Journal of Biotechnology 95 (2002) 249–256

Extraction of extracellular polymeric substances (EPS) of
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
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, formaldehyde–NaOH 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 formaldehyde–NaOH), 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
and energy reserves during starvation. EPS were
found crucial to the flocculation (Frølund 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 sub-
stances (Frølund 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
* Corresponding author. Tel.: +852-2859-2660; fax: +852-
E-mail address: (H.H.P. Fang).
0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S0168- 1656( 02) 00025- 1
H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249–256 250
was found in substantial quantities in the
sludges of many wastewater treatment reactors
(Fang and Jia, 1996; Veiga et al., 1997). Humic
substance (Frølund 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 concen-
tration in EPS is scarce. The EPS normally con-
tain 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 centrifuga-
tion, ultrasonication and heating, whereas com-
mon chemical extractions include uses of
alkaline, ethylenediamine tetraacetic acid
(EDTA) and cation exchange resin. However,
there is no standard extraction procedure estab-
lished so far, making it very difficult to mean-
ingfully compare and interpret published results.
This study was conducted to compare the ef-
fectiveness of six extraction procedures for EPS
of sludges sampled from three wastewater treat-
ment 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% BOD
, and 85–90% ni-
trogen from wastewater. The acidogenic sludge
was sampled from a fermentor (Biostat B, B.
Braun Biotech) treating a sucrose-rich wastewa-
ter. 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 de-
graded 97% phenol in wastewater and produced
a biogas comprising 69% methane and 31% car-
bon 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 high-
speed centrifugation (20 000 G) for 20 min, fol-
lowed 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 ex-
tracted 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 ultrasoni-
cation (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. Frølund et al. (1996)
recommended 0.5–1 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) 249–256 251
2.3. Chemical analysis of extracted EPS
The total quantity of extracted EPS was mea-
sured 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 (Frølund 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-hy-
droxydiphenyl sulfuric acid method (Blu-
menkrantz and Asboe-Hansen, 1973) as modified
by Kintner and Van Buren (1982) using glu-
curonic 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).
2.4. Quantification of EPS using CLSM
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 mi-
crobial 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 con-
tent. The EPS contents in the aerobic and
acidogenic sludges were also measured using this
method for comparison. The instrument, proce-
dures and image analytical software used were the
same as previously reported (Zhang and Fang,
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) 249–256 252
Table 1
EPS extracted from various sludges
Extraction Acidogenic (mg per g-VS) Aerobic (mg per g-VS) Methanogenic (mg per g-VS)
179.093.2 164.993.9 102.191.5 Formaldehyde–NaOH
104.990.7 EDTA 73.290.5 146.891.1
100.090.3 77.991.1 36.890.8 Formaldehyde-ultrasound
58.491.1 Cation exchange resin 30.190.9 57.891.0
56.991.2 49.791.2 32.590.4 Formaldehyde
36.990.6 15.890.2 Control 25.790.3
Mean value (n=3) 9S.D.
Table 2
Comparison of EPS extracted from aerobic and anaerobic sludges
Extraction method Sludge Total EPS (mg per g-VS) Reference
165 Aerobic Present study Formaldehyde–NaOH
Aerobic Cation exchange resin 109 Rudd et al., 1984
58 Liu et al., 2001 Aerobic 80 °C
179 Formaldehyde–NaOH Present study Acidogenic
102 Methanogenic Present study Formaldehyde–NaOH
91 Phenol Veiga et al., 1997 Methanogenic
74 Fang and Jia, 1996 Methanogenic Formaldehyde at 100 °C
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 extrac-
tion from all sludges in descending order is: form-
aldehyde–NaOH, EDTA, formaldehyde-ultra-
sound, cation exchange resin, formaldehyde, and
control. For each gram of volatile solids, the
formaldehyde–NaOH process extracted 165, 179,
and 102 mg of EPS for the aerobic, acidogenic and
methanogenic sludges, respectively. For compari-
son, 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 formal-
dehyde alone, cation exchange resin and formalde-
hyde-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 depend-
ing on the extraction method. Corresponding val-
ues in literature for aerobic sludge varied
drastically, from 6–254 mg (Liu et al., 2001) to 437
mg (Frølund 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 formaldehyde–NaOH 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
formaldehyde–NaOH 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 formaldehyde–NaOH 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 pro-
cesses. 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) 249–256 253
Table 3
Constituents of EPS in each gram of aerobic activated sludge
Protein Humic substance Carbohydrate Extraction Uronic acid Unknown DNA
(mg) (mg) (mg) (mg) (mg) (mg)
54.692.0 40.591.7 50.493.7 4.290.4 0.3590.05 14.893.3 Formaldehyde
2.190.4 0.4790.03 49.793.1 59.292.5 22.990.5 EDTA 12.491.2
20.491.0 28.990.9 18.991.5 1.890.1 0.1390.02 7.890.5 Formaldehyde-
Cation exchange 17.690.9 16.490.8 1.290.2 0.1490.02 9.891.0 12.790.4
1.190.1 0.0790.01 12.390.3 9.490.3 10.990.6 15.991.0 Formaldehyde
0.590.1 Control 0.0690.01 7.790.1 3.190.2 7.990.1 6.490.3
Mean value (n=3) 9S.D.
Table 4
Constituents of EPS in each gram of acidogenic sludge
Protein Carbohydrate Extraction Humic substance Uronic acid Unknown DNA
(mg) (mg) (mg) (mg) (mg) (mg)
Formaldehyde 15.191.0 110.994.0 5.590.3 0.1590.03 21.591.1 25.891.0
2.390.2 0.2290.03 38.391.1 EDTA 41.791.2 6.590.4 15.991.0
5.090.4 2.590.3 0.0590.01 10.890.7 10.090.4 71.691.3 Formaldehyde-
3.090.2 2.290.1 0.0890.01 Cation exchange 8.290.3 38.790.8 6.290.3
1.790.2 0.0290.00 5.990.4 7.490.3 2.590.3 39.491.0 Formaldehyde
Control 1.090.1 23.790.7 0.0290.00 5.990.2 4.490.2 1.990.2
Mean value (n=3) 9S.D.
Table 5
Constituents of EPS in each gram of methanogenic sludge
Protein Humic substance Carbohydrate Extraction Uronic acid Unknown DNA
(mg) (mg) (mg) (mg) (mg) (mg)
Formaldehyde 23.392.0 19.190.8 2.190.2 0.1990.01 15.391.45 42.191.7
1.290.1 0.2690.03 12.090.7 28.390.52 24.390.7 6.890.5 EDTA
5.690.2 1.090.1 0.0490.00 Formaldehyde- 5.190.78 12.090.4 13.190.7
5.590.3 0.990.1 0.0590.01 7.990.4 5.190.95 10.690.4 Cation exchange
11.990.8 4.690.3 0.890.1 0.0390.00 5.590.44 Formaldehyde 9.790.7
0.390.1 0.0390.00 2.590.17 Control 4.190.2 3.190.1 5.890.2
Mean value (n=3) 9S.D.
H. Liu, H.H.P. Fang / Journal of Biotechnology 95 (2002) 249–256 254
all sludges were primarily composed of carbohy-
drate, protein and humic substance, plus small
quantities of uronic acid and DNA. About 34–
39% of EPS extracted by EDTA remained uniden-
tified, 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 carbohy-
drate, 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 carbohy-
drate, protein and humic substance (ranging
25–33%) fromaerobic 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 0.2–0.3 (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 con-
tained substantial amounts of humic substance. In
the EPS extracted from aerobic sludge by formal-
dehyde–NaOH, 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 formalde-
hyde–NaOH process extracted the highest amou-
nts of EPS from all the sludges. Formaldehyde
could fix the cell, and thus prevent cell lysis, by
reacting with amino, hydroxyl, carboxyl and sulf-
hydryl groups of proteins and nucleic acids of the
cell membrane (Alcamo, 1997). The presence of
NaOH increased the pH, resulting in the dissocia-
tion of acidic groups in EPS and the repulsion be-
tween the negative-charged EPS. This also increas-
ed 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 1.1–1.2%
were found in the EPS extracted by formaldehyde–
NaOH for the three sludges. In addition, DNA
accounted for only 0.1–0.2% of the EPS extracted
by all processes, except EDTA. All of these evi-
dences suggested that the formaldehyde–NaOH
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
0.2–0.4% 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) 249–256 255
extracted by EDTA had the highest unidentified
constituents (34.0–39.0%), as compared with 9.0–
19.1% in EPS extracted by the other five pro-
cesses. 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 re-
mained 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 re-
cently by Zhang and Fang (2001). In a 5 ml
aerobic sludge sample having a VS content of
4100 mg l
, bacterial cells occupied 0.00262 ml
of volume based on a series of cofocal laser
scanning microscopic images. The cell concentra-
tion was accordingly estimated as 2310 mg l
Thus each gram of VS contained 437 mg of EPS,
more than doubling the 165 mg extracted by
formaldehyde–NaOH. 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 metabo-
lites, 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 formal-
dehyde–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 formaldehyde–NaOH from
the two flocculent sludges were comparable, and
both were substantially higher than that extracted
from the granular methanogenic sludge. Micro-
scopic observations have shown that the microor-
ganisms in the flocculent sludges were loosely
aggregated, whereas those in the granular sludge
were densely packed (Fang, 2000). EPS appar-
ently play an important role in the microstruc-
tures 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.
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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