~ Pergamon War Sci T«h Vol. 39. No.7, pp. 211-218.

1999
ro 1999/AWQ
Published by Elsevier SCIence LId
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0273-1223/99 $2000 + 0.00
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COMPARISON OF EXTRACTION
METHODS FOR QUANTIFYING
EXTRACELLULAR POLYMERS IN
BIOFILMS

Xiaoqi Zhang"', Paul L. Bishop'" and Brian K. Kinkle"''''

• Department o/Civil and Environmental Engineering, University o/Cincinnati,
Cincinnati, OH 4522/·0071. USA
.. Department 0/ Biological Sciences, University 0/ Cincinnati. Cincinnati.
OH 4522/-0006. USA

ABSTRACT

Five commonly used extraction methods - regular ceninfugation. EDTA extraction. ultracentrifugation,
steaming extraction and regular centrifugation With formaldehyde (ReF) - were selected to study their
effectiveness and repeatability in extracting extracellular polymeric substances (EPS) from aerobic/sulfale
reducing and mtrifyingldenitrifying biofilm samples. Biofilm EPS extraction yields were represenled by
carbohydrate and protein concentrations; the amount of cell lysIS during the extractions was indicated by
DNA concentratlOn. The results showed !hat analyzing wash walers is essential in quantifying biofilm EPS;
the contribution of this step varied from 8-50% of the total carbohydrate yield. dependIng on the extracllOn
method. Among the extraction methods, the RCF extraction gave the greatest carbohydrate yield, the
steaming extraction gave the greatest protem yield, and the other three extraction methods gave
approximately equivalent amounts of carbohydrale and proteins for both types of biofilm. DNA in the EPS
was 27 times smaller than in the pellets, indicating no significant cell lysis occurred during the extractions.
~ I999lA WQ Published by Elsevier Science Ud. All rights reserved

KEYWORDS

Aerobic/sulfate reducing; biofilms; carbohydrate; DNA; extracellular polymeric substances (EPS);

extraction methods; nitrifying/denitrifying; proteins.

INTRODUCTION

Microbial extracellular polymeric substances (EPS) are high molecular-weight mucous secretions of bacteria
and micraalgae. They range from tight capsules, which closely bind cells, to the loosely attached slime
material. Biofilm EPS possesses many important functions in water and wastewater treatment. including
anchoring the microorganisms near food sources, protecting them from dehydration and toxic substances,
and providing ion exchange properties due to negatively-charged surface functional groups which allow
them to bind cationic species such as heavy metals (Sutherland. 1977).

The EPS composition determines many important properties ofbiofilm such as density. porosity, difTusivity,
strength, elasticity, frictional resistance. thermal conductivity, and metabolic activity. More information
about their compositions will contribute to a better understanding of the physical and physiological behavior
ofbiofilms in environmental systems.
21 I
212 X. ZHANG et al.

Many ~ethods have been used to extract the EPS from different bacterial cultures and activated sludges for
analysIs. These extraction methods have included use of ammonium hydroxide, sodium hydroxide,
ethylene-diaminetetraacetic aCId (EDTA), sulfuric acid, trichloroacetic acid, boiling benzene,
ultrasonication. blending, high-speed centrifugation, and extraction by boiling or autoclaving (Brown and
Lester. 1980). However, most of these extraction studies were perfonned on activated sludge (Brown and
Lester, 1980, Gehr and Henry, 1983, Morgan et al., 1990, Urbain et al., 1993, Jia et al., 1996, Fr01und et al.,
1996); very little research has been conducted on biofilm samples.

The chemical compositions of the EPS are usually reported in the literature to be very heterogeneous.
Carbohydrate predominates and represents up to 65% of extracellular materials (Horan and Eccles, 1986);
other substances are also present, such as proteins. nucleic acids and lipids (Goodwin and Forster, 1985).
Their exopolymer component ratios vary, depending on the sample source and extraction technique (Morgan
et al., 1990). For example, Horan and Eccles (1986), Forster and Clarke (1983) and Morgan et al. (1990)
found more carbohydrate than proteins in activated sludges, with a ratio range of 0.16-0.70. Karapanagiotis
et al., (1989), Forster (1982) and Morgan et al. (1990) found more proteins than carbohydrate in digested
sludges, with a ratio range of 1.1-5.1.

In this study, five extraction methods, which are currently used for activated sludge, were evaluated for the
extraction of EPS from biofilm samples grown under aerobic/sulfate reduction and
nitrification/denitrification conditions. The five extraction methods are: (I) regular centrifugation, (2)
EDTA extraction, (3) ultracentrifugation, (4) steaming extraction, and (5) regular centrifugation with
fonnaldehyde. The five extraction methods were compared on the same samples to examine their
effectiveness and repeatability and to detennine to what degree they may cause cell lysis. Minimum cell
lysis is desired during the extractions to avoid contamination by intracellular material. To evaluate the
degree of cell disruption that may be caused during the extractions, pellets in the biofilm were intentionally
lysed and the amounts of DNA from the EPS and the pellets were compared.

Researchers dealing with the EPS of activated sludge nonnally wash their activated sludge samples first and
then discard the wash solution. Gehr and Henry (1983) proposed, though, that an initial washing step will
remove exopolymer material from activated sludge. Presumably, the washing step will wash out some
loosely attached extracellular polymers. The contribution of the washing step to the EPS collected from
biofilm samples was verified and the amount of its contribution was further examined in this research.

METHODS

Bio.,films Biofilrns are typically heterogeneous structures. Many biofilms may essentially be considered to
be layered. with an aerobic layer overlying anoxic or anaerobic layers. This is true ofbiofilms in which both
nitrification and denitrification occur, and in ones where sulfate reduction occurs in lower, anoxic layers.

Aerobic/sulfate reducing biofilms and nitrifying/denitrifying biofilms were grown in laboratory-scale

rotating drum biofilm reactors (RDBRs). Biofilm growth occurs on the surface of the RDBR rotating drum
and removable sampling slats which arc coated with shrink tubing. A more detailed description of the
reactor system can be found in Hanner (1991) and Harmer and Bishop (1992). The aerobic/sulfate reducing
biofilm reactor was initially seeded with wastewater and digested sludge from a municipal/industrial
wastewater treatment plant in Cincinnati, Ohio. The reactor was continuously fed with: yeast extract, 50
mg/I; sodium lactate (C 3Hs0 3Na), 156 mg/I; KH2P04, 120 mg/l; NaHC03, 180 mg/l; NH4CI, 100 mg/I;
CaCI 2 -2H 20, 15 mg/I; MgS04 -7H 20, 60 mg/I; FeCh- 4H 20, 1.5 mg/l; Na2S04, 200 mg/l. The pH was
maintained at 7.2-7.3 and dissolved oxygen (DO) was kept at 1.5-3.5 mg/1. The nitrifying/denitrifying
biofilm reactor was seeded with nitrifiers and secondary effluent from a trickling filter wastewater treatment
plant in Dayton, Ohio. It was continuously fed with: bacto peptone, 25 mg/l; yeast extract, 25 mg/I; NH4C1,
95 mg/I; K2C0 3, 320 mg/I; K2HP04. 25 mg/I; KH 2P04, 10 mg/I; MgS04 -7H 20, 11.25 mg/l; CaCh-2H20,
13.75 mg/I; FeCh -6H20, 0.125 mg/I; MnS04-H20, 0.0112 mg/1; CUS04. 0.0007 mg/I; Na2Mo04-2H20,
0.0004 mg/I; 2n504-71-1 20, 0.0120 mg/1. The pl-l and DO were kept at 7.5 and 1.5-3.5mg/I, respectively.
Biofilm samples were collected from the sampling slats and total solids (TS) were measured to represent
biofilm mass.
 Quantifying extracellular polymers in biofilms
213
BPS e~tractiQn meth~s All biofilm samples were analyzed in triplicate. Four steps were involved in
extractmg and collectmg the EPS from the biofilm samples (Figure I).

Biofilm samples
Washing, recover slime material (A)

Figure J. Procedure for the extrachon method compansons.

Step J: Washing (recover slime material). Biofilm samples (about 1 g) were put into centrifuge tubes along
with 25 ml MilIiQ water (Millipore). The tubes were shaken gently, and then centrifuged at 3,500 rpm
(6,ooOg) for 10 minutes. The liquid was decanted from the centrifuge tubes, and collected as the slime
material.

Step 2: Stripping (recover capsule-bound material). After the washing step, the tubes containing the biofilm
pellets were filled with another 25 ml MilIiQ water. The contents were blended in a vortex blender (Oster,
867-28L) at high speed for 1 minute to recover the capsule-bound material.

For purposes of extraction method comparisons, liquid from the washing step and the stripping step were
combined and brought to a volume of 50 mt.

Step 3: Extraction (separate the stripped materialfrom the cells). Five extraction methods were applied and
compared. These included:

(1) Rel:U1ar centriful:atjon (lia et al., 1996, with modification). 10 ml of the combined sample were
centrifuged at 12,000 rpm (lI,227g) for 30 minutes at room temperature.

(2) forA extraction (Brown and Lester, 1980). 10 ml of 2% EDTA was added to 10 ml of the combined
sample and left quiescently for 3 hours at 4°C. The centrifuge tubes were then centrifuged at 14,000 g for
20 minutes at 4°C.

(3) U1tracentriful:atjon (Brown and Lester, 1980). 10 ml of the combined sample were centrifuged at
33,000 g for 10 minutes at 4°C. The pellets obtained were resuspended with another 10 ml MilliQ water,
and centrifugation was repealed for another 10 minutes to enhance the extraction.

(4) Slearnjnl: extraction (Brown and Lesler, 1980). 10 ml of the combined samples were sleamed in an
autoclave at 80 aC under I bar pressure for 10 minutes and then centrifuged while slill hot at 8,000 g for 10
minutes; during centrifugation, the temperature was reduced to 15°C. The steaming treatment was used in an
214 X. ZHANG et al.
autoclaving under normal autoclave
attempt to reduce the disruptive effects on the cells of boiling or
conditions (120°C, 16 bars).

The same extraction procedure

(5) ~e~ular centriful:atjon with foona1dehyde (RCF) (Jia et al., 1996).
25 ml 8.5% NaCI containin g 0.22% formaldehyde was
descn~ed above was used on I g samples, except that
water in the stripping step. 10 ml of the combine d sample was centrifuged at 12,000
added Instead ofMilliQ
Refrigera ted Superspe ed Centrifu ge with an SS-34 rotor
rpm (11,227 g) for 30 minutes in a Sorvall RC-5B
(Du Pont).

centrifugation after each extraction

Step 4: Filtering and collectmg the EPS. The supernatant obtained on
that samples were free of cells. The pellets
was filtered through 0.22 j.!m cellulose acetate filters to ensure
were discarded ifnot used for the cell lysis study.

yields. the same procedure described

In order to evaluate the contribution of the washing step to biofilm EPS
the capsule material were treated separately to
above was replicated. except that the slime material and
obtain separate EPS yields.

ChemIcal composition qna[ysiy The EPS yields in the extracts

were represented by carbohydrate and
protein concentrations, and cell lysis was indicated by DNA concentr ation. A HP 8453 UV-Vis diode-array
hotomete r was used. A phenol-s ulfuric acid (Gerhardt et al., 1994) method was
(190 - 1100 nm) spectrop
. A modifica tion of the Bradford (1976) method,
used to quantitY Carbohydrate, with glucose as the standard
ie procedur e (Pierce Chemica l), was used to quantify proteins, with Bovine Serum
called the Coomass
method (Gerhard t et al., 1994) was used to quantitY DNA,
Albumin (BSA) as the standard. A fluorometric
with salmon sperm as the standard .

Protein dialysis. Formaldehyde used in the RCF extraction interfere

s with protein analysis. Theref~re, a~1
n were put through a Spectra/p or I Molecularporous DialySIS
the samples treated after the RCF extractio
e with MWCO 6 - 8,000 (Spectru m) to remove the formalde hyde. The dialysis membranes were
Membran
An optimum dialysis time of 5-7
placed in a beaker containing 2 liter of continuously stirred MilliQ water.
was determin ed by monitori ng protein concentr ations over an 88 hour time period.
hours

Biofilm samples
Collect pellets
Washing , recover (A)

Grind in liquid N" freeze-th aw three times

Add extracllo n buffer and proteinas e K

Carbohy drate
DNA

Figure 2. Procedure for lysing the pellets from biotilms.

during the extractions, the pellets

Celll.l'Sis stuc[y In order to evaluate the degree of cell lysis that occurred
a freeze-thaw method (Zhou et aJ., 1996). Figure 2
were collected after the RCF extraction and lysed using
 Quantifying eXllacellular polymers in biofiIms 215

shows the procedure for lysing the pellets from biofilms. DNA, carbohydrate and proteins from the pellets
were quantified. In order to verifY the accuracy of results, the EPS and the lysed pellets were combined and
analyzed for DNA, carbohydrate and proteins.

RESULTS AND DISCUSSION

Contribution of the wasbinK step to the BPS yields It was found that the carbohydrate extraction yield
contributed by the washing step varied from 8-50% among the different extraction methods (see Figure 3),
which verifies that collecting the wash water from the washing step is essential in biofilm EPS analysis
(Gehr and Henry, t 983). Ultracentrifugation (33,000 g) contributed the greatest amount of carbohydrate to
the EPS yields from the washing step. Depending on the method used, the more the carbohydrate collected
from the washing step, the less the carbohydrate recovered from the stripping step.
M
(:,
70
*en
f- 60
Oll
::l.
On
::l. 50
c::"
.~
t> 40
..='" 30
CIl
CIl

'"
E 20
eOJ

-0 10
>-
..c:
0
.D
.... 0
'"
U

~~~p-.

M
b
- 70
*
en
f-

On
Oll
::l.
::l.
c::
60
50
--
.~
t> 40
<b '"
CIl
30
~
E
C 20
OJ
.E 10
Vi
c::
0

en
u 0
0..
lJ.J

Figure 4. Exllactlon method comparison on sulfite/reducing biofiIms.

216 X ZHANGetal

70
I

d=
arb hydrate
60 Proleins T
I
SO
I) A
J I
40

"'0

5 20
~
Vi 10 '
c:
o
u
til
a..
o
UJ

FIgure 5. ExtractIon method companson on nilrifying/denitrifymg blotilms.

Extraction method cowpansons Figures 4 and 5 show the extracted carbohydrate, protein and DNA
concentrations measured by each extraction method. Among the methods, for both types of biofilms, the
RCF extraction provided the greatest amount of carbohydrate, but only relatively small amounts of proteins
because formaldehyde cross-links proteins and make them hard to detect. The steammg extraction
procedure helps to release the stripped material from the pellets; it gave the second greatest amount of
carbohydrate and the greatest amount of proteins. The other three extraction methods gave approximately
equivalent amounts of carbohydrate and proteins. In both types of biofilms, the protein yield was lower than
the carbohydrate yield In the EPS. The RCF method gave the highest yield ratio of carbohydrate to proteins:
13 7 for the aerobic/sulfate reducing biofilms, and t t.O for the nitrifying!denitrifying biofilms; the rest of the
methods gave a range of ratios: 1.54-2.23 for the aerobic/sulfate reducing biofilms, and 2.99-4.64 for the
nitrifyingldemtrifying biotilms.

A perequlslte for a good extraction method is to remove exopolymer material effectively, with minimal cell
lysis (Gehr and Henry, 1983). DNA was used as the cell lysis indicator. The amounts of DNA after
extraction may be due either to its release from the exopolymer matrix or from cell disruption. DNA
concentrations were very low in both aerobic/sulfate reducing and nitrifymgldenitrifying biofilms, the
highestbeing(4.17± 1.53) x 1O"llglllgTS.

Cell lySIS study. Although the amounts of DNA in the extracted EPS were very small, it is still difficult to
conclude that there was no cell lysis during any of the extractions without knowing the DNA amounts in the
pellets. Therefore, an experiment was designed to study the DNA amounts in the pellets, together with a
comparison of DNA amounts in the EPS and the blOfilms. Carbohydrate and protein concentrations were
«
measured as well (FIgure 6). A great amount of DNA 18.52 ± 1.55) x 10'3 IJgllJg TS) was detected in the
pellets. There was only (0,68 ± 0.15) x 10'3 1Jg!1!8 TS in the EPS. DNA amounts in the pellets were 27
hmes higher than in the EPS on a total solids basis. Generally, greater than 90 % of the cells will be lysed
using the freezc-thaw method (Zhou et al., 1996). Apparently, no significant cell lysis occurred during any
extractions. The sum of the DNA from the EPS and the pellets almost exactly equaled the DNA amounts in
the blOfilms, which verified the accuracy of the measurement.
 Quantifymg extracellular polymers in biotilms 217

80,---------------

70 I- Carbohydrate
c=:::J Proteins
~:1.
"Cb
:1.
60

50
I_DA

40

30

20

10

0'--- Lo,• ~ I..,.. '---

EPS Pellets Bi films

Figure 6. Blofibn composition (sulfate/reducmg biofilms, RCF extraction method),

CONCLUSIONS

Collecting the wash solutions is essential in analyzing the EPS from biofilm; its contribution varied from
8-50% of total carbohydrate yields, depending on the extraction method. The five extraction methods
presented a general trend in their ability to extract the EPS from aerobic/sulfate reducing and
nitrit)'ing/denitrit)'ing biofilms. The RCF extraction gave the greatest carbohydrate yield and steaming
extraction gave the greatest protein yield for extracted EPS from both types of biofilms. The other three
extraction methods gave approximately equivalent amounts of carbohydrate and proteins. DNA
concentrations in the EPS were 27 times smaller than in the pellets, indicating no obvious cell lysis occurred
during the five different extractions.

ACKNOWLEDGMENT

We gratefully acknowledge Tong Yu, Jin Li, Qingzhong Wu and Mark Kramer of the University of
Cincinnati for their assistance with reactor operation. This study was funded by the Superfund Basic
Research Programs of the National Institutes of Environmental Health Sciences (NIEHS), USA.

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