Acta hydrochim. hydrobiol. 2006, 34, 411  424 A. Raszka et al. 411
Review
The role and significance of extracellular polymers in activated
sludge. Part I: Literature review
Anna Raszkaa, Monika Chorvatovab, Jiri Wannerb
a
Silesian University of Technology, Department of Environmental Biotechnology, Gliwice, Poland
b
Institute of Chemical Technology, Department of Water Technology and Environmental Engineering, Prague,
Czech Republic
In biological wastewater treatment, bacteria tend to aggregate by forming flocs, biofilms and
even granules. A large part of the floc structure in activated sludge is composed of extracellular
polymers. The extracellular polymeric substances (EPS) are mainly responsible for the structural
and functional integrity of biofilms/flocs and they are considered to be important for the phys-
ico-chemical properties of activated sludge. This review considers the composition of EPS and
proportion between the constituents. Moreover it includes the relationship between the EPS con-
stituents and sludge properties such as sludge retention time, hydrophobicity and surface
charge.
Keywords: Extracellular polymeric substances / polysaccharides / proteins / humic substances / nucleic acids / acti-
vated sludge /
Received: November 29, 2005; accepted: April 24, 2006
DOI 10.1002/aheh.200500640
Tenney and Stumm [4] proposed that polymers
1 What are the extracellular polymeric
exposed on the microbial surface might adsorb and
substances?
bridge between cell surfaces and therefore initiate floc
formation. The EPS are mainly responsible for the struc-
In biological wastewater treatment, bacteria tend to
tural and functional integrity of biofilms/flocs and deter-
aggregate forming flocs, biofilms and even granules. The
basic of floc structure are filamentous bacteria [1, 2]. Jen- mine its physico-chemical and biological properties. The
EPS form a three-dimensional, gel-like, highly hydrated
kins et al. [1] and Wanner [2] postulated that filamentous
and often charged biofilm matrix and play a key role in
microorganisms form a backbone within the floc, to
which the floc-formers are firmly attached. The floc-for- the understanding of structure, function, properties and
development of microbial aggregates [3, 5]. The EPS are
mers are represented by various chemoorganotrophic
highly charged polymers that interact with water in a
bacteria groups, genera and species being able to convert
similar way as gels [6].
organic substrates to specific extracellular material. They
Hydrogels are formed with a three-dimensional (3D)
form an extracellular polymeric substances (EPS) matrix
network of polymer chains, where some parts are sol-
to which the bacteria are enmeshed [2]. The variety of
microbial aggregates, constituents of prokaryotic (bac- vated by water molecules but the other parts are chemi-
teria, archaea) and eukaryotic (algae, fungi) microorgan- cally or physically linked with each other. They swell, but
do not dissolve in aqueous environment. Therefore,
isms, are kept together by EPS [3].
hydrogels can come from a cross-linked network of
hydrophilic polymers in water as the meaning of the pre-
Correspondence: Anna Raszka, Silesian University of Technology, De- fix hydro is aqueous and they maintain their 3D struc-
partment of Environmental Biotechnology, Ul. Akademicka 2, 44-100 Gli-
ture after absorbing large amounts of water and swelling
wice, Poland
[7 12]. Hydrophilic molecules are polar or charged while
E-mail: [e-mail]
Fax: [phone]
hydrophobic molecules are non-polar. Since non-polar
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
,
,
412 A. Raszka et al. Acta hydrochim. hydrobiol. 2006, 34, 411  424
molecules less readily mix in water compared to polar a covalent C C bond. However, the large number of func-
molecules, they should contribute to the binding tional groups of a macromolecule may result in an over-
together of the sludge flocs [13]. all binding energy which is well in the range of several
Hydrophobic cells would attach both on the surface covalent C C bonds [22, 24].
and within a floc structure whereas hydrophilic cells As polymers may glue and bridge the adjacent cells
would not. Thus, the microbial and macromolecular they are believed to be responsible for bioflocculation
composition of the matrix changes over time [14, 15]. process. There are three theories which explain the
Increasing the hydrophobicity of cell surfaces would pro- mechanisms of bioflocculation: DLVO theory (or double
mote cell-to-cell interaction and further serves as indu- layer theory), alginate theory, and divalent cation brid-
cing force for cell to aggregate out of hydrophilic liquid ging theory.
phase [16]. The DVLO theory describes that charged particles have
Hydrophilic interactions and hydrogen bond forces a double layer of counterions that surround the particle.
were considered as the mechanisms of binding biopoly- The first layer (known also as Stern layer) is comprised of
mers and water in the study of Jin et al. [13], who investi- a tightly associated counterion layer, and the second
gated the flocculating ability of activated sludge. The layer (known as diffuse layer) is made of less tightly asso-
bound water is not a well-defined parameter and various ciated counterions [25, 26]. The concentration of ions in
methods have been used to characterize it (e.g. drying, the diffuse layer decreases with distance from the parti-
centrifugal settling) [13, 17 19]. The water content of the cle surface until the concentration of ions equals to that
sludge can be divided into two categories:  free water of the bulk solution and thus an electric potential devel-
and  bound water [17, 20]. The free water can be easily ops around the particle. This double layer or cloud of
removed by thickening or weak mechanical means and ions surrounding the particle results in repulsion of adja-
behaves thermodynamically as pure water. The bound cent particles and inhibits aggregation. The size of the
water, on the other hand, is kept firmly in the floc double layer decreases while the ionic strength increases.
matrix, bound to the sludge or trapped between sludge With decreasing of the double layer thickness the repul-
particles, cannot be removed by mechanical means, and sion between particles also decreases and allows short-
represents a small proportion of the total water. Further- range attractive forces to promote aggregation [26].
more, the bound water has a different chemical potential In the alginate theory bioflocculation results from bac-
in comparison to the one in the bulk water. The molecu- terial production of alginate, a polysaccharide made of
lar mechanisms of water binding are of crucial impor- repeating mannuronic and gluronic acids. The unique
tance for a rational basis of the improvement of dewater- composition of this polysaccharide results from the for-
ing techniques. Schmitt et al. [21] investigated water bind- mation of alginate gels in the presence of calcium ions.
ing by measuring the exchange of D2O against H2Oinan The authors report that the settling and dewatering prop-
FTIR-ATR study. They confirmed existence of the water erties were significantly impacted by the concentration
bands and found no contribution of other molecules of calcium, but not magnesium [26, 27]. In turn the diva-
with  OH and  NH bonds and suggest that the fine struc- lent cation bridging theory claim that calcium and mag-
ture of the EPS matrix is shown to be more complex than nesium were important to the bioflocculation process. In
the usual concept of only two classes of water: pore water the theory divalent cations bridge charged functional
and bound water. In a broader sense, it can be assumed groups negatively within the EPS and this bridging helps
that the EPS matrix displays a fine structure that will to aggregate and stabilize the matrix of biopolymer and
influence not only the properties of water but also those microbes and therefore promote bioflocculation [26, 28].
of dissolved molecules and ions, and thus influences In short, biopolymer flocculants cause aggregation of
mass transport in biofilms [21 23]. particles and cells by bridging and charge neutralization.
Flemming [24] considered two types of binding When the bioflocculant extends from the particle's sur-
mechanisms between water molecules and the EPS-struc- face into the solution for a distance greater than the dis-
ture: i.e. electrostatic interactions and hydrogen bonds. tance over which the interparticle repulsion acts then
Within electrostatic interactions carboxylate groups and bridging occurs, while charge neutralization occurs
hydroxyl groups are expected to play the most important when the biopolymer flocculant is oppositely charged
roles among the functional groups present in EPS, while compared to the particles [29 31].
hydrogen bonds are mainly active between the EPS The extracellular localization of the EPS and their com-
hydroxyl groups (particularly frequent in polysacchar- position may be the result of different processes: active
ides) and water molecules. The individual binding force secretion, shedding of cell surface material, cell lysis and
of any type of weak interaction is very small compared to adsorption from the environment [3].
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Acta hydrochim. hydrobiol. 2006, 34, 411  424 Extracellular polymers in activated sludge 413
Table 1. Components of soluble and bound EPS.
The functions of the EPS matrix are mainly [3, 32]:
 adhesion to surfaces,
Soluble EPS Bound EPS
 aggregation of bacterial cells in flocs and biofilms,
 stabilization of the biofilm (flocs) structure, Macromolecules Sheaths
Colloids Capsular polymers
 formation of a protective barrier that provides resis-
Slimes Condensed gel
tance to biocides or other harmful effects,
Loosely bound polymers
 retention of water, Attached organic material
 sorption of exogenous organic compounds for the
accumulation of nutrients from the environment,
Table 2. Differences between sheath and slime in EPS.
 accumulation of enzymatic activities, such as diges-
tion of exogenous macromolecules for nutrient
Sheath Slime
acquisition.
Character of association firmly looser
Polymers are believed to be responsible for the stability
with cell
and permeability of biofilms and this can happen by a
Effect on centrifugation retained with cells stayed in
two different mechanisms: the polymers may influence supernatant
Localisation of enzymes ectoenzymes exoenzymes
the association of individual cells and their adhesion to a
support or during excretion they can form a continuous
matrix within which cells multiply and daughter cells
are imbedded [33]. When two smooth surfaces approach of association nature of polymer with the cell.  Sheath
is associated with the cell closely while  slime has no
to each other some interactions appear between them.
The interactions can be divided into long- and short- direct contact with the cell. These two fractions can be
range forces. Among the long-range forces electrostatic separated by centrifugation. Polymers that stay in super-
and van der Waals forces can be distinguished while natant we call  slime , whereas those that are retained in
cells as  sheath . Sometimes for activated sludge the
hydrogen bonds and coordination bonds belongs to the
name  slime is used for loosely bound EPS and the name
forces of short range [33, 34].
The adhesion process is considered as creating a new  sheath for firmly bound EPS [38, 39].
interface, cell-support, by disrupture of two preexisting Next Nielsen et al. [40] subdivide EPS into  bound EPS
and  soluble EPS (Table 1) with a view to significant
interfaces, cell-liquid and support-liquid. This involves
thus a molecular contact between the two adhering sur- dependence of the aggregation of polymer gel in environ-
faces. If one surface alone is covered by polymers or if the mental conditions (e.g. pH). As associated with flocs, the
bound EPS settles together with activated sludge, while
two surfaces are partly covered, freely moving polymeric
the soluble part of EPS is component of effluent (Table 2).
chains may adsorb on the opposite surface and form a
Laspidou and Rittman [32] found in their review that
bridge between the cell and the support [33, 34].
The EPS can be located on or outside the cells surface. researchers are divided in two different schools of think-
ing about microbial products. The  EPS school has con-
The extracellular enzymes are either released in free
sidered active biomass and EPS, while the  SMP (soluble
form (exoenzymes) in the medium or bound to the cell
surface (ectoenzymes) [35, 36]. The release of enzymes by microbial products) school has considered active bio-
microorganisms into their external environment forms mass, SMP and inert biomass. In order to conciliate these
the basis for the interaction between cells and high mole- two kinds of consideration, they developed a unified the-
ory that couples them and reconciles apparent contradic-
cular weight exogenous substrates [3]. Extracellular
enzymes are complexed by EPS, which is demonstrated tions. They propose that  all soluble EPS are both utiliza-
tion-associated products (UAP) and biomass-associated
by Frolund et al. [37]. They report that a large proportion
products (BAP). It means that soluble EPS and SMP are the
of the exoenzymes is immobilized in the activated sludge
by adsorption in the EPS matrix, and that exoenzymes same, bound EPS are hydrolyzed to form BAP and both
should be considered to be an integrated part of the EPS BAP and UAP are formed to substrate, which is used as an
matrix. Complex formation allows the electrostatic inter- electron donor at the biomass synthesis (Fig. 1, Fig. 2).
Bound EPS is divided between active and inert biomass
action to occurs between enzymes and EPS and prevents
the enzymes wash out [3]. and hydrolyzed to BAP. By active biomass they mean liv-
According to the dissertation by Lansky [38], EPS can be ing cells that have an energy demand for maintenance
divided into two types:  sheath and  slime . The forma- while residual (non-biodegradable) inert biomass is pro-
duced during endogenous biomass decay. Soluble EPS
tions produced in the pure cultures are defined in view
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414 A. Raszka et al. Acta hydrochim. hydrobiol. 2006, 34, 411  424
They report that because production of EPS costs energy,
too high production rate leads to a decrease in the
growth rates of the producers. Probably the EPS accumu-
lation can be reduced by too high production rates since
less energy is available to make more cells. The authors
mentioned that the patchiness and roughness of the bio-
Figure 1. Scheme of interrelations between constituents of
EPS. film decreased and the porosity of the biofilm increased
due to EPS formation. Also EPS production by one species
stimulates growth of the other due to competition [47].
Synthesis of ribonucleic acid (RNA) and deoxyribonu-
cleic acid (DNA) by cells was found to be proportional to
the growth rate. RNA concentrations varied considerably
according to physiological cell state, whereas those of
DNA were relatively constant [46, 48].
While the polymeric matrix surrounds the biofilms
and activated sludge flocs a preliminary extraction step
is required in order to identify and quantify the EPS
Figure 2. Scheme of interrelations between constituents of
matrix. The aim of any extraction method is to remove as
EPS.
much of the biopolymer as possible without damaging
the integrity of bacterial cell. Abundance of the polymer
constituents depends on the extraction method and up
equals with SMP or with the sum of UAP and BAP (see the
equations below). to now there is no universal method for quantitative
extraction of biopolymers. The comparative study of the
cells + substrate (electron donor) fi active biomass
extraction methods done by Novak and Haugen [49],
Rudd et al. [50], Gehr and Henry [51] and Comte et al. [52]
hydrolysis
+ bound EPS + UAP bound EPS gggggs BAP
can be useful for comparison among different authors'
endogenous decay
results. There are several methods for EPS extraction: cen-
active biomass ggggggggs residued dead cells
trifugal stripping [53], alkali stripping [54], ethanolic
extraction [55] and cation-exchange resin [35, 52, 56 62].
BAP + UAP fi substrate (electron donor)
However the CER method seems to be the most popular
The authors do not provide any analytical methods to during recent years and it was inspected in great details
measure the considered elements but they created a by Frolund et al. [63].
mathematical model to quantify the relationships The amount and composition of EPS depend on bio-
among three solid species: bacteria, EPS, and residual film (activated sludge flocs) growth conditions. Different
inert biomass and three soluble species: original sub- structure and properties of biofilm depends on diffusion
strate, utilization-associated products (UAP), biomass- coefficient cause differences in the qualitative and quan-
associated products (BAP), and an electron acceptor, such titative composition of EPS [46, 64]. Many researchers
as oxygen. This theory is confirmed by modeling investi- found proteins, carbohydrates, nucleic acids and lipids
gations described in [41]. as the main EPS components. The amount of extracted
EPS and its components and ratio between each EPS con-
stituent vary, depending on the sample source, extrac-
tion technique and also on analytical method. While
2 What is the composition of the EPS?
implementing analytical methods for measuring EPS
As was mentioned above the EPS originate from lysis and constituents it is important to know whether they have a
hydrolysis products, excretion by the microorganisms high sensitivity to the compound and a low sensitivity to
and organic matter adsorption from the wastewater. The interfering substances [65]. The most doubts with choos-
composition of the EPS matrix depends on the growth ing a right analytical method seem to be around proteins
conditions of the biofilm or sludge flocs. Several studies and saccharides. The advantages and disadvantages of
have shown that the adsorption of organic polymers and several methods for protein and carbohydrates measure-
proteins improves biofilm growth stronger than nutrient ments are presented in Table 3.
adsorption [42 46]. Interesting conclusions about rela- The Lowry method is considered as the most appropri-
tions between EPS and bacteria growth are given by [47]. ate by many authors who compared several analytical
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Acta hydrochim. hydrobiol. 2006, 34, 411  424 Extracellular polymers in activated sludge 415
Table 3. Analytical methods and their features to protein and carbohydrates measurement.
Compound Method Methodology A (advantages)
D (disadvantages)
Protein analysis Kjeldahl-N acid digestion of the organic solids D: applicable with assumption that pro-
followed by digestion and measure- teins contain 16% of nitrogen, while ni-
ment of ammonia released trogen content varies in different pro-
teins,
D: organic nitrogen compounds as
urea, humic substances are also in-
cluded
biuret colorimetric method based on a D: low sensitivity
purple color development in the
reaction between proteins and
copper sulfate in alkaline solution
Bradford colorimetric method based on D: wide variability in sensitivity to vari-
reaction between coomassie ous proteins
brilliant blue G-250 with active
basic and aromatic functional groups
of proteins
Lowry color development in two steps: D: interfering substances: detergents,
first step is based on biuret reaction, humic substances, lipids, urea, nucleic
in the second one the proteins reacts acids and sugars
with the active acids contained in A: high sensitivity
the Folin-Ciocalteau reagent A: slightly variable in sensitivity to vari-
ous proteins
Carbohydrates analysis anthrone green color development in the D: wide variability in sensitivity to vari-
reaction between carbohydrates and ous carbohydrates
anthrone reagent in a strong sulfic
acid environment
phenol color development in the reaction A: high specificity to carbohydrate with
between carbohydrates and phenol an equal color intensity
reagent in an acid environment
methods for protein measurements [22, 57, 61, 66 74]. 27 different proteins resemble the color developed by
Despite the fact there are many influencing substances, BSA [65, 78].
most of the compounds are known to be in tolerable con- For carbohydrates measurements there is no such
centrations in wastewater. The humic substances con- problems with choosing standard as with protein. The
tained in wastewater can be in higher concentrations most popular and suitable compound is glucose, checked
than the tolerable limit but the modification of the previously by Raunkjaer et al. [65].
Lowry method can be used for humic substances meas- In the bacterial composition of EPS are accounted:
urement only and after using of correction factor polysaccharides (monosaccharides, uronic acids, amino
(described by Frolund et al. [63]) the amount of protein sugars), proteins, nucleic acids, (phospho)lipids (fatty
uninfluenced by humic substances can be calculated [65, acids, glycerol, phosphate, ethanolamine, serine, cho-
75]. line, sugars) and humic substances [3, 5, 57].
The results of the particular compounds of EPS can dif- While the EPS constitute 50& 60% of the organic frac-
fer between the authors also with standard used for each tion, the cell biomass makes up only 2& 20% of the
method. For protein measurement the most common organic fraction [62, 63]. Wilen et al. [62] in their study
standard is a bovine serum albumin [60, 67, 76, 77]. Davis found that the amount of protein is more than 43% of
[78] compared a color development of gamma globulin the total amount of polymeric material in the sludge.
(GG) and bovine serum albumin (BSA) in Bradford and Horan and Eccles [79] postulated that the main consti-
Lowry method. He found that GG developed 38% of the tuents of the extracellular matrix are polysaccharides,
color produced by using the Bradford method and 105% which are represented by up to 65% of extracellular mate-
using Lowry method. The main reason is that for Brad- rials whereas the dominant character of protein in acti-
ford method a macromolecular structure of the protein vated sludge and biofilm EPS is confirmed in studies of
is required while the Lowry method develops smaller Goodwin and Foster [80], Urbain et al. [81], Frolund et al.
molecules (dipeptides). In the study of the same author [63, 66], Bura et al. [58], Liao et al. [57], Wilen et al. [62].
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416 A. Raszka et al. Acta hydrochim. hydrobiol. 2006, 34, 411  424
The composition of the EPS matrix was found to be
dependent on the wastewater composition. Wilen et al.
[62] investigated seven different full-scale activated
sludge wastewater treatment plants (five domestic and
two industrial). They found that extracted EPS consist of
45& 55% proteins, 30& 33% humic substances and 10%
carbohydrates. That is in agreement with the studies of
Frolund et al. [63, 66], Jorand [56] and Urbain [81]. Never-
theless, the different order of identified EPS constituents
was reported by Jia et al. [82], Laspidou et al. [32] and Liao
et al. [57]. They found that the proteins and carbohydrates
(instead of humic substances) are the major EPS constitu-
ent. In contrast to the authors mentioned previously
Ford et al. [83] showed that the proteins constituted only
Figure 3. The major EPS components of activated sludge from
different WWTPs.
10& 15% of the extracellular matrix. Some of the
researchers have used a ratio between the proteins and
carbohydrates which usually ranges between 0.2 and 5.0
[38, 57]. study of four full-scale treatment systems: a poultry pro-
The DNA content in the EPS of the studied sludge was cessing plant, a potato processing facility, a petroleum
highly variable and constituted about 2& 15% of the EPS refinery and a municipal treatment system. The data
content in the sludge samples. Vallom and McLoughin from Wilen et al. [62] and Bura et al. [58] studies are show
[84] proposed that an excess of DNA could originate from in Figure 3. It is clear that for each activated sludge the
lysis, but the lysis cannot be justified only by DNA level. A dominant polymer is protein. In the study of Bura et al.
more precise explanation has been given by Palmgren [58] no humic substances were measured. Most differ-
and Nielsen [59]. They measured DNA in a mixed culture ences between sludges they obtained for the amount of
of activated sludge flocs and a pure culture of Pseudomo- DNA and acidic polymers. The DNA content was highest
nas putida. The main part of the DNA was found in the in the poultry and municipal systems both of which
extracellular slime of the bacterial culture and extrac- showed the best settling characteristic.
tion of the slime fraction inducted no lysis. Thus prob- Activated sludge from plants with different processes
ably the DNA originates more from excretion than from design can also give different EPS extracts [66, 81]. Niel-
lysis, as a means of transforming DNA from one bacter- sen et al. [40] investigated biofilters and trickling filters
ium to another [85]. However other researchers [22, 68, where they also found the proteins as the largest fraction
86] recognized the origin of DNA in EPS matrix from lysis of the EPS. Anaerobic sludge tends to have higher concen-
only. Jorand et al. [56] found that the entire DNA assayed tration of protein in its extracted polymers [88, 89]. Foster
after thirty seconds sonication was from lysis. High levels [88] mentioned that the ratio between proteins and car-
of DNA in EPS were found while the number of living bac- bohydrates ranges from 0.2& 0.7 for activated sludge,
teria decreased. Lysis of cells, which likely consists of whereas for digested sludge this ratio is 2& 5. Cloete and
enzymes originating from the nuclear material is a con- Oosthuize [5] studied activated sludge from an enhanced
sequence of the mortalities of bacteria [22, 52, 58, 66, 68]. biological phosphorus removal (EBPR) system. The
A positive correlation (r = 0.65) between proteins and sludges consisted not only of free bacteria and filamen-
DNA contents in the EPS was obtained by Palmgren and tous organisms, but mostly clustered bacteria encapsu-
Nielsen [59]. That indicates that cell lysis was one of the lated by the EPS. Cell clusters with associated EPS from
mechanisms contributing to the presence of proteins in two EBPR sludges contained between 57% and 59% phos-
the EPS. Some authors use the presence of extracellular phorus, while the EPS alone contained between 27% and
DNA as an indicator of cellular lysis [84] and others as a 29% phosphorus (percentages represent averages of phos-
measure of biomass [87], but in fact it is not clear whether phorus content of ten view fields analyzed by scanning
DNA is extracellular or only exists as cellular DNA. electron microscopy per sample). Results suggest that
The sludge from an oil refinery contained a larger pro- phosphorus removal in activated sludge might be not
portion of proteins (70%) and a lower fraction of humic only due to phosphate accumulating organisms (PAO),
substances (15%), while the sludge from a leachate but also by the EPS acting as a phosphorus reservoir.
WWTP contained a higher concentration of humic sub- A mineral composition of anaerobic and granular
stances (42%) [62]. Bura et al. [58] made a comparative sludge with high concentrations of Ca, Mg, K, Fe, S and P
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Acta hydrochim. hydrobiol. 2006, 34, 411  424 Extracellular polymers in activated sludge 417
was revealed by Debourquier et al. [90]. Among these ele- cular weight compounds was reported to be limited by
ments, phosphorus and calcium took up 25%. High con- bacterial aggregates in particular by the presence of the
centrations of phosphorus may possibly originate from EPS matrix [35, 58]. Despite the fact that high molecular
the phosphate groups abundant in biopolymers such as weight compounds are metabolized by enzymes outside
phospholipids and nucleic acids [91]. of cells the diffusion of high molecular weight com-
Wilen et al. [62] obtained a strong correlation between pounds is reported to be limited through bacterial aggre-
the sum of the cations Ca2+ and Mg2+ and total extracted gates in particular by the presence of the extracellular
EPS (r = 0.8). Correlation was also found for trivalent matrix [35]. Cadoret et al. [35] studied in comparative
cations (Fe3+ and Al3+) and a mixture of di- and trivalent study the hydrophilic activities between whole and dis-
cations but not at the same strength. The authors found persed activated sludge in order to establish how the
that correlations could appear because of the extraction aggregation state and the diffusion through the EPS
method used by which divalent cations could be released matrix limit substrate availability to the extracellular
easier than trivalent cations. matrix. They found that the availability of large com-
pounds for the extracellular enzymes is assumed to be
enhanced in the dispersed samples in comparison with
whole sludge [35].
3 How does the EPS interact with controlling
The characteristic feature of biofilm and activated
parameters of activated sludge?
sludge is the ability of microorganisms to live and grow
Sludge retention time has a great impact on the distribu- in aggregates. Due to aggregation in flocs, the microor-
tion of individual microbial species in activated sludge ganisms gain an advantage in comparison to free-living
according to their growth and decay rates [2]. Since bacteria due to the protection against predators. More-
results of several studies [42 44] confirmed the influence over, free leaving cells are washed out from the system,
of EPS presence on bacterial growth also the impact on while the microorganisms in flocs are kept in. The ability
sludge volume index (SVI) has been examined. of activated sludge microorganisms to create flocs is the
Investigations of the relationship between EPS and most important feature of activated sludge. An appropri-
sludge retention time (SRT) were carried out by Kiff [92], ate velocity of sedimentation can be then achieved.
Gulas et al. [93] and Morgan et al. [76]. The total amount of Thanks to that biomass can be separated by gravity from
EPS was independent of the SRT, but the most significant a purified wastewater (the most economic solution) [2].
effect of SRT on EPS they found as a change in the propor- Polymers exposed on the microbial surface may act to
tion of components but not the total EPS content. absorb and bridge between cell surfaces and therefore
Liao et al. [57] investigated the influence of SRT on the initiate floc formation [4, 76]. Because biopolymers have
EPS and physico-chemical properties such hydrophobi- influence on formation of flocs (size and structure), they
city and surface charge of sludge. They found that the car- are believed to play a key role in the flocculation process.
bohydrate content was significantly higher and the con- Most of the proposed mechanisms for bioflocculation are
tent of protein was significantly lower at lower SRTs (4 based on the complex interactions between these high
and 9 days). From the behavior of the proteins:carbohy- molecular weight polymers, which bond electrostatically
drates ratio they suggested that at lower SRTs not all the and physically to microbial surfaces [76, 97]. In the work
carbon sources available for growth were consumed. of Jia et al. [82] EPS is believed to be responsible not only
Excess carbon substrates could have been converted to for the aggregation of activated sludge flocs but also for
polymers that accumulated as EPS. That is confirmed by anaerobic granules. Ross [97] suggested that the biofloc-
Harris and Mitchell [94], Dugan [95] and Andreadakis culation mechanisms proposed for the aerobic process
[96]. A strong positive correlation between DNA and SVI might also be applicable to the formation of anaerobic
was found by Bura et al. [58], however, the authors does granules. Harada et al. [98] during investigation on two
not present how strong the correlation is and any other types of UASB (upflow anaerobic sludge blanket) granules
authors have not been obtained similar relations. They found that granules larger in size and characterized by
also have shown that the COD:N:P ratio of the waste- higher mechanical strength produce more EPS. On the
waters can influence the biopolymer composition of other hand Sam-Soon et al. [99, 100] speculated that high
microbial flocs in the activated flocs matrix and that hydrogen pressure would induce hydrogen-utilizing
there is no correlation between SVI and carbohydrates methanogens to produce EPS, thus boosting sludge gran-
content. Microbial EPS may hinder the diffusion of sub- ulation. In this case Jia et al. [82] examined EPS produc-
strates in the activated sludge flocs and their availability tion and characteristic of two types of enrichment cul-
to the extracellular enzymes. The diffusion of high mole- tures: one was composed only of hydrogenotrophic
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
418 A. Raszka et al. Acta hydrochim. hydrobiol. 2006, 34, 411  424
methanogens (HM), the second one was a mixture of HM concentrations of the multivalent cations in their investi-
and hydrogenotrophic sulfate reducing bacteria (HSR). A gated sludge were significant. The measurements were
glucose degrading sludge was used for comparing pur- performed on seven activated sludges (five sewage treat-
pose. Production of EPS was dependent upon substrate. ment plants treating domestic wastewater and two
More EPS was discovered in the glucose degrading industrial activated sludge  treatment processes for oil
sludge, but from two enriched hydrogenotrophic anae- refinery and leachate effluent) and the relation obtained
robes, the HM culture contained more EPS than the HSR between concentration of Ca2+ and Mg2+ and SVI and ZSV
culture. The investigations showed also that more pro- was  0.73 and 0.67 respectively, while for Fe3+/Al3+ 0.92
teins than carbohydrates were produced at the begin- and 0.86 respectively. However, the oil refinery sludge
ning of the batch test when the concentration of hydro- contained low concentrations of divalent and trivalent
gen was high. The reason is not clear but for the HM cul- ions but had well settling and compaction properties.
ture with the decreasing amount of hydrogen the Since the biopolymers have high negative charge they
amount of proteins and carbohydrates returned have a high affinity for cations [13]. Perhaps this can be
(decreased) to their original levels. For the HM/HSR mixed explained by the results from effect of mineral materials
culture the increase of EPS at a high concentration of and cations on activated and alum sludge settling [109].
hydrogen was not as noticeable as for the enriched HM They found that not the concentration of soluble cations
culture probably because of lower dependence on the but the ratio between particular cations explains the
initial hydrogen concentration [82]. effect of mineral materials on settleability. Mineral mate-
Since the EPS are the major components of flocs, and rials which improved the activated sludge settling most,
have charged functional groups, it is reasonable to had high Ca/Mg-ratios, and mineral materials which
assume that they contribute to the net surface properties improved the alum sludge settling most had low mono-
of the flocs and then the flocculation properties [101]. valent to divalent cation ratios [109].
Biopolymers act in conjugation with divalent metal ions The function of biopolymers on bioflocculation and
such as calcium to help the formation and settlement of sludge physicochemical properties as such e.g. settleabil-
sludge flocs in both aerobic and anaerobic treatment sys- ity, dewatering, floc strength and charge have been
tems [102]. Microbial flocculation is generally attributed intensively studied during last years. As an organic poly-
to exopolymeric bridging of bacterial cells [53, 94]. Floc mer, the EPS increase water viscosity, which helps a sin-
components bridging involves ionic interactions gle cell to form a microenvironment necessary for the
between charged functional groups of biopolymers and activity of the extracellular enzymes [2, 57, 62]. The
divalent cations [27, 28], and may also be mediated absorbed water reduce the osmotic pressure difference
through hydrophobic moieties of exopolymers [81, 103], between the biological aggregate and the surrounding
specific protein polysaccharide interactions [60], or sim- liquid so it is therefore not surprising that an increase in
ple physical enmeshing [104]. In addition, electrical dou- total amount of the extracted EPS corresponds to an
ble layer effects have been proposed to play an important increase of SVI [6, 13, 62, 110, 111].
role in floc stability [105, 106]. In the polymer bridging The proteins are the most important EPS component
model, the EPS which contain different negatively for flocculation. A positive correlation between floccula-
charged groups, are bound together by means of divalent tion ability (FA) and increasing concentration of proteins
and/or trivalent cations [27, 102, 105, 107] to form large (r = 0.8) in both sludge and EPS extract was noticed by
polymeric networks in which the different floc constitu- Wilen et al. [62], Higgins and Novak [102] and Jorand et al.
ents such as single bacteria and bacterial colonies are [112]. It can be explained by the fact that the proteins in
embedded [27, 62]. the EPS are mainly created by hydrophobic amino acids
Cations bound on the floc surfaces partially neutralize that should promote flocculation [112]. Paul et al. [85]
the negative charged groups and affect the surface found that the strongly flocculated activated sludge had
charge [108]. Thus, the improved compressibility and set- a higher content of DNA in the flocs matrix than the
tleability could be due to bridge binding interactions more loosely flocculated and not flocculated sludges.
occurring among negatively charged polymeric groups Bura et al. [58] have shown that the COD:N:P ratio of the
and cationic ions and/or surface charge reduction due to wastewaters can influence hydrophobicity and surface
binding to negatively charged sites on the floc surface charge. Also during the investigation of the effect of
[13]. Increasing concentrations of the multivalent cations nutrients on the EPS composition and structure of flocs
Ca, Mg, Fe and Al in the sludge flocs contributed to they discovered that while the surface charge decreased
improve compressibility and settleability. The relation- an increase of uronic acids and DNA within the EPS
ships between SVI and ZSV (zone settling velocity) and matrices occurs [58].
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
Acta hydrochim. hydrobiol. 2006, 34, 411  424 Extracellular polymers in activated sludge 419
Table 4. Correlation between EPS constituents and activated
Hydrophobicity and hydrophilicity are usually used to
sludge parameters.
describe a molecule or a structure possessing the feature
of being rejected from an aqueous medium (i.e. hydro-
Sludge Flocculation Surface Relative
phobicity), or being positively attracted (i.e. hydrophili-
retention ability charge hydro-
city) [16]. Hydrophobicity (or the surface energy) and the
time (SRT) (FA) (SC) phobicity
surface charge, which are relevant to short- and long-
Proteins + + + +
range forces respectively, must be taken into account in
Carbohydrates + + +
order to describe adhesion of microorganisms to a sup-
DNA +
a b c
port [33]. Increasing the hydrophobicity of cell surfaces Total EPS  +/  +/ 
would promote cell-to-cell interaction and further serves
+ correlation  no correlation
as inducing cell force for aggregating out of hydrophilic
a
The effect of SRT on EPS is a change in the proportion
liquid phase [16]. In bacteria, hydrophobicity is directly
between components but not the total EPS composition.
related to the surface concentration of carbon present as b
The effect of SC on total EPS is dependent on the proportion
hydrocarbon moieties and inversely related to oxygen
between the constituents of activated sludge  positive
concentration or to the N/P ratio [33, 113]. charge of the protein is neutralized by negative charge of
carbohydrate.
The prevalence of proteins over carbohydrates in the
c
The effect of relative hydrophobicity on total EPS is depen-
extracted material is coherent with the high hydrolytic
dent on the proportion between the constituents of acti-
capacity of the EPS [35, 60]. Jorand et al. [60] studied
vated sludge  the proteins contribute hydrophobicity of
hydrophobic/hydrophilic properties of activated sludge
activated sludge while carbohydrates hydrophilic nature.
EPS. They found that the urban sludge contains about
60% more EPS than the sludge from brewery and pilot
industrial scale reactor fed with urban wastewater. The hydrophobicity. The significant negative correlation to
urban sludge EPS carries high amounts of proteins. the relative hydrophobicity was obtained for humic sub-
Hydrophobic interactions of the secreted polymer (i.e. stances and proteins contributed hydrophobicity of acti-
the behavior of particles and molecules that are unable vated sludge and a weak correlation between relative
to interact with water, either via hydrogen bonds or elec- hydrophobicity and carbohydrates [62] (the main correla-
trostatically, to draw together in an aqueous environ- tions between EPS constituents and activated sludge
ment) had received attention of Houghton and Quar- parameters are collected in Table 4).
amby [114] and Zita and Hermansson [91]. Zita and Her- It should be emphasized that cell hydrophobic or
mansson [91] observed a strong correlation between the hydrophilic interactions, to its great extent, are also
hydrophobicity of cells and degree of their attachment to dependent on the surface tension of the bulk solution in
activated sludge flocs. Jorand et al. [60] state that the which bacteria live. Consequently, the adhesion of hydro-
hydrophobicity is made up of proteins but not carbohy- philic cells is enhanced at low liquid surface tension,
drates. They fractioned the activated sludge EPS into inso- while the opposite is true for hydrophobic cells [16].
luble and precipitated EPS. They showed that within the Generally hydrophobicity and surface charge were
unprecipitated fraction of activated sludge EPS the found not to be influenced by the total EPS content in
hydrophobic substances cover 7% of the dissolved sludge. However the level of total carbohydrate had a
organic carbon (DOC) while hydrophilic acids take a part negative influence on the hydrophobicity and surface
in 13% of the DOC. charge while the protein content of EPS had a weak posi-
The influence of EPS constituents on hydrophilic/ tive influence on the surface charge. It is suggested that
hydrophobic properties was investigated also by Wilen et hydrophobicity and surface charge, rather than the
al. [62]. They measured relative hydrophobicity (RH) i.e. quantity of ECP, should be used for control of the floccu-
the average value of both hydrophilic and hydrophobic lating ability of sludge [22, 57]. The proportion of ECP
groups in the sludge and which reflected both the prop- components (proteins/total carbohydrates) was more
erties at the surface and within the interior of the flocs important in determining the surface charge of both
[62]. They wanted to confirm the results from a few pre- anaerobic and aerobic sludge flocs than the quantities of
vious studies indicating that mainly proteins, humic sub- individual ECP components [22, 76]. Furthermore the
stances and uronic acids in the EPS contribute to the positive influence of protein on hydrophobicity was
hydrophobicity of activated sludge while carbohydrates reported also by Jorand et al. [60]. This was explained by a
contribute more to their hydrophilic nature [60, 102]. neutralization of positive charges on the proteins and
Both in the extracted EPS and sludge the total amount of negative charges on carbohydrates. It means that not the
polymers showed the negative effect on the relative quantities but the proportions of the EPS components
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
420 A. Raszka et al. Acta hydrochim. hydrobiol. 2006, 34, 411  424
can be important in controlling hydrophobicity and sur- water and cause a high viscosity [81, 117]. The compo-
face charge [57]. nents in the EPS influence the water binding properties,
A strong inverse linear correlation (r2 =  0.88) between and thus it has also effect on the dewaterability of the
the surface charge and water contact angles (contact sludge. The individual binding forces among functional
angle measurements are used for determination of the groups in macromolecule are affected by the water con-
surface energy  hydrophobicity is evaluated by water tent and increase together with increasing polymer con-
contact angle; hydrophobicity/hydrophilicity) of sludge centration, which is an argument to degrade the EPS to
for all studied floc samples was obtained by Sponza [69]. improve dewaterability [22, 24].
This is explained since the more positively charged floc Bound water and capillary suction time (CST) have
surfaces indicate higher hydrophobicity while the more been used previously for the characterization of the
negatively charged floc surfaces show the higher hydro- dewaterability of activated sludge. The CST had good sta-
philicity [68]. Hydrophilicity and surface charge were tistical correlations with the polymeric constituents
found to be influenced by the EPS components mainly measured in both sludge and the extracted EPS. The
from proteins and DNA depending on the composition of bound water was well correlated only with the individual
wastewater [69]. polymers measured in the sludge. High amount of the
According to Wilen et al. [108], all the measured acti- individual and total polymers in the extracted EPS corre-
vated sludge samples carried a negative surface charge. sponded to a good dewaterability determined by the CST
This is mainly due to ionization of functional groups of [13]. Chen et al. [67] studied the influence of acid and sur-
the EPS such as carboxylic, sulfate and phosphate. The factants on dewaterability of activated sludge. They
surface of microorganisms is negatively charged under found that both acid and surfactant treatment improved
usual pH conditions. This is the case for many materials activated sludge dewatering: it makes it easy to pack the
that can be considered as potential supports, including sludge aggregates and reduce the water content of dewa-
organic polymers. The density of adhering cells increases tered sludge. Moreover, the more surfactant was used the
as the pH decreases from 6 to 4 and the net negative more EPS was released. This information is in accordance
charge of the cells decreases [33]. with studies of Gehr and Henry [51] and Thomas [118].
It should be mentioned that adhesion is very slow Rosenberger and Kraume [61] found in their eight
when cells and support are negatively charged. Although membrane bioreactors study (treating municipal, domes-
when the support is positively charged the cell-support tic, or industrial wastewaters) that increasing suspended
attraction allows initially a quick adhesion [3]. Hence the EPS concentrations always resulted in decreasing sludge
initial microbial adhesion can be promoted by chemical filterability.
modification of the surface properties and thereby
decrease electrical repulsion or achieve attraction [33,
113]. In both sludge and extracted EPS the more poly-
4 Conclusions
meric materials appear the more negative turned surface
charge. The most important factor responsible for such Under natural conditions EPS production seems to be an
changes are proteins and humic substances, which con- important factor of survival, as most bacteria occur in
tribute most significantly to the surface charge. Mikkel- microbial aggregates such as flocs and biofilms, whose
sen and Keiding [110] found a positive correlation structural and functional integrity is based essentially on
between the charge density of extracted EPS and its con- the presence of an EPS matrix. The EPS matrix may serve
tent of proteins, which further supports the findings that as a multipurpose functional element of microbial com-
proteins have a significant influence on the surface munities, including adhesion, protection, structure,
charge. Several types of sludge (activated sludge, digested recognition and physiology [3]. Physical conditions, avail-
sludge, UASB granular sludge) examined by Morgan et al. ability of nutrients, amount of microbial cells and their
[76] and their EPS extract carry a negative charge. How- physiological status influence the structure of the
ever, the charge carried by both the granular sludges and matrix. The cells within the matrix can be dispersed and
their EPS extract was found to be less negative than that may form a thin layer with surrounding EPS. Several
of the other examined activated sludges [76]. viewpoints around structure of the EPS matrix are
On the one hand, the presence of EPS affects floccula- described in the manuscript.
tion ability and the effect is positive. On the other hand, The yield of extracted polymeric material was found to
the presence of EPS can cause difficulties with dewater- differ significantly depending on the nature of the sludge
ing and filterability [61, 112, 115, 116]. Proteins, polysac- sample, extraction method and EPS composition anal-
charides and DNA, which are enclosed in EPS, entrap ysis. Number of extraction methods and their different
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
Acta hydrochim. hydrobiol. 2006, 34, 411  424 Extracellular polymers in activated sludge 421
efficiency and number of EPS components analysis meth- [4] Tenney, M. W., Stumm, W. J.: Chemical flocculation of
microorganisms in biological waste treatment. J. Water
ods provide difficulties with comparability of various
Pollut. Control Fed. 39, 1370  1388 (1965).
researchers' results. As the EPS matrix composition is
[5] Cloete, T. E., Oosthuize, D. J.: The role of extracellular exo-
very heterogeneous and not predictable to the end the
polymers in the removal of phosphorus from activated
number of interference factors to particular analytical
sludge. Water Res. 35, 3595  3598 (2001).
methods will lead to wrong results interpretations. In
[6] Keiding, K., Wybrandt, L., Nielsen, P. H.: Remember the
this case the further studies to develop more precise ana-
water: comment on EPS colligative properties. Water Sci.
lytical methods are required.
Technol. 43, 17  23 (2001).
The researchers are in agreement that proteins are the
[7] Yoshida, R., Uchida, K., Kaneko, Y., Sakai, K., Kikuchi, A.,
most dominant constituent of EPS and that the rest are
Sakurai, Y., Okano, T.: Comb-type grafted hydrogels with
mainly carbohydrates, DNA and humic substances. The rapid de-swelling response to temperature changes. Na-
ture 374, 240  242 (1995).
EPS influence on the activated sludge process was studied
[8] Varga, I., Gilanyi, T., Meszaros, R., Filipcsei, G., Zrinyi, M.:
intensively. Correlations between amount of total EPS or
Effect of cross-link density on the internal structure of
its constituents to flocculation ability, hydrophobicity,
poly(Nisopropylacrylamide) microgels. J. Phys. Chem.
and surface charge were found in many studies. It was
B105, 9071  9076 (2001).
also suggested that the EPS improve a product quality in
[9] Gupta, P., Vermani, K., Garg, S.: Hydrogels: from controlled
wastewater treatment by positive effect on dewaterabil-
release to pH-responsive drug delivery. Drug Discov.
ity and filterability of the activated sludge.
Today 7, 569  579 (2002).
Our knowledge about the role of microorganisms and
[10] Diez-Pena, E., Quijada-Garrido, I., Barrales-Rienda, J. M.: On
the biopolymers extracted by them in a wastewater treat-
the water swelling behaviour of poly(N-isopropylacryla-
ment is being elucidated, but there are still  a few ques- mide) [P(N-iPAAm)], poly(methacrylic acid) [P(MAA)], their
random copolymers and sequential interpenetrating
tions for future research. For example, which cells are
polymer networks (IPNs). Polymer 43, 4341  4348 (2002).
responsible for production of which EPS component or
[11] Annaka, M., Tanaka, C., Nakahira, T., Sugiyama, M., Aoyagi,
can we determine which cell within the matrix is active
T., Okano, T.: Fluorescence study on the swelling behavior
in changing its composition and structure or can we
of comb-type grafted poly(N-isopropylacrylamide) hydro-
determine exactly how the matrix is ordered. [101].
gels. Macromolecules 35, 8173  8179 (2002).
[12] Gil, E. S., Hudson, S. M.: Stimuli-reponsive polymers and
The interest in extracellular polymers of the first author
their bioconjugates. Prog. Polym. Sci. 29, 1173  1222
appears andrises duringscientific internship supported by EU
(2004).
V FP project EVK1-CT-2002-80009 in the Centre of Excellence
[13] Jin, B., WilØn, B. W., Lant, P.: A comprehensive insight into
 Environmental Biotechnology Research Centre DEMETER. floc characteristics and their impact on compressibility
and settleability of activated sludge Chem. Eng. J. 95,
The first author of the article is grateful to the scientific staff of
221  234 (2003).
The Prague Institute of Chemical Technology, Department of
[14] Freeman, C., Lock, M. A.: The biofilm polysaccharide
Water Technology and Environmental Engineering (Czech
matrix: a buffer against changing organic substrate sup-
Republic) for the scientific guiding and help with better under-
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