Journal of Biotechnology 95 (2002) 249 256

www.elsevier.com/locate/jbiotec

Extraction of extracellular polymeric substances (EPS) of

sludges
Hong Liu, Herbert H.P. Fang *
Department of Ci6il Engineering, Centre for En6ironmental Engineering Research, The Uni6ersity of Hong Kong,
Pokfulam Road, Hong Kong
Received 1 October 2001; received in revised form 28 December 2001; accepted 14 January 2002

Abstract
The efficacies of extracting extracellular polymeric substances (EPS) from aerobic, acidogenic and methanogenic
sludges using EDTA, cation exchange resin and formaldehyde under various conditions were compared. Results show
that formaldehye plus NaOH was most effective in extracting EPS for all sludges; only 1.1 1.2% of DNA in the
sludge samples were detected, suggesting the EPS extracted were not contaminated by intracellular substances. For
each gram of volatile solids, formaldehydeNaOH extracted 165, 179 and 102 mg of EPS from aerobic, acidogenic
and methanogenic sludges, respectively. All EPS were mainly composed of carbohydrate, protein and humic
substance, plus small quantities of uronic acid and DNA. Carbohydrate was predominant in the acidogenic sludge
(62% in the EPS extracted by formaldehydeNaOH), whereas protein was predominant in the methanogenic sludge
(41%). Humic substance, which has often been overlooked, accounted for 30.6, 8.4 and 22.8% of the extracted EPS
from aerobic, acidogenic and methanogenic sludges, respectively. However, judging from EPS quantities estimated
from confocal laser scanning microscopic observations, formaldehyde NaOH extracted only a limited portion of
EPS. Optimization of extraction procedures and/or development of a more effective extraction method are warranted.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Extracellular polymeric substances (EPS); Extraction; Formaldehyde; Quantification; Sludge

1. Introduction
Extracellular polymeric substances (EPS) are
metabolic products accumulating on the bacterial
cell surface (Morgan et al., 1990). They form a
protective layer for the cells against the harsh
external environment, and also serve as carbon
* Corresponding author. Tel.: + 852-2859-2660; fax: + 8522559-5337.
E-mail address: hrechef@hkucc.hku.hk (H.H.P. Fang).

and energy reserves during starvation. EPS were

found crucial to the flocculation (Frlund et al.,
1996; Rudd et al., 1984) and dewatering (Nielsen
et al., 1996) of activated sludge, as well as to the
microstructure of methanogenic granular sludge
(Schmidt and Ahring, 1996).
EPS are composed of a variety of organic substances (Frlund et al., 1996). Carbohydrate was
identified as the predominant constituent in the
EPS of many pure cultures (Cescutti et al., 1999;
Sutherland and Kennedy, 1996), whereas protein

0168-1656/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.
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H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249256

was found in substantial quantities in the

sludges of many wastewater treatment reactors
(Fang and Jia, 1996; Veiga et al., 1997). Humic
substance (Frlund et al., 1995), uronic acid and
deoxyribonucleic acids (DNA) (Tsuneda et al.,
2001; Zhang et al., 1999) were also detected in
EPS; however, information about their concentration in EPS is scarce. The EPS normally contain small quantities of DNA, which are
released from the dead cells after lysis. Large
quantities of DNA in the EPS could be an
alarming indication that the cells were lysed
during the harsh extraction process.
Quantification of EPS is strongly dependent
upon the extraction methods (Wingender et al.,
1999). Physical extractions include centrifugation, ultrasonication and heating, whereas common chemical extractions include uses of
alkaline,
ethylenediamine
tetraacetic
acid
(EDTA) and cation exchange resin. However,
there is no standard extraction procedure established so far, making it very difficult to meaningfully compare and interpret published results.
This study was conducted to compare the effectiveness of six extraction procedures for EPS
of sludges sampled from three wastewater treatment reactors, two of which were anaerobic
(acidogenic and methanogenic, respectively) and
one aerobic (activated sludge). The contents of
carbohydrate, protein, humic substance, uronic
acid and DNA in the extracted EPS samples
were analyzed for comparison. EDTA, cation
exchange resin, and formaldehyde were used as
extractants in combination with ultrasonication,
NaOH and centrifugation.

2. Material and methods

2.1. Source of sludges

The aerobic activated sludge was sampled
from a local municipal wastewater treatment
plant (Shatin, Hong Kong) using the four-stage
Bardenpho process (Shiskowski and Mavinic,
1998). With 13 days of sludge age and 10 h of
hydraulic retention, the process typically re-

moved 90% COD, 95% BOD5, and 85 90% nitrogen from wastewater. The acidogenic sludge
was sampled from a fermentor (Biostat B, B.
Braun Biotech) treating a sucrose-rich wastewater. It converted over 95% sucrose with 6 h of
hydraulic retention into butyrate and acetate,
plus a biogas containing 63% hydrogen, 35%
carbon dioxide and 2% nitrogen. The granular
methanogenic sludge was sampled from an
upflow reactor treating phenolic wastewater with
12 h of hydraulic retention. The reactor degraded 97% phenol in wastewater and produced
a biogas comprising 69% methane and 31% carbon dioxide (Fang et al., 1996).

2.2. Extraction of EPS

EPS of three sludge samples were extracted
under six conditions. Fig. 1 illustrates detailed
procedures of each extraction process. For the
control, EPS was physically extracted, without
adding any chemical extractant, simply by highspeed centrifugation (20 000 G) for 20 min, followed by two membrane separations to remove
microbial cells (through a 0.2 mm membrane)
and low molecular-weight metabolites (through
a dialysis membrane of 3500 Da; Pierce, USA),
before lyophilization at 50 C for 48 h. For
the other five processes, sludges were first extracted by chemicals; the extracted solutions
were then treated following the same procedures
as the control. The extractants used in this
study included EDTA (2%; at 4 C for 3 h),
cation exchange resin (Dowex 50 8, Fluka,
USA; at 4 C for 1 h), formaldehyde (at 4 C
for 1 h), formaldehyde plus NaOH (1 N; at
4 C for 3 h), and formaldehyde plus ultrasonication (60 W for 2.5 min). Extractant residues
in the solution were removed by the dialysis
membrane filtration in the subsequent treatment.
The effect of time on EPS extraction was not
examined in this study. Frlund et al. (1996)
recommended 0.51 h for EPS extraction with
minimum risk of induced cell lysis, and results
of another study (Fang and Jia, 1996) also
showed that EPS extraction was completed in
less than 1 h.

H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249256

251

2.3. Chemical analysis of extracted EPS

2.4. Quantification of EPS using CLSM

The total quantity of extracted EPS was measured by the weight of solids after lyophilization.
The carbohydrate content in EPS was measured
by the anthrone method (Gaudy, 1962) using
glucose as the standard. The contents of protein
and humic substance in EPS were measured by
the modified Lowry method (Frlund et al., 1995)
using bovine serum albumin and humic acid
(Pract., Fluka, USA) as the respective standards.
Uronic acid content was measured by the m-hydroxydiphenyl sulfuric acid method (Blumenkrantz and Asboe-Hansen, 1973) as modified
by Kintner and Van Buren (1982) using glucuronic acid as the standard. The DNA contents
in both EPS and sludge were measured by the
diphenylamine colorimetric method (Sun et al.,
1999) using E. coli DNA as the standard. The
DNA were extracted from the sludge following
the procedures described previously (Zhang and
Fang, 2000).

A technique was recently developed to quantify

EPS in a microbial community using confocal
laser scanning microscopy (CLSM) (Zhang and
Fang, 2001). After staining, total volume of microbial cells could be measured by CLSM. The
weight difference between the volatile solids (VS)
and the cells, which could be estimated from the
cell volume and density, represents the EPS content. The EPS contents in the aerobic and
acidogenic sludges were also measured using this
method for comparison. The instrument, procedures and image analytical software used were the
same as previously reported (Zhang and Fang,
2001).
3. Results

3.1. EPS quantities extracted from sludges

Table 1 summarizes the amounts of EPS ex-

Fig. 1. Procedures for six EPS extraction processes

H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249256

252

Table 1
EPS extracted from various sludges
Extraction

Aerobic (mg per g-VS)

Acidogenic (mg per g-VS)

Methanogenic (mg per g-VS)

FormaldehydeNaOH
EDTA
Formaldehyde-ultrasound
Cation exchange resin
Formaldehyde
Control

164.99 3.9
146.89 1.1
77.9 91.1
57.8 91.0
49.7 91.2
25.7 9 0.3

179.09 3.2
104.9 90.7
100.09 0.3
58.4 91.1
56.9 9 1.2
36.9 90.6

102.1 9 1.5
73.2 9 0.5
36.8 9 0.8
30.1 9 0.9
32.5 9 0.4
15.8 9 0.2

Mean value (n =3) 9S.D.

Table 2
Comparison of EPS extracted from aerobic and anaerobic sludges
Sludge

Extraction method

Total EPS (mg per g-VS)

Reference

Aerobic
Aerobic
Aerobic
Acidogenic
Methanogenic
Methanogenic
Methanogenic

FormaldehydeNaOH
Cation exchange resin
80 C
FormaldehydeNaOH
FormaldehydeNaOH
Phenol
Formaldehyde at 100 C

165
109
58
179
102
91
74

Present study
Rudd et al., 1984
Liu et al., 2001
Present study
Present study
Veiga et al., 1997
Fang and Jia, 1996

tracted from the three sludge samples by the six

processes. Results show that the amount of EPS in
a sludge sample was strongly dependent upon the
extraction method. The effectiveness of EPS extraction from all sludges in descending order is: formaldehydeNaOH, EDTA, formaldehyde-ultrasound, cation exchange resin, formaldehyde, and
control. For each gram of volatile solids, the
formaldehydeNaOH process extracted 165, 179,
and 102 mg of EPS for the aerobic, acidogenic and
methanogenic sludges, respectively. For comparison, the corresponding quantities of EPS extracted
by the control were only 26, 37 and 16 mg,
respectively. The formaldehyde NaOH process in
general extracted about six times more EPS than
the control, two to three times more than formaldehyde alone, cation exchange resin and formaldehyde-ultrasound, and 10 30% more than EDTA.
Table 1 shows that the EPS content in each gram
of aerobic sludge varied from 26 to 165 mg depending on the extraction method. Corresponding values in literature for aerobic sludge varied
drastically, from 6254 mg (Liu et al., 2001) to 437
mg (Frlund et al., 1996).

Table 2 lists the reported amounts of EPS

extracted from aerobic and methanogenic sludges
for comparison; there was no EPS data for
acidogenic sludge reported in literature. Results
also show that formaldehydeNaOH extracted
more EPS in this study than those extracted by
other means reported in literature. For the aerobic
activated sludge, the amount of EPS extracted by
formaldehydeNaOH was about 50% more than
that extracted by cation exchange resin and nearly
two times more than that extracted by heating at
80 C. For methanogenic sludge, the amount of
EPS extracted by formaldehydeNaOH was 10%
higher than that extracted by phenol, and nearly
40% more than that by formaldehyde alone at
100 C.

3.2. Constituents of EPS extracted from sludges

Table 3 compares the EPS constituents in the
aerobic activated sludge extracted by various processes. Tables 4 and 5 compare the corresponding
results, respectively, for the acidogenic and
methanogenic sludges. Results show that EPS in

H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249256

253

Table 3
Constituents of EPS in each gram of aerobic activated sludge
Extraction

Carbohydrate
(mg)

Protein
(mg)

Humic substance
(mg)

Uronic acid
(mg)

DNA
(mg)

Unknown
(mg)

Formaldehyde
NaOH
EDTA
Formaldehydeultrasound
Cation exchange
resin
Formaldehyde
Control

40.5 91.7

54.6 9 2.0

50.4 9 3.7

4.2 90.4

0.35 90.05

14.8 93.3

12.491.2
28.9 90.9

22.9 9 0.5
20.4 9 1.0

59.29 2.5
18.9 9 1.5

2.1 90.4
1.8 90.1

0.47 90.03
0.13 90.02

49.7 9 3.1
7.8 90.5

12.7 90.4

17.6 9 0.9

16.4 9 0.8

1.2 90.2

0.14 90.02

9.8 91.0

15.9 91.0
7.7 90.1

12.3 9 0.3
7.990.1

10.99 0.6
6.4 90.3

1.1 90.1
0.5 9 0.1

0.07 9 0.01
0.06 9 0.01

9.4 90.3
3.1 9 0.2

Mean value (n = 3) 9S.D.

Table 4
Constituents of EPS in each gram of acidogenic sludge
Extraction

Carbohydrate
(mg)

Protein
(mg)

Humic substance
(mg)

Uronic acid
(mg)

DNA
(mg)

Unknown
(mg)

Formaldehyde
NaOH
EDTA
Formaldehydeultrasound
Cation exchange
resin
Formaldehyde
Control

110.99 4.0

25.89 1.0

15.1 9 1.0

5.5 9 0.3

0.15 90.03

21.5 91.1

41.79 1.2
71.6 91.3

6.5 90.4
10.890.7

15.9 9 1.0
5.09 0.4

2.3 9 0.2
2.5 9 0.3

0.22 90.03
0.05 90.01

38.3 91.1
10.0 90.4

38.7 90.8

6.2 90.3

3.090.2

2.2 9 0.1

0.08 90.01

8.2 90.3

39.4 91.0
23.7 90.7

5.990.4
4.49 0.2

2.59 0.3
1.9 9 0.2

1.7 9 0.2
1.0 9 0.1

0.02 90.00
0.02 90.00

7.4 90.3
5.9 90.2

Mean value (n =3) 9 S.D.

Table 5
Constituents of EPS in each gram of methanogenic sludge
Extraction

Carbohydrate
(mg)

Protein
(mg)

Humic substance
(mg)

Uronic acid
(mg)

DNA
(mg)

Unknown
(mg)

Formaldehyde
NaOH
EDTA
Formaldehydeultrasound
Cation exchange
resin
Formaldehyde
Control

19.19 0.8

42.19 1.7

23.3 9 2.0

2.1 9 0.2

0.19 90.01

15.3 91.45

6.89 0.5
12.0 9 0.4

12.090.7
13.1 90.7

24.39 0.7
5.690.2

1.2 9 0.1
1.0 9 0.1

0.26 90.03
0.04 90.00

28.3 90.52
5.1 90.78

7.99 0.4

10.69 0.4

5.590.3

0.9 9 0.1

0.05 90.01

5.1 90.95

9.7 90.7
4.1 90.2

11.990.8
5.890.2

4.6 9 0.3
3.1 9 0.1

0.8 9 0.1
0.3 9 0.1

0.03 90.00
0.03 90.00

5.5 90.44
2.5 90.17

Mean value (n =3) 9 S.D.

254

H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249256

all sludges were primarily composed of carbohydrate, protein and humic substance, plus small
quantities of uronic acid and DNA. About 34
39% of EPS extracted by EDTA remained unidentified, but only 9 19% of EPS extracted by the
other five processes were unidentified, most of
which might be lipid or phenols (Schmidt and
Ahring, 1996).
Results in Tables 3 5 show that formaldehyde
NaOH was most effective in extracting carbohydrate, protein and uronic acid for all sludges,
whereas EDTA was most effective in extracting
humic substance and DNA. Ultrasound enhanced
the extraction of all the constituents of EPS by
formaldehyde. Table 3 shows that formaldehyde
NaOH extracted about equal amounts of carbohydrate, protein and humic substance (ranging
2533%) from aerobic activated sludge. Table 4, on
the other hand, shows that carbohydrate accounted
for 62% of the EPS in the acidogenic sludge; this
could be due to the conversion of substrate sucrose
into levans by the levansucrase (Sangiliyandi and
Gunasekaran, 2001).

4. Discussion

4.1. Constituents of EPS

For years, carbohydrate was considered the main
constituent of EPS in pure cultures (Sutherland,
1997; Sutherland and Kennedy, 1996). Recent
studies of mixed cultures in wastewater treatment
systems found that protein was also an important
constituent in EPS, possibly due to the large
quantities of exoenzymes entrapped in the EPS
(Dignac et al., 1998). In this study, the protein
content was greatest in the methanogenic sludge.
Table 5 shows that 41.3% of EPS extracted from
the methanogenic sludge by formaldehyde NaOH
were protein and only 18.7% were carbohydrate.
The ratio of carbohydrate and protein for the
methanogenic sludge was 0.45, as compared with
the reported ratios of 0.2 0.5 in previous studies
(Fang and Jia, 1996; Veiga et al., 1997). The same
ratio for aerobic sludge was 0.74 in this study,
which is substantially higher than the reported
ratios of 0.20.3 (Liu et al., 2001; Rudd et al.,

1984). The discrepancy could be due to the failure

to take account of humic substance in the previous
studies, leading to the overestimation of protein
content. This was because the presence of humic
substance could increase the color intensity when
the Lowry method was used for protein analysis.
Results of this study also found that, in addition
to carbohydrate and protein, the EPS also contained substantial amounts of humic substance. In
the EPS extracted from aerobic sludge by formaldehydeNaOH, 30.6% were humic substance. The
corresponding values were 8.4% for acidogenic
sludge and 22.8% for methanogenic sludge. These
results clearly indicate that humic substance is a key
constituent of EPS and should not be overlooked.

4.2. Effecti6eness of EPS extraction

Results of this study show that the formaldehydeNaOH process extracted the highest amounts of EPS from all the sludges. Formaldehyde
could fix the cell, and thus prevent cell lysis, by
reacting with amino, hydroxyl, carboxyl and sulfhydryl groups of proteins and nucleic acids of the
cell membrane (Alcamo, 1997). The presence of
NaOH increased the pH, resulting in the dissociation of acidic groups in EPS and the repulsion between the negative-charged EPS. This also increased the EPS solubility in water and thus allowed
more EPS to be extracted (Wingender et al., 1999).
Analytical results show that the each gram of
volatile solids contained 17.8, 13.8 and 28.7 mg of
DNA in aerobic, acidogenic and methanogenic
sludges, respectively. Of this DNA, only 1.11.2%
were found in the EPS extracted by formaldehyde
NaOH for the three sludges. In addition, DNA
accounted for only 0.10.2% of the EPS extracted
by all processes, except EDTA. All of these evidences suggested that the formaldehydeNaOH
extraction process did not cause cell lysis, and thus
the extracted EPS were not contaminated by the
intracellular substances.
The EPS extracted by EDTA constituted
0.20.4% DNA, the highest among all extraction
processes. This could be due to the removal of
cations by EDTA from the cell membrane, causing
cell lysis and the release of intracellular DNA
(Wingender et al., 1999). In addition, the EPS

H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249256

extracted by EDTA had the highest unidentified

constituents (34.039.0%), as compared with 9.0
19.1% in EPS extracted by the other five processes. This might be due to the formation of
complex between EDTA and the EPS. The
EDTA EPS complexes were not removed by
membrane separations, and thus the EDTA remained in the EPS after lyophilization.

4.3. Comparisons of extracted EPS with those

estimated by CLSM
The quantities of EPS in the aerobic and
acidogenis sludges were also analyzed using
CLSM following the methodology developed recently by Zhang and Fang (2001). In a 5 ml
aerobic sludge sample having a VS content of
4100 mg l 1, bacterial cells occupied 0.00262 ml
of volume based on a series of cofocal laser
scanning microscopic images. The cell concentration was accordingly estimated as 2310 mg l 1.
Thus each gram of VS contained 437 mg of EPS,
more than doubling the 165 mg extracted by
formaldehydeNaOH. Similarly, based on the
CLSM analysis each gram of acidogenic sludge
contained 536 mg of EPS, as compared with 179
mg extracted by formaldehyde NaOH. Even
though the quantities of EPS estimated by CLSM
might include the low-molecular weight metabolites, the substantial differences suggested that
using formaldehyde NaOH, the most effective
process so far, might still only extract a limited
portion of EPS. Thus, optimization of the formaldehyde NaOH extraction procedures, or further
development of a more effective extraction
method, is still warranted.

4.4. EPS in flocculent and granular sludges

The aerobic activated sludge and the acidogenic
sludge were both in flocculent form, whereas the
methanogenic sludge was granular. The total EPS
contents extracted by formaldehydeNaOH from
the two flocculent sludges were comparable, and
both were substantially higher than that extracted
from the granular methanogenic sludge. Microscopic observations have shown that the microorganisms in the flocculent sludges were loosely

255

aggregated, whereas those in the granular sludge

were densely packed (Fang, 2000). EPS apparently play an important role in the microstructures of flocculent and granular sludges. However,
the mechanisms of floc and granule formation and
the exact role of EPS remain unclear. Further
studies on these aspects are also warranted.

Acknowledgements
The authors wish to thank the Hong Kong
Research Grants Council (HKU 7004/00E) for
the financial support of this study, and to Tong
Zhang for the CLSM analysis.

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