
bacterial cells and transporting in environments (Toner et al., 2005;
Guine et al., 2006; Hu et al., 2007).
The presence of many functional groups in EPS, such as carboxyl,
phosphoric, sulfhydryl, phenolic and hydroxyl groups, can complex
with heavy metals (Liu and Fang, 2002b; Joshi and Juwarkar, 2009; Ha
et al., 2010). Based on the estimated numbers of the available carboxyl
and hydroxyl groups, the EPS are regarded to have a very high binding
capacity (Flemming and Leis, 2002; Guibaud et al., 2003; 2006).
Proteins, carbohydrates and nucleic acids in EPS all have the abilities
to complex with heavy metals (Dignac et al., 1998; Priester et al.,
2006; Zhang et al., 2006). The binding capability and strength of the
bonds between EPS and heavy metals were known to be high, and the
adsorption obeyed the Langmuir or Freundlich equations (Bhaskar
and Bhosle, 2006; Moon et al., 2006; Zhang et al., 2006). Furthermore,
the soluble EPS might have a greater adsorptive ability for heavy
metals than the bound EPS from sludge (Comte et al., 2006a).
The binding between EPS and divalent cations, such as Ca2+ and
2+
Mg , is one of the main intermolecular interactions in maintaining
the microbial aggregate structure (Mayer et al., 1999). In the
adsorption of heavy metal onto activated sludge, Ca2+ and Mg2+
were found to release into the solution simultaneously, indicating that
the ion exchange mechanism was involved (Yuncu et al., 2006).
EPS can also adsorb organic pollutants, such as phenanthrene (Liu
et al., 2000), benzene (Spath et al., 1998), humic acids (Esparza-Soto
and Westerhoff, 2003), and dye (Sheng et al., 2008). It might be
attributed to the fact that there are some hydrophobic regions in EPS
(Spath et al., 1998; Flemming and Leis, 2002). Spath et al. (1998)
reported that more than 60% of benzene, toluene and m-xylene were
adsorbed by EPS, and that only a small fraction of these pollutants was
adsorbed by cells. The EPS are always negatively charged, and could
bind with the positively charged organic pollutants through electro-
static interaction (Esparza-Soto and Westerhoff, 2003). Moreover,
proteins have a high binding strength and capability than humic
substances. The soluble EPS have a higher fraction of proteins than the
bound EPS, and thus they may have a greater binding capacity than
the bound EPS (Pan et al., 2010).
4.2. Biodegradability of EPS
EPS can also be used by bacteria as sources of carbon and energy.
Usually, the main components of the EPS are carbohydrates and
proteins, and in biological wastewater treatment reactors the
enzymes for the degradation of these polymers are abundant. The
bacteria in activated sludge can utilize the EPS that are excreted by
other bacteria for metabolic activity (Boyd and Chakrabarty, 1994;
Zhang and Bishop, 2003). However, Laspidou and Rittmann (2002)
argued that certain parts of EPS cannot be degraded by microorgan-
isms. Wang et al. (2005, 2007) also regarded that part of EPS from
aerobic granular sludge were biodegradable and elaborated that the
EPS that were present in the outer layer of aerobic granular sludge
could not be biodegraded, although those located in the inner layer
were biodegradable. Park and Novak (2007) reported that the EPS
extracted using various methods had different biodegradability. For
887
instance, the EPS extracted using the CER method were aerobic
biodegradable, while the EPS extracted using the sulfide method were
anaerobic biodegradable. The small molecular substances that are
produced as a result of EPS degradation can be used as carbon and
energy sources for cell growth in conditions of nutrient shortage. EPS
degradation can also result in the deflocculation of sludge flocs. The
non-degradable portion of EPS may flow with the effluent from
reactors and deteriorate the quality of the effluent.
4.3. Hydrophilicity/hydrophobicity of EPS
The EPS in microbial aggregates have many charged groups (e.g.,
carboxyl, phosphoric, sulfhydryl, phenolic and hydroxyl groups) and
apolar groups (e.g., aromatics, aliphatics in proteins, and hydrophobic
regions in carbohydrates) (Flemming and Leis, 2002). The formation of
hydrophobic areas in EPS would be beneficial for organic pollutant
adsorption (Spath et al., 1998). The presence of hydrophilic and
hydrophobic groups in EPS molecules indicates that EPS are amphoteric.
The relative ratio of these two groups is related to the composition of
EPS. Jorand et al. (1998) used XAD resin to separate the hydrophilic and
hydrophobic EPS fractions and found that approximately 7% were
hydrophobic and mainly comprised proteins, whereas the hydrophilic
fraction mainly consisted of carbohydrates. The analysis of monosac-
charide and amino acid contents in EPS after hydrolysis revealed
that approximately 25% of the amino acids were negatively charged
and approximately 24% were hydrophobic (Dignac et al., 1998). The
hydrophilicity/hydrophobicity of EPS is likely to significantly influence
the hydrophobicity of microbial aggregates and their formation
in bioreactors (Liu and Fang, 2003). It also demonstrates the importance
of the EPS as the sorption sites for organic pollutants (Flemming and
Leis, 2002).
5. Factors influencing EPS production
5.1. Substrate type
Substrate type has a substantial effect on the microbial commu-
nities in sludge, and the microbial metabolism, thus influences the
production of EPS. Li and Yang (2007) reported that the sludge fed
with glucose had more EPS production than that fed with acetate.
Sponza (2002, 2003) examined the EPS productions of the sludge
from continuous stirred tank reactors treating various types of
wastewaters. They found that under steady-state conditions the
protein content was higher in the EPS from the sludge treating winery
and municipal wastewaters than that treating pulp-paper, textile and
petrochemical wastewaters. Sheng et al. (2006c) compared the EPS
production from a photosynthetic bacterial strain using various
substrates, and found that a bacterium would produce more EPS
using benzoate as the substrate than using acetate, propionate and
butyrate. This suggests that bacteria would excrete more EPS under
unfavorable conditions.
5.2. Nutrient content
EPS can be degraded by bacteria as carbon and energy sources
when there is a substrate shortage. Nutrient levels have a significant
effect on EPS production and composition. The EPS content of the
sludge increased with an increase in food to microorganism ratio
(Jang et al., 2007). EPS production could be promoted when the
phosphor was in short supply (Liu et al., 2006). Bura et al. (1998) and
Hoa et al. (2003) also found that the carbohydrate content in EPS
extracted from activated sludge increases when phosphorus is in
short supply. Durmaz and Sanin (2001) found EPS in activated sludge
to be rich in proteins but low in carbohydrates at a carbon to nitrogen
ratio of 5, but as the carbon to nitrogen ratio increased to 40, the
amount of protein decreased sharply whereas the amount of
888 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894
carbohydrates increased. Other researchers have found that activated
sludge growing on wastewater with a low carbon to nitrogen ratio
tends to produce EPS with a high proteins/carbohydrates ratio (Bura
et al., 1998; Liu and Fang, 2003).
5.3. Growth phase
Jia et al. (1996) investigated the effects of cultivation time on EPS
production in activated sludge, and found the EPS content to be
closely related to the bacterial growth phase. During the exponential
growth phase, the EPS content increased with cultivation time, but
during the stationary phase it decreased with increasing cultivation
time. In contrast, the EPS content from a photosynthetic bacterial
strain decreased with cultivation time during the exponential growth
phase, but remained almost unchanged during the stationary phase
(Sheng et al., 2006b; Sheng and Yu, 2006b).
Solid retention time (SRT) also has a considerable effect on the
production of EPS, but the results reported in literature are somewhat
contradictory. Many researchers have found that the EPS in various
microbial aggregates increases with an increasing SRT, which implies
that bacteria produce more EPS in endogenous conditions. Sesay et al.
(2006) found that an increase in SRT had a significant and positive
correlation with the total quantity of EPS in activated sludge as well as
the contents of proteins and carbohydrates in EPS. The ratio of
proteins to carbohydrates also increased from 1.5 to 2.5 with an
increase in SRT from 4 to 20 days. However, some researchers had
suggested that EPS is independent of SRT. Liao et al. (2001) found that
the EPS content did not change significantly with a longer SRT, and the
protein/carbohydrate ratio increased as the SRT was increased from 4
to 12 days, but remained unchanged as the SRT was further increased
from 12 to 16 days. Li and Yang (2007) reported that the TB-EPS of
activated sludge had no relationship with the SRT, but that the LB-EPS
decreased with an increasing SRT.
5.4. External conditions
As EPS are bound with cells mainly through ion bridging with
multivalent metals, metal concentration may also influence the EPS
content. Turakhia and Characklis (1989) and Sheng et al. (2006c)
reported that the Ca2+ concentration had no effect on the EPS,
whereas Higgins and Novak (1997) found that the protein content in
the sludge EPS increased at higher Ca2+ or Mg2+ concentrations, and
that higher Na concentrations led to a lower protein content. With the
increasing concentration of Fe fed to sludge, the EPS characteristics
and components could also be altered (Li, 2005).
In the presence of toxic substances such as heavy metals, microbial
cells in activated sludge and biofilms produced more EPS to protect
themselves from the harsh environment (Fang et al., 2002; Aquino
and Stuckey, 2004; Priester et al., 2006). Furthermore, under toxic
conditions the increase of the protein content far exceeded that of
other components in EPS. As the concentration of toxic substances
went over a threshold, its effect on the promotion of EPS production
became less significant (Sheng et al., 2005b). However, some toxic
substances, e.g., bismuth dimercaptopropanol, can also inhibit the
production of EPS. At a level just below the minimum inhibitory
concentration, the production of EPS of Brevundimonas diminuta could
be significantly reduced. Total carbohydrates and proteins decreased
by approximately 95% over a 5-day period after being exposed to
bismuth dimercaptopropanol at a level near the minimum inhibitory
concentration (Badireddy et al., 2008).
The shear rate of reactors can also influence the composition of
EPS. An increase in shear rate or aeration intensity in SBRs could
increase the EPS content in sludge (Adav et al., 2007). Research also
found that the carbohydrate contents in EPS extracted from activated
sludge increased with increasing air flow rate in a sequence batch
reactor (SBR), whereas the protein contents remained almost
unchanged at various air flow rates (Shin et al., 2001), which
indicated that shear may stimulate bacteria to produce more
carbohydrates. Ramasamy and Zhang (2005) found that a sudden
increase in shear rate led to an increased carbohydrate content in EPS,
but after a period of cultivation the carbohydrate content returned to
the original level. The EPS in sludge also could be released by
hydrodynamic or mechanic shear force, which would lead to an
increase in the content of soluble EPS (Aquino and Stuckey, 2006;
Sheng et al., 2006a).
The aerobic or anaerobic conditions also can influence the
production of EPS. The EPS content of sludge would decrease under
anaerobic conditions (Nielsen et al., 1996). It is reported that activated
sludge flocs tend to disintegrate under oxygen limitation or depletion
conditions. Such a disintegration might be caused by the suppression
of EPS production or the hydrolysis of EPS as well. Shin et al. (2001)
compared the EPS production of activated sludge in three bioreactors
and found that at a high dissolved oxygen level the production of
carbohydrates in EPS increased with time, whereas the protein
content remained unchanged. At a low dissolved oxygen level, the
concentrations of both carbohydrates and proteins are kept the same.
6. Relationship between EPS and functions of microbial
aggregates
6.1. Mass transfer in microbial aggregates
In wastewater treatment reactors, EPS cover the surface or fill in
the interior of cells of microbial aggregates. Li and Ganczarczyk (1990)
observed the presence of plenty of EPS in the interior of activated
sludge flocs with amorphous-phase surrounding cells. This suggests
that substrate must pass through the EPS layer for transferring to cells.
The pores in granular sludge can also be clogged by the EPS, which
decreases the mass transfer efficiency of the substrates (Mu et al.,
2006; Zheng and Yu, 2007). Generally, the component diffusion
coefficients of EPS are lower than those of water, which means that
EPS may influence the import of nutrients and the export of metabolic
products. Characklis et al. (1990) claimed that as impermeable
substances, EPS may prevent the permeation of dye to cells. EPS
significantly influence the effective diffusion coefficients of substrates.
A high level of EPS is not beneficial for substrate mass transfer. The
permeability of anaerobic granules was found to be lower at a higher
level of EPS (Mu and Yu, 2006). However, as EPS can adsorb organic
substances and increase their concentration in the region of the cell
surface, the role of EPS in mass transfer must be carefully considered
(Hinson and Kocher, 1996).
6.2. Surface charge of microbial aggregates
As there are many charged functional groups in EPS, their content
and composition influence the surface charge of microbial aggregates.
However, the physicochemical characteristics of the different compo-
nents in EPS are not identical, and thus they have different effects on
the surface charge of aggregates. Usually, the EPS content had a
positive effect on the net negative surface charge of sludge (Jia et al.,
1996; Mikkelsen and Keiding, 2002a). Wilen et al. (2003) found that
the total EPS content and the individual components both had a
positive effect on the negative charge of sludge, and that the effects of
proteins and humic substances were the most significant. Liao et al.
(2001) reported that the carbohydrate content of EPS had a positive
relationship with the net surface charge. On the contrary, Wang et al.
(2005) investigated changes in EPS and the surface characteristics of
sludge in the aerobic sludge granulation process, and found that the
total EPS content and the protein and carbohydrate contents had a
negative effect on the net surface charge of sludge. On the contrary,
the DNA content had no significant effect on either the surface charge
or the hydrophobicity of the sludge.
 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894
The ratios among EPS components have a more significant effect
on surface charge of microbial aggregates than the content of
individual components (Morgan et al., 1990). The proteins/carbohy-
drates ratio was found to have a negative effect on net surface charges
of sludge, while total EPS content was found to have no influence (Liao
et al., 2001). This attributed to the unique charge properties of
proteins. The amino groups in proteins are positive and can neutralize
the negative charges from carboxyl and phosphate groups, and thus
decrease the net negative surface charges of sludge.
6.3. Flocculation ability of microbial aggregates
The flocculation ability of microbial aggregates is a key to the
achievement of a low turbidity and a high quality of effluent. The
interactions between EPS and cells have a significant effect on
microbial flocculation ability (Morgan et al., 1990). In a study of the
deflocculation of activated sludge under anaerobic conditions,
bacteria and EPS were found to make up the main part of the
deflocculated matter (Wilen et al., 2000), which indicated that EPS
play an important role in flocculation.
There are two flocculation mechanisms induced by cations: double
layer compression and ion bridging through EPS. A high ionic
concentration would promote the bacterial flocculation and that
such a phenomenon was attributed to the compressing double electric
layer (Liu et al., 2007a). The ion bridging interactions between
multivalent cations and EPS also play a key role in microbial
flocculation (Sobeck and Higgins, 2002; Nguyen et al., 2007).
Multivalent cations (e.g., Ca2+ and Mg2+) tend to bridge with EPS
and thus improve the flocculation of microbial aggregates (Higgins
and Novak, 1997; Liu et al., 2007a). An increase in the monovalent
cations in activated sludge would deteriorate the sludge character-
istics and floc structure (Kara et al., 2008). The dispersed cells in
activated sludge tended to reflocculate with the addition of Ca2+ (Zita
and Hermansson, 1994). To improve the flocculation ability of sludge,
direct addition of multivalent cations could be one useful approach
(Higgins et al., 2004).
The role of EPS composition in relation to flocculation has also
been studied. Microbial aggregates tend to deflocculate after the
removal of their surface proteins. Addition of a small amount of
protein-hydrolyzing enzyme to a reactor would lead to the sludge
deflocculation, while dose of a carbohydrate-degrading enzyme
caused much less deflocculation (Higgins and Novak, 1997). Nucleic
acids may also play an important role in flocculation. The bacterial
flocculation ability worsened after the degradation of nucleic acids in
the EPS of Rhodovulum sp., which was treated by nucleic acid
hydrolase (Watanabe et al., 1998). Wilen et al. (2003) found that the
flocculation ability of sludge increased with an increase in the protein
content or a decrease in the humic substance content, and decreased
with an increase in the total EPS content. These results imply that the
influence of individual EPS components on the flocculation of
microbial aggregates is complex. The ratios of the main EPS
components may be more influential on the microbial flocculation
(Liao et al., 2001). The authors also emphasized that readily extracted
EPS are more beneficial for flocculation. Li and Yang (2007) indicated
that the LB-EPS had a negative effect on sludge flocculation and
excessive EPS in the form of LB-EPS could weaken cell attachment
and result in poor flocculation.
The microbial cell flocculation can be described using the classical
Derjaguin–Landau–Verwey–Overbeek (DLVO) or extended DLVO
theories (Bos et al., 1999; Liu et al., 2007a). In the DLVO theory the
total energy of adhesion is the result of the van der Waals attractive
forces and the generally repulsive interactions due to the interpen-
etration of the electrical double layers (Rijnaarts, et al., 1999), while
the van der Waals forces, polar interactions, electrical double layer
interaction and Brownian movement forces are taken into consider-
ation by the extended DLVO theory. If the cell kinetics could overcome
889
the total energy barrier in the DLVO curves, the cell could aggregate
and flocculation would occur. The DLVO theory provides an effective
way to evaluate the contribution of EPS to the sludge flocculation
(Fig. 2) (Liu et al., 2010).
6.4. Settleability of microbial aggregates
Many studies have demonstrated EPS to have a negative effect on
the settleability of microbial aggregates (Jin et al., 2003). As EPS are
negatively charged, a high concentration of EPS increases the surface
charge of microbes, which results in an increase in the repulsive forces
between cells and a decrease in the settleability of microbial
aggregates (Morgan et al., 1990). The LB-EPS are also found to have
a negative effect on the sludge settleability, due to the fact that an
increase in LB-EPS content may bring more bound water into the
aggregates, and therefore produce highly porous flocs with a low
density (Yang and Li, 2009). The sludge volume index (SVI) is often
used to characterize the settleability of sludge, with a low SVI value
denoting good settleability.
Generally, the SVI of microbial aggregates increases as the EPS
content increases. However, up to now, the effects of the main
components of EPS on the settleability of microbial aggregates have
not been well elucidated. Proteins and DNA contents in EPS would
have more significant effects on the settleability of microbial
aggregates. The DNA and protein contents in EPS were found to
have a positive relationship with the SVI (Bura et al., 1998; Martinez
et al., 2000; Liao et al., 2001), while the effect of the carbohydrate
content in EPS on SVI was not significant (Liao et al., 2001; Hoa et al.,
2003).
6.5. Dewatering ability of microbial aggregates
EPS can be regarded as key factors in the thickening and
dewatering processes of sludge (Houghton et al., 2001; Mikkelsen
and Keiding, 2002a). Two types of binding mechanisms between
water molecules and EPS are involved: electrostatic interactions and
hydrogen bonds. The former are active between the permanent
dipole of the water of the functional groups of the EPS, and the latter
are active between EPS hydroxyl groups and water molecules (Neyens
et al., 2004).
Generally, the pressure-filtration is the main means for sludge
dewatering. The specific resistance to or capillary suction time is
commonly used to characterize the dewatering ability of sludge
through press. An increase in EPS generally leads to a poorer sludge
dewatering ability, possibly because the steric force that is generated
by EPS prevents contact between cells. In addition, the
200
TB-EPS
160
Gpi
120 EPS
Gpi
G (KT)
80
LB-EPS
40 Gpi
0
-40
0 4 8 12 16 20
Separation distance H (nm)
Fig. 2. Total interaction energy profiles of EPS as a function of interparticle distance (Liu
et al., 2010).
890 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894
macromolecules in EPS cause the retention of much water in sludge
flocs and increase the amount of interstitial water in such flocs. EPS
can also form a stable gel that prevents water seepage from the pores
of flocs, which deteriorates the dewatering ability of sludge. After EPS
were removed, the sludge dewatering ability would be improved
(Chen et al., 2001).
However, some studies have shown that sludge dewatering ability
improves as the EPS content increases (Mikkelsen and Keiding, 2002a;
Jin et al., 2004). With a higher EPS content, activated sludge had a
lower shear sensitivity and lower degree of dispersion, leading to a
good dewatering ability (Mikkelsen and Keiding, 2002a). Houghton
et al. (2001) proposed that the effect of EPS on the dewatering ability
of sludge depended on the content of EPS in sludge. Using sludge
samples from eight wastewater treatments, they found that the
dewatering ability of activated sludge initially increased with the EPS
content, but then decreased once the EPS content exceeded a certain
threshold. This suggests that a lower EPS content is more beneficial for
flocculation, as this causes the cells to bind more tightly. As the EPS
content further increased and exceeded a certain threshold, the water
that was retained by the EPS significantly increased, which resulted in
a lower sludge dewatering ability (Houghton and Stephenson, 2002).
The various components in EPS have different effects on the
dewatering ability of microbial aggregates. The proteins had a high
water-holding capacity. Thus, a reduced protein fraction in sludge EPS
improved the sludge dewatering ability (Sponza, 2002; Cetin and
Erdincler, 2004). Increasing carbohydrate fraction in EPS would
enhance the sludge dewatering (Cetin and Erdincler, 2004; Jin et al.,
2004). The effect of humic substances on dewatering ability of sludge
was insignificant.
6.6. Stability of microbial aggregates
The stability of microbial aggregates is essential to the solid/liquid
separation process in biological wastewater treatment systems (Seka
and Verstraete, 2003). Stability is defined as the ability of microbial
aggregates to resist hydrodynamic and mechanical shear (Liao et al.,
2002; Mikkelsen and Keiding, 2002b; Sheng and Yu, 2006c). Particles,
bacteria, and EPS are eroded from the surface of aggregates as a result
of hydrodynamic shear force (Mikkelsen and Keiding, 2002b). The
structure of microbial aggregates is an important factor that governs
the stability of flocs and the solid/liquid separation process (Chu and
Lee, 2004; Sheng et al., 2006a). The EPS are involved in the structure of
microbial aggregates and the interactions between cells. In addition,
the main intermolecular interactions between cells, including poly-
mer entanglement (Mikkelsen and Nielsen, 2001), ion bridging
through EPS, and electrostatic interaction (Klausen et al, 2004;
Sheng et al., 2006a), as well as van deer Waal's force and hydrogen
bonds (Mayer et al., 1999), contribute to the stability of microbial
aggregates. EPS are thus expected to govern to a certain extent the
stability of microbial aggregates (Stoodley et al., 2002; Adav et al.,
2008). A higher EPS content in sludge would result in greater sludge
stability (Mikkelsen and Nielsen, 2001). Sheng et al. (2006a) proposed
that the sludge had a multiple-layer structure with two distinct
regions, as illustrated in Fig. 3. The outer region was a dispersible part,
in which the dispersible sludge cells were glued by the readily
extractable EPS. The inner region was a stable one, where the residual
sludge cells were glued by other non-readily extractable EPS. After
exposed to shear, the outer region would disperse. Thus, the readily
extracted EPS also have a close relationship with sludge stability.
6.7. Adhesion ability of microbial aggregates
The microbial surface properties are important to the interfacial
interactions between the cells and the solid surface, which is of crucial
importance for biofilm formation in the aquatic environment. The
adsorption of EPS to a material surface would alter the substrata
Fig. 3. A proposed multi-layer structural model for the sludge (Sheng et al., 2006a).
physicochemical characteristics and hence influence the initial
bacterial adhesion process (Gomez-Suarez et al., 2002; Omoike and
Chorover, 2006). The presence of EPS on cell surfaces could enhance
cell deposition in solid surface (Long et al., 2009; Tong et al., 2010).
The carboxylate, phosphate and amine functional groups were found
to contribute to the adhesion of bacteria to solid surface (Leone et al.,
2006; Parikh and Chorover, 2006). After EPS were removed from the
sludge surface, the number of attached microbial cells decreased (Park
et al., 2000). With a similar surface charge density the EPS-rich strains
had a much greater bacterial adhesion potential in porous media over
the EPS-deficient strains (Liu et al., 2007b). The adhesion of microbial
cells to a surface resulted from EPS deposition would also lead to
biocorrosion or biofouling (Sand and Gehrke, 2006).
Both electrostatic and chemical bonding interactions are associat-
ed with the bacterial adhesion (Poortinga et al., 2002). Tsuneda et al.
(2003) found that if the EPS amount was relatively small, cell
adhesion onto solid surfaces was inhibited by the electrostatic
interaction, and that if it was relatively large, cell adhesion was
enhanced by the polymeric interactions. Furthermore, the suppres-
sion of electro-repulsive forces promotes the adhesion (Tsuneda et al.,
2004). These interface force could be explored using classical DLVO
or extended DLVO theories (Bos et al., 1999). The adhesion of EPS to
solid surface was controlled by the non-DLVO forces, in addition to the
DLVO interactions (Zhu et al., 2009). However, in both theories,
the polymeric interactions and ion bridging are not taken into
account, which are also the dominant interactions in microbial
adhesion (Ong et al., 1999; Rijnaarts et al., 1999). Thus, more studies
should be carried out to evaluate the contribution of EPS onto the
bacterial adhesion.
6.8. Formation of microbial aggregates
Sludge granulation refers to the self-immobilization of microbes in
biological wastewater treatment reactors, which results in a compact
structure of aerobic and anaerobic granules. Microscopic pictures
show that there are plenty of EPS in the interior of these aerobic and
anaerobic granules (de Beer et al., 1996; Tay et al., 2001; McSwain
et al., 2005). The interactions between EPS and microbial cells can
influence the formation of granular sludge. Quarmby and Forster
(1995) found that the carbohydrate content in EPS and the granular
strength both decreased simultaneously, which suggested that EPS
played crucial roles in sludge granulation and the maintenance of the
structure of granular sludge. Zhou et al. (2006) found that a slight
overloading in UASB reactors would stimulate the EPS production and
shorten the period of granulation. Addition of exogenous EPS to
anaerobic reactors with deteriorated granules would lead to a
significant recuperation of operational performance of the reactors
 G.-P. Sheng et al. / Biotechnology Advances 28 (2010) 882–894
(Vivanco et al., 2006). In the aerobic sludge granulation process, the
EPS content increased with cultivation time at the initial stage, but
remained constant after the granules had matured (Wang et al.,
2005). All of these indicate the essential roles of EPS in microbial
granulation. The composition and distribution of EPS influence the
formation of microbial granules. EPS can bind cells closely through ion
bridging interactions, hydrophobic interactions, and polymer entan-
glement, which serves to enhance and promote the formation of
microbial granules, as shown in Fig. 4.
Bacterial biofilms are structured communities of cells enclosed in
self-produced hydrated EPS matrix adherent to an inert surface. Large
amounts of EPS are associated with the cells and contribute to the
biofilm heterogeneity (Sutherland, 2001b). EPS are viewed as
important mediators in the adhesion of bacteria to surfaces. They
are involved in the adhesion process during biofilm formation,
maintaining the structural integrity of biofilms and therefore the
overall stability of biofilm communities (Wolfaardt et al., 1999).
Recently, extracellular DNA was found to be a key component for
bacterial biofilm formation and was required for the initial establish-
ment of Pseudomonas aeruginosa biofilms (Whitchurch et al., 2002).
The extracellular DNA served as a cell-to-cell interconnecting matrix
component in biofilms (Allesen-Holm et al., 2006). It also has a spatial
filamentous network structure, allowing access to suspended partic-
ulate organic matters and cooperative behavior and interactions in a
community (Bockelmann et al., 2006).
7. Future work needed
EPS are very important for microbial aggregates in biological
wastewater treatment systems. However, there is still much to learn
regarding the roles of EPS in the functions and characteristics of
microbial aggregates. The following areas should be studied to gain a
greater understanding of EPS:
Development of EPS extraction methods. Extraction of EPS from
microbial aggregates is the foundation to study the EPS character-
istics and the roles of EPS played in bioreactors. As some of the
fractions of EPS in microbial aggregates could not be extracted
using the commonly used methods, new EPS extraction methods
with a high efficiency should be pursued. Such methods should be
mild to avoid the lysis of cells and the disruption of EPS.
Establishment of EPS in-situ analytical methods. The characteristics
of EPS are expected to be changed after extraction, such as
molecular structure and conformation. With the development of
modern analytical techniques, it is possible to identify the EPS
compounds using one or a combination of some in-situ innovative
analytical techniques. These methods should be able to analyze the
hydrated samples without dewatering.
Fig. 4. Sketch of microbial granulation enhanced by EPS (adapted from Liu et al., 2004).
891
Identifying the sub-fractions of EPS. In previous studies little
attention has been paid on the extraction, distribution and
characteristics of TB-EPS, LB-EPS and soluble EPS, which have
been proven to play different roles in microbial aggregates and
biological wastewater treatment systems. Identification and
elucidation of the origins, compositions and characteristics of
these sub-fractions of EPS could be useful to clarify the contra-
dictions about EPS in previous studies. Also, their functions would
be well evaluated.
Elucidation of the key roles of EPS. In previous studies, the
relationships between EPS and the functions of microbial aggre-
gates (e.g., flocculation, settlement, dewatering, adsorption etc)
are often controversial. The controversies on the roles of EPS might
be attributed to their complicated compositions. Each component
shows its own special effect and thus the overall effect might
become unpredictable and case-dependent. This also causes some
problems in manipulating the EPS content and improving the
performances of microbial aggregates. Thus, it is essential to
elucidate the roles of each major component in EPS and their sub-
fractions, i.e., LB- and TB-EPS.
Identification of the key factors influencing the production of EPS. As
the origins of EPS are complex, many factors could influence the
production of EPS. After the roles of EPS components become
known, identification of such key factors would then be very useful
to manipulate the EPS compositions and contents in microbial
aggregates and thus to improve the functions of microbial
aggregates, e.g., flocculation, settlement and dewatering abilities.
Acknowledgements
The authors wish to thank the Natural Science Foundation of China
(50625825, 50708106 and 50978243), and the Outstanding Young
Scientists Foundation of Anhui Province, China (10040606Y27) for the
partial support of this study.
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