wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 8

Avai lab le a t www.sc iencedi rec t .com

journa l homepage : www.e lsev ie r . com/ loca te /wat res

Extracellular polymeric substances diversity of biofilms grownunder contrasted environmental conditions

Monique Ras a, Dominique Lefebvre a, Nicolas Derlon b,c,d, Etienne Paul b,c,d,Elisabeth Girbal-Neuhauser a,*a LBAE, Laboratoire de Biologie appliquee a l’Agro-alimentaire et a l’Environnement, Institut Universitaire de Technologie, Universite Paul

Sabatier Toulouse III, 24 Rue d’Embaques, 32000 Auch, FrancebUniversite de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, Francec INRA, UMR792 Ingenierie des Systemes Biologiques et des Procedes, F-31400 Toulouse, FrancedCNRS, UMR5504, F-31400 Toulouse, France

a r t i c l e i n f o

Article history:

Received 2 September 2010

Received in revised form

15 November 2010

Accepted 15 November 2010

Available online 24 November 2010

Keywords:

Microbial biofilm

Autotrophic

Extracellular polymeric substances

Size distribution

Extraction strategy

* Corresponding author. Tel.: þ33 5 62 61 28E-mail address: elisabeth.neuhauser@iut

0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.11.021

a b s t r a c t

Extracellular Polymeric Substances (EPS) analysis was undertaken on three biofilms

grown under different feeding conditions and offering diverging microbial activities and

structural characteristics. EPS were extracted by a multi-method protocol including soni-

cation, Tween and EDTA treatments and were characterized by size exclusion chroma-

tography (SEC). Tween and sonication extracts presented higher EPS size diversity

compared to EDTA extracts. EPS size diversity also increased with microbial functions

within the biofilms and a specific 25e50 kDa cluster was identified only in extracts from

biofilms presenting autotrophic activity. Another specific size cluster (180 kDa) occurred in

Tween extracts provided from the mechanically stable biofilms. Such specific EPS appear

as potential indicators for describing microbial and structural properties of biofilms.

This study brings new elements for designing EPS fractionation and shows that size

distribution analysis is an interesting tool to relate EPS diversity with macro-scale char-

acteristics of biofilms.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction treatment facilities generally use suspended floc forming

Biofilms are described in literature as fixed micro-organisms

on an interface and immobilized in a matrix of extracellular

polymeric substances (or EPS) of microbial origin. The stable

environment offered by the EPS matrix cradles the develop-

ment of a large span of microbial communities of which

several can be deleterious. The microbial heterogeneity of

biofilms can also be of great interest in the environmental

sector since such concentrated and diversified microbial

activities can be beneficially exploited for treating organic and

inorganic water pollutants. However, municipal wastewater

13; fax: þ33 5 62 61 63 01-tlse3.fr (E. Girbal-Neuhauier Ltd. All rights reserved

biomasses which are often washed out from the system (Liu

et al., 2004; Matsumoto et al., 2007) and hence experience

low microbial diversity functions. Fixed biomass such as

biofilms can prevent such losses by retaining bacterial diver-

sity inside the system and particularly slow-growing bacterial

populations, such as nitrifiers. Such configurations can

hence increase the treatment efficiency.

The EPSmatrix is often stated as consolidatingmaterial for

the entire biofilm. Indeed, the extracellular compartment

can reach 98% of the total organic carbon fraction of biofilms

(Jahn and Nielsen, 1998). EPS compounds are excreted by the

.ser)..

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 81530

microbial population, but can also result from natural cell

lysis or from hydrolytic activities. A wide variety of polymers

are reported within the matrix, where a major proportion is

attributed to proteins and polysaccharides, while lipids and

nucleic acids are rather found in minor proportions (Azeredo

et al., 1999; Jahn and Nielsen, 1995). The influence of envi-

ronmental conditions on the composition of EPS compounds

has already been suggested in literature (Branda et al., 2005).

Regarding the use of carbon and nitrogen elements for EPS

production, the carbon/nitrogen ratio of the influent is liable

to impact the type of produced EPS, i.e. carbohydrate and

protein production (Durmaz and Sanin, 2001; Li et al., 2008).

In addition, the carbon/nitrogen ratio can specify the micro-

bial ecology of the biofilm (Ohashi et al., 1995), by promoting

either heterotrophic growth (high ratio) or autotrophic micro-

organisms (low ratio). Regarding the biochemical responses

to environmental and microbial parameters, characterizing

the EPS fraction of biofilms could thus be a relevant procedure

for describing relations between EPS and biofilm structure.

In literature, studies undertaken on molecular character-

ization of EPS in biofilms are few. Under axenic conditions,

exopolysaccharides and more specifically uronic acids con-

taining polymers extracted from biofilms are described as

essential for providing the matrix framework through strong

anionic interactions (Chen and Stewart, 2002; Davies et al.,

1993). For multi-species biofilms, the conditions are even

more complex since a wide range of other molecular inter-

actions have to be considered. Proteins are characterized

by ionic, hydrophobic and neutral amino-acids and a large

range of chemical interactions (electrostatic, hydrophobic

and low energy hydrogen bonds) are able to link proteins to

the biological matrix (Mayer et al., 1999). Proteins also include

functionally active enzymeswhich take part in the production

and degradation of the matrix. Therefore, inherent chemical

properties of EPS and especially proteins can offer qualitative

information on both physical and dynamic properties of the

biofilm.

However, the structural heterogeneity and complex func-

tional properties in environmental associated biofilms make

EPS characterization somewhat difficult. Several analytical

methods including physical and chemical techniques are

used (Denkhaus et al., 2007) but with care depending on the

aim of investigation as well as the type of studied biofilm.

Microscopic and optical methods which involve EPS staining

techniques are not always appropriate for visualizing these

components in thick and complex biofilms due to light

attenuation or probe penetration problems. Infrared spec-

troscopy is also widely used in biofilm analysis with similar

limitations relative to the penetration capacity of the IR radi-

ations (Boualam et al., 2002). Considering these technical

restrictions, molecular characterization of complex biofilms

can be achieved by extracting the EPS from the biofilm and

then characterizing the soluble extract by chromatography or

electrophoresis separationmethods. Although widely used on

activated sludge samples (Comte et al., 2007; Garnier et al.,

2005), this molecular scale investigation strategy was never

applied for biofilm EPS characterization.

Molecular weight (MW) distributions of extracted EPS can

offer global characteristics of the sample and has been sug-

gested as a useful tool for fingerprint identification. Garnier

et al. (2005) evidenced different MW profiles depending on

the origin of activated sludge. Authors showed that proteins

where generally found in the high MW fractions (10e600 kDa)

while sugars were rather found in the lower MW fractions

(1 kDa). However, studying the size distribution of EPS in

complex bacterial aggregates reveals to be tricky since such

analysis implies prior extraction methods which can affect

not only the proportion of extracted EPS (Ras et al., 2008a;

Zhang et al., 1999) but also the qualitative aspect of these

polymers (Comte et al., 2007; Simon et al., 2009).

The present paper explores EPS size distributions within

biofilms in order to figure out specific molecular characteris-

tics which could explain particular biofilm biological and/or

physical properties. In order to validate such an approach,

the investigated biofilms were grown under contrasted envi-

ronmental conditions to promote diverging microbial activi-

ties within each biofilm. A multi-method protocol previously

described for extracting EPS from activated sludge (Ras et al.,

2008a) was used to sample EPS compounds from the bio-

films. This protocol, based on mechanical, hydrophobic and

ionic extraction methods, offers a globally diversified EPS

extract which can be consistent of the studied biofilms. The

distribution of EPS contents as well as EPS molecular weight

profiles were investigated in order to relate specific molecular

EPS characteristics to biofilm growth conditions and/or

microbial populations. The impact of EPS extraction proce-

dures on this molecular fingerprint diagnosis was also

considered. According results are expected to help improve

knowledge on biofilm growth control which is lacking in the

wastewater and water distribution sectors.

2. Methods

2.1. Experimental setup

Three biofilms were grown in hydrodynamic controlled Cou-

ette Taylor reactors as described by Coufort et al. (2007). For

a fixed gap between the two concentric cylinders, the rota-

tional speed of the inner cylinder was fixed in order to have

a wall shear stress of 0.5 Pa during the growth period. Biofilms

grew on 25 polyethylene plastic plates (100 � 50 � 5 mm)

distributed around the external cylinder.

2.2. Biofilm growth conditions

A mixed carbon source composed of ethanol, propionic acid,

glucose and sodium acetate was used as organic substrate

for the development of the biofilms. Reactors were inoculated

with conventional activated sludge sampled from the aeration

tank of a local municipal wastewater treatment plant. Two

biofilms were developed under organic substrate-limiting

conditions and with a constant surface loading rate of

2.5 g COD m�2 d�1 (COD: Chemical Oxygen Demand). In order

to obtain either a heterotrophic biofilm (B1) or a mixed auto-

trophic/heterotrophic biofilm (B2), the feed diverged in COD/

NH4eN ratios. The feed for B1was fixed at 73 g COD g�1 NeNH4

(9.5 mg NH4eN L�1) and the feed for B2 at 4 g COD g�1 NH4eN

(175 mg NH4eN L�1). For these two cases, the oxygen

concentration in the bulk liquid was kept constant at a value

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 8 1531

closed of the oxygen saturation concentration. A third biofilm

(B3) was grown under a high substrate loading rate of

25 g COD m�2 d�1 and with a ratio of 4 g COD g�1 NH4eN

(175 mg NH4eN L�1). In this case, the oxygen concentration

was kept constant between 6 and 7 mg O2 L�1, inducing

oxygen-limiting growth conditions.

During the overall characterization period, ammonia

(NH4þ), nitrite (NO2

�), nitrate (NO3�) and COD were measured

daily in the inlet and in the outlet of the Couette Taylor

reactors. The ammonia concentration was measured using

the Nessler method, nitrite and nitrate concentrations were

determined by spectrometry and the COD was obtained with

the method based on the oxidation by potassium dichromate

(Standard Methods, 1995). Biofilm at steady state was defined

as a biofilm characterized by stable COD removal, nitrification

and denitrification rates. Stable COD removal, nitrification

and denitrification and thus steady state were usually reached

after 60 days of biofilm development.

2.3. Biofilm characterization

The average biofilm thickness was measured by image anal-

ysis as described in Coufort et al. (2007). Mean accumulated

mass was measured after biofilm detachment from the poly-

ethylene plastic plates by gentle scraping and suspension in

the liquid reactor. Detached biomass was then recovered

by centrifugation (1500�g; 15 min) and measured in terms

of Suspended Solids and Volatile Suspended Solids concen-

tration (g VSS L�1) according to the standard procedures

(Standard Methods, 1995).

Total COD removal, nitrification efficiency and denitrifi-

cation efficiency were evaluated by comparing the inlet

and outlet values of COD, ammonium, nitrate and nitrite

concentrations.

2.4. EPS extraction by the multi-method protocol

Bound EPS were extracted according to the previously

described multi-method protocol validated on activated

sludge samples (Ras et al., 2008a). Biofilm samples were

centrifuged (10 000�g; 20 min) and pellets were washed twice

in Phosphate Buffer Saline (PBS) pH 7. Each biofilm sample

was subdivided in three 10 mL aliquots containing around

5 g VSS L�1 for triplicate extractions. One protocol involved

three extractionmethods in sequence: sonication (3� 2min in

PBS), Tween (0.25% in PBS, 1 h) and then EDTA (2% in Tris-HCl

0.3 mol L�1, pH 8.5, 1 h), with intermediate centrifugation

steps (10 000�g; 20 min). EPS extracts were measured the

same day for protein and polysaccharide contents as well

as for G6P-DH activity, and then stored at �20 �C for further

analysis.

The protocol extraction efficiency was evaluated after

repeating three times the protocol sequence on the same

biofilm sample. The decrease of the protein content recovered

after each protocol sequence fitted an exponential curve as

described in Ras et al. (2008a). The total protein content in

biofilm extracts obtained by repeating the extraction protocol

reached 246 mg eq. BSA g�1 VSS, with 116 mg eq. BSA g�1 VSS

obtained by applying the protocol only once (results not

shown). The extraction yield performed on the biofilm was

hence 47%, which is similar to yields obtained on activated

sludge samples (Ras et al., 2008a).

Protein measurements were performed on all soluble

extracts from B1, B2 and B3 biofilms using the Bicinchoninic

Acid (Smith et al., 1985) or BCA reagent (SigmaeAldrich),

according to Ras et al. (2008b) procedure. This quantification

method was chosen according to its better tolerance towards

chemicals used during extraction compared to modified

Lowrymethod (Ras et al., 2008a). Bovine SerumAlbumin (BSA)

was used as standard. Polysaccharide concentrations were

determined using the Anthrone method (Dreywood, 1946).

Glucose was used as standard. Each measurement was

undertaken on duplicate samples.

2.5. Cell lysis control

The activity of the intracellular G6P-DH was measured

according to Ras et al. (2008a). Enzyme substrate solution

was prepared with 0.2 M Tris-HCl pH 8.5, 0.2 M 2-mercaptoe-

thanol (Acros), 0.0005 M Nicotine Adenine Dinucleotide (NAD,

Acros) and 0.01 M D-glucose-6-phosphate (Fluka). Enzyme

activity was evaluated after incubating 200 mL of sample with

800 mL of the enzyme substrate solution at room temperature

and measuring NADH production at 340 nm during 30 min

G6P-DH activity was expressed as units (U) per mg of VSS, one

unit corresponding to the number of nmol of NADH produced

per min in the assay conditions.

In order to correlate the G6P-DH activities measured in

the extracts or in the whole biofilms with a number of lysed

cells, a preliminary calibration was performed using Cupria-

vidus necator DSM 545 suspensions. C. necator was cultured as

previously described by Ramsay et al. (1990). Briefly, the

culture was first grown for 12 h in a liquid Nutrient Broth

medium (Merck) under agitation (200 rpm) and at 30 �C. 10 mL

of the suspension was then inoculated to 150 mL of a Mineral

Medium supplemented with glucose (10 g L�1) and incubated

for 12 h at 30 �C at 100 rpm. Every 4 h, 10 mL of a culture

medium sample was filtered on a cellulose 0.2 mm filter then

dried and weighed in order to determine the total biomass

concentration. Bacterial population was also evaluated by

serial dilution of the samples and numeration on TCA agar

plates: a value of 4.08 10�6 g of dry biomass per 106 cells was

determined. After 12 h, bacteria were harvested by centrifu-

gation (10 000g, 10 min) and resuspended in a equal volume

of TES buffer (Tris-HCl 50 mM pH 8, EDTA 0.29 g L�1, saccha-

rose 25%).

Cell lysis was then induced by adding 50 mL of a lysozyme

solution at 47 000 U/mg (SigmaeAldrich) to 1 mL of the TES

bacterial suspension. After 1 h at 37 �C, numeration was per-

formed on the suspension and the G6P-DH measured on the

supernatant. Data obtained on three independent samples

indicated that 0.2 U were released per 106 disrupted cells,

also corresponding to 49,020 U per g of dry cells.

2.6. Chromatography analysis

Chromatography was performed using a high-performance

liquid chromatography system (AKTA Purifier, GE Healthcare)

equipped with a 1 mL injection loop, a UV detector and

a conductivity cell. Size exclusion chromatography (SEC) used

Fig. 1 e Microscopic side views of B1 (A), B2 (B) and B3 (C)

biofilms. S: Substratum ; B: Biofilm.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 81532

a 24 mL sepharose gel filtration column (Superose 6, GE

Healthcare). Elution was carried out at room temperature

using PBS at constant 1 mL min�1 flow rate. According to

manufacturer information, size fractionation is performed

between 5 and 5000 kDa. Calibration of the column was

undertaken by injecting ten different size protein standards

(high and low molecular weight calibration kits GE Health-

care: aprotinin (6500 Da), ribonuclease (13 700 Da), chymo-

trypsin (25 000 Da), carbonic anhydrase (29 000 Da), bovine

serum albumin (67 000 Da), conalbumin (75 000 Da), aldolase

(158 000 Da), catalase (232 000 Da), ferritin (440 000 Da) and

thyroglobulin (669 000 Da)).

The calibration curve revealed the following equation: log

(MW) ¼ �0.2939V þ 9.8481 with Molecular Weight (MW)

expressed in Da and the elution volume V in mL. The total

exclusion volume was determined after injection of Blue

Dextran 2000 (GE Healthcare, 2$106 Da) andwas found at 8mL.

Chromatogram profiles were recorded with UNICORN 5.1

software (GE Healthcare). Peak retention times and peak areas

were directly calculated and delivered by the program.

3. Results

3.1. Global characteristics of the developed biofilms

Three biofilms (B1, B2 and B3) were developed under different

feeding conditions in terms of COD/NH4eN ratios as well as

surface organic loading rates. These experimental conditions

were chosen according to previous results which reported

the influence of growth conditions on biofilm structure and

biological activity (Coufort et al., 2007; Derlon et al., 2008;

Wijeyekoon et al., 2004). The B1 biofilm grew under a high

COD/NH4eN ratio of 73 (nitrogen limitation) while B2 and B3

biofilms grew under a low COD/NH4eN ratio of 4 (excess

nitrogen). This carbon/nitrogen ratio varied by modifying

ammonium concentration in the feed. Both B1 and B2 biofilms

were grown under a low surface loading rate of 2.5 g CODm�2

per day, while B3 biofilm received a high surface loading rate

of 25 g COD m�2 per day.

Physical measurements (Table 1) and microscopic observa-

tions (Fig. 1) of all biofilms were undertaken when steady state

COD and nitrogen removal rates were reached. B1 and

B3 biofilms were characterized by a particularly thick and fila-

mentous structure inopposition to the thinanddenseraspect of

Table 1 e Physical and structural characteristics of B1, B2 and Bthickness, accumulated biomass and natural cell lysis measur

Biofilm B1

COD/TKN 73

Surface loading (g COD m�2 d�1) 2.5

Aspect Homogeneous

Filamentous

Surface colonization Complete

Average biofilm thickness (mm) 4.4 � 1.1

Mean accumulated mass (g VSS m�2) 8.5

a Heterogeneous biofilm thickness due to sloughing events.

B2 biofilm (Fig. 1). B1 and B2 biofilms were fed under a low

organic load and exposed a homogeneous colonization over the

surface plates. B3 biofilm, on the other hand, was fed under

a high organic load and experienced sloughing events which

caused partial colonization of the surface plates. Thickness

measurementsweredifficult to proceedonB3biofilmdue to the

strong surface heterogeneity (values ranging from 0.5 to 4mm).

B1 accumulatedmore biofilmmass (8.5 g VSSm�2) compared to

B2(4.2gVSSm�2),andinspiteofdetachmentevents,B3revealed

the highest accumulatedmass (16.6 g VSSm�2) (Table 1).

3.2. Biofilm microbial activities

Microbial activities were investigated in B1, B2 and B3 biofilms

after reaching steady state conditions. Fig. 2 reveals that

heterotrophic activity was found in all biofilms. However,

carbon removal efficiencies were higher for B2 and B3 biofilms

(respectively 93%and97%) compared toB1biofilmgrownunder

a higher COD/NH4eN ratio (84%). Nitrogen removal activities

where only be measured in the B2 and B3 biofilms grown

under low COD/NH4eN ratios. Indeed, B2 and B3 biofilms per-

formed simultaneous nitrification and denitrification activi-

ties, while B1 biofilm did not express any nitrogen removing

activity. However, nitrification efficiency was found to be

higher in B2 biofilm compared to B3 biofilm (85% versus 66%),

and denitrification efficiency on the other hand was two fold

higher in B3 biofilm compared to B2 biofilm (100% versus 50%).

According to these results, B1 biofilm, fed on a high carbon/

nitrogen ratio, was identified as a single heterotrophic biofilm

while both B2 and B3 biofilms, fed on low carbon/nitrogen

3 biofilms. Growth conditions, colonization aspect, biofilmed on B1, B2 and B3 biofilms.

B2 B3

4 4

2.5 25

Homogeneous

Dense

Heterogeneousa

Filamentous

Complete Partial

1.6 � 0.4 0.5e4a

4.2 16.6

Fig. 2 e Carbon removal (-), nitrification ( ) and

denitrification (,) efficiencies measured in B1, B2 and B3

biofilms, when reached steady state conditions.

0

100

200

300

400

500 US Tw EDTA TOTAL

0

50

100

150

Prot

eins

(mg.

gVSS

)

Suga

rs (m

g.gV

SS )

5

6

B 1 B 2 B 3

B 1 B 2 B 3

A

B

C

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 8 1533

ratios, were identified asmixed autotrophic and heterotrophic

biofilms performing simultaneous nitrification and denitrifi-

cation. The developed biofilms appeared as mature and thick

structures, potentially offering a large range of microbial

populations. Biochemical properties of these three diverging

biofilms were then investigated in terms of EPS contents and

size characteristics.

0

1

2

3

4

B 1 B 2 B 3

P/S

Fig. 3 e Protein content (A), Sugar content (B) and Protein to

Sugar (P/S) ratio (C) in soluble extracts obtained by the

multi-method extraction protocol. Soluble extracts were

harvested after each extractionmethod (ultrasonic, Tween,

or EDTA) and both proteins and sugars were assayed. Error

bars are evaluated from doubled extractions and duplicate

measurements.

3.3. EPS content in biofilms

A multi-method extraction protocol, previously described for

quantifying EPS from activated sludge (Ras et al., 2008a), was

applied on each B1, B2 and B3 biofilm. The extraction protocol

involved three different extraction methods (sonication,

Tween and EDTA) applied sequentially on the same sample in

order to collect a consistent fraction of EPS. Soluble extracts

were harvested by intermediate centrifugation steps and

quantified in terms of proteins and polysaccharides.

Fig. 3 shows that protein contents in all extracts were

systematically higher compared to polysaccharide contents,

and thus independently of the applied extraction method

(sonication, TweenorEDTA) aswell as thebiofilm (B1, B2orB3).

Extraction yields diverged between the applied methods, but

revealed similar trends between the biofilms. Indeed, both

protein andpolysaccharide contentswere alwayshigher in the

extracts obtained by EDTA and sonication steps, while Tween

steps always appeared as the least efficient extractionmethod.

Total EPS contents in each biofilm were defined by

summing the amounts of proteins and polysaccharides

obtained by each extraction method (sonication þ Twe-

en þ EDTA). Fig. 3A and B show that B1 biofilm had the lowest

amount of proteins (43 mg g�1 VSS) and polysaccharides

(15 mg g�1 VSS) whilst protein and polysaccharide contents

was four fold higher in B2 and 10 fold higher in B3 biofilms.

As shown in Fig. 3C, protein/polysaccharide ratios in the

various extracts varied between 1.8 and 5.4 but protein to

polysaccharide ratio of the total extracted EPS were similar

for both B1 and B2 biofilms (2.9 � 0.2) and slightly higher in

B3 biofilm (3.7 � 0.2).

In order to control potential cell lysis during the extraction

procedure, G6P-DH activity was systematically measured in

each soluble extract. The measured units obtained in each

extract were added in order to evaluate the total released G6P-

DH activity per biofilm. Table 2 shows that some G6P-DH

activity was detected in B2 and B3 but not in B1 biofilm

extracts. G6P-DHunits can be related to a number of disrupted

cells and hence to a mass of organic cell compounds. This

conversion is possible by using experimental correlation

factors established with a C. necator culture (described in the

Material and Methods). G6P-DH units measured in B2 extracts

Table 2 e Controls of cell lysis during the extractionperformed on B1, B2 and B3 biofilm and evaluation of therelated contamination level of the EPS extracts.

B1extract

B2extract

B3extract

G6P-DH activitya

(U g�1 VSS)

0 82 2547

Number of eq. lysed cellsb

(106 g�1 VSS)

0 412 12735

Released cellular

compoundsc (mg g�1 VSS)

0 2 52

Total extracted proteins and

sugars (mg g�1 VSS)

58 217 539

Level of extract contamination

by released cellular

compoundsd (%)

0 0.8 9.7

a Total G6P-DH activity as the sum of the G6P-DH units measured

in sonication, Tween and EDTA extracts.

b Evaluated by measurement of the G6P-DH activity released after

lysis of cupriavidus necator pure suspensions: 0.2 U per 106 equiva-

lent lysed cells.

c Evaluated using the correlation factor of dry biomass per number

of cells: 4.08$10�6 g per 106 cupriavidus necator cells.

d Released cellular compounds after extraction/total extracted

proteins and sugars. Qu

an

titative E

PS

d

istrib

utio

n

(%

p

eak area)

0%

20%

40%

60%

80%

100%

B1 B2 B3

-100

400

900

1400

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Elution volume (mL)

Ab

so

rb

an

ce (m

AU

).g

VS

S

B1B2B3

1000

10000

100000

1000000

13 16 17 20 22Elution volume (mL)

Mol

ecul

ar w

eigh

t (kD

a)

7 - 3 kDa

(5)

(4)

(3)

(2)

(1)

< 0.5 kDa(> 24 mL)

25 - 20kDa(17 – 18 mL)

(20-22 mL)

2 - 0.5 kDa(22 -24 mL)

(8 mL)> 5000 kDa

A

B

Fig. 4 e Global size distribution profiles at 280 nm of total

EPS extracted from each B1, B2 and B3 biofilm (A) by size

exclusion chromatography. Linear semi-logarithmic

relation between molecular weight of standard proteins

and elution volume (A, insert). Five different EPS size

clusters (1 to 5) were identified between 0.5 kDa and 5000

kDa and their relative distribution inside each biofilm was

evaluated by peak integration of the 280 nm signal (B).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 81534

are equivalent to 412$106 C necator disrupted cells, which is

liable to the release of 1.7 mg of cellular components per g of

biofilm VSS. Comparing this amount with the amount of

proteins and sugarsmeasured in the soluble extracts indicates

that the multi-method protocol did not induce significant

cell breakage in B2 biofilm since the level of contamination of

the extracted EPS by released cellular molecules was esti-

mated to 0.8%. However, by performing similar determination

for B3 biofilm extracts results indicate a higher level of intra-

cellular compounds that was estimated as 9.7% of the total

extracted sugars and proteins.

3.4. Global EPS size distribution in biofilms

A global EPS fingerprint investigation was undertaken by

pooling each EPS extract obtained from each extraction step of

the protocol (sonication, Tween and EDTA), and this for B1, B2

and B3 biofilms individually. Fig. 4 shows the size distribution

of the pooled fractions from each individual biofilm. Since

proteins were predominant in all extracts (Fig. 3), the absor-

bance signal was chosen at 280 nm. Moreover, results are

expressed in mAU g�1 VSS in order to standardize the signal

between each biofilm sample and to compare the relative

predominance of size fractions between each other, by direct

evaluation of their peak area. The column was previously

calibrated by injecting standard size proteins, which led to

a linear semi-logarithmic relation between molecular weight

of proteins and elution volume (Fig. 4A).

Chromatographicprofilesobtained fromthepooledextracts

highlight qualitative and quantitative differences between B1,

B2 and B3 biofilms. Nevertheless, three fractions occurred

systematically in all biofilmprofiles: (i) a highmolecularweight

fraction eluted inside the exclusion volume of the column

(8 mL) indicating size fractions above 5000 kDa, (ii) an inter-

mediate size fractionelutedbetween20and22mL, represented

by 3e7 kDa size molecules and (iii) a range of small size mole-

cules eluted beyond 24 mL, which corresponds to the total

inclusion volume of the column. These latter small fractions

are not in the optimal separation range offered by the column

but are expected to be under 0.5 kDa and are grouped in one

single category. Fig. 4A also shows that these three recurring

size fractionscomposealone theB1biofilmprofile.On theother

hand, additional peaks were identified in B2 and in B3 biofilm

profiles. Indeed, both B2 and B3 biofilm profiles revealed

a fraction eluted at 17e18 mL (i.e. 20e25 kDa), and B3 biofilm

alone revealed a fraction eluted at 22e24 mL (i.e. 0.5e2 kDa).

A total of five different size clusters were identified among

the three studied biofilms: cluster 1 (>5000 kDa), cluster 2

(20e25 kDa), cluster 3 (3e7 kDa), cluster 4 (0.5e2 kDa) and

cluster 5 (<0.5 kDa). The relative abundanceof EPS size clusters

between each other and between each biofilmwas undertaken

by peak integration of each chromatographic profile. Fig. 4B

compares size clusters between each biofilm, and highlights

the predominance of the three recurring EPS size clusters (1,

3 and 5). Cluster 3 (3e7 kDa) was the most represented and

with 86%, 60% and 46% occurrence of the total peak areas

eluted from B1, B2 and B3 chromatograms respectively. The

cluster 2 was specifically found in B2 and B3 biofilms, and

in the same proportions (3%). The cluster 4 appeared in B3

-20

180

380

580

780

980

1180

1380

elution volume (mL)

Abso

rban

ce (m

AU)

-10

0

10

20

30

40

50

60

70

80

90

elution volume (mL)

Abso

rban

ce (m

AU)

-20

0

20

40

60

80

100

120

140

160

180

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

elution volume (mL)

Abso

rban

ce (m

AU)

B1 B2 B3A

B

C

Fig. 5 e Specific size distribution profiles at 280 nm of EPS

extracted from each step of the multi-method protocol,

sonication (A), Tween (B) and EDTA (C) from B1 biofilm

( ), B2 biofilm ( ) and B3 biofilm ( ).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 8 1535

biofilmalone and represented 5%of the total peak areas. These

data indicate that EPS diversity was higher in the mixed

autotrophic/heterotrophic biofilms (B2 and B3) compared to

the simple heterotrophic biofilm.

3.5. EPS size distribution versus extraction methods

A more specific EPS fingerprint investigation was undertaken

on the three biofilms by identifying the previously described

size clusters in individual extracts provided by the extraction

protocol (sonication, Tween and EDTA extracts). Fig. 5 shows

size distribution of EPS obtained by each extraction method

individually.

Fig. 5C shows that EDTA-extract profiles were generally

poor in EPS size diversity and offered similar profiles between

the three biofilms. Indeed, this EDTA extraction step revealed

the three recurring EPS clusters alone (clusters 1, 3 and 5) with

a predominance of cluster 3, i.e. EPS belonging the 3e7 kDa

fraction eluted between 20 and 22 mL. Fig. 5Aand B show

that sonication and Tween extract size profiles, were more

diversified and diverged between biofilm samples. Indeed,

only the recurring clusters (1, 3 and 5) were found in B1 bio-

film, whilst all clusters (1e5) were found in B2 and B3 biofilms.

This result shows that cluster 2 was found only within the

heterotrophic/autotrophic B2 and B3 biofilms independently

on the extraction method. This latter result confirms the

global analysis performed previously. On the other hand,

cluster 4 which was identified in B3 biofilm alone in the global

analysis is finally identified by this specific analysis, in the

sonication and Tween extracts of B1 and B2 biofilms. Inter-

estingly, a new size cluster not yet identified in previous

profileswas only visualized in Tween extract profiles provided

from B1 and B2 biofilms. This latter fraction was eluted at

15.6 mL, indicating a specific size of 180 kDa (Fig. 5B).

4. Discussion

The aim of this study was to evaluate whether molecular

diversity of EPS are potential markers for biofilm macro-scale

characteristics. In order to validate such an approach,

molecular investigations were undertaken on three biofilms,

each being differentiated by their growth conditions, i.e.

different substrate loading rates or different nitrogen content

in the supply.

4.1. Relating feed to biofilm properties

The COD/NH4eN ratio was first chosen as a key parameter

to promote the development of carbon or nitrogen remov-

ing micro-organisms. This ratio was decreased from 73

(nitrogen limitation) for B1 biofilm, to 4 (excess nitrogen) for

B2 biofilm, by increasing the NH4þ content in the supply.

Neither nitrification, nor denitrification activity was

measured in this B1 biofilm indicating that the small

amount of NH4þ in the feed was consumed for heterotrophic

growth only. On the other hand, B2 biofilm which grew

under excess nitrogen conditions, showed simultaneous

autotrophic and heterotrophic activities. These observations

are in agreement with other findings (Matsumoto et al.,

2007) which showed that in spite of carbon deficiency

heterotrophic bacteria can out-compete other communities

such as autotrophic ammonium-oxidizing bacteria, and this

due to their higher growth rate (Elenter et al., 2007;

Morgenroth and Wilderer, 2000; Okabe et al., 1995). Whilst

the heterotrophic B1 biofilm exposed a filamentous structure

with a high accumulated mass, B2 biofilm grew into a dense

granular type biofilm with a lower accumulated mass. This

type of structure is also in agreement with theories valuable

for biofilm or granule formation involving slow-growing

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 81536

nitrifiers which seem to affect the mass density of biological

matrixes (Derlon et al., 2008; Elenter et al., 2007; Liu et al.,

2004).

Changing the organic loading rate by 10 fold between B2

and B3, without modifying the COD/NH4eN ratio, also

affected the structural and microbial properties of biofilms.

As expected, the high organic load applied for B3 biofilm

promoted bacterial growth which was confirmed by the

high accumulated biomass measurements. However, this B3

biofilm presented partial filaments with sloughing events

which caused heterogeneous colonization. It is probable

that this thick biomass developed by B3 biofilm might have

been more exposed to hydrodynamics, which could have

triggered localized detachment events (Ohashi et al., 1995),

compared to the thinner and homogeneous structure

described for B2 biofilm. In addition, such a thick structure

promoted anaerobic zones inside the B3 biofilm, which was

confirmed by a two fold higher denitrification activity

compared to B2 biofilm. Moreover, oxygen deficiency in B3

biofilm could have promoted bacterial mortality and nitri-

fiers, who often lose out when competing heterotrophic

bacteria for oxygen, might have been particularly affected.

This hypothesis is supported by the fact that nitrification

efficiency was lower in the thick B3 biofilm (65%) compared

to the thin B2 biofilm (85%) and by the detection of G6P-DH

activity in B3 biofilm prior to EPS extraction (results not

shown).

Results clearly show that controlled environmental

conditions can pilot microbial activities inside growing bio-

films, and alsomodify their macro-scale structural properties.

4.2. Influence of environmental conditions on EPSproduction

In order to harvest a representative pool of biofilm EPS,

a multi-method protocol based on both mechanical and

chemical extraction steps was applied on the three biofilms.

Quantitative analysis of extracted proteins and poly-

saccharides suggests that excess nitrogen in the feed (B2 and

B3 biofilms) triggered more EPS production than nitrogen

limitation (B1 biofilm). These results do not join those reported

by Miqueleto et al. (2010) who related decreasing values of

soluble and bound EPS to decreasing carbon/nitrogen ratios

in the feed of an anaerobic sequence batch biofilm reactor.

EPS were produced only when oxygen, even at very low

concentration was available, suggesting that microaerophilic

micro-organisms were the main secretors. Li et al. (2008)

showed that different thicknesses of membrane-aerated bio-

films in which counter-gradients of oxygen and substrate

existed, led to different EPS distributions. These authors

reported a maximum EPS content (120e140 mg EPS g�1 VSS

extracted by formaldehyde and NaOH) in the aerobic region

of the studied biofilm where carbon limitation occurred and

autotrophic ammonia oxidizing bacteria developed. This

latter content can be compared to the nitrifying B2 biofilm

where the extractible EPS reached 210 mg g�1 VSS.

The total amount of extracted EPS was 2.5 times higher in

B3 biofilm (high organic load) compared to B2 biofilm (low

organic load) and several theories can be quoted for this

increase in EPS content. Firstly, the higher substrate load

applied on B3 biofilm might have promoted bacterial growth

rates, forming a thick and less cohesive structure as confirmed

by sloughing events. This is in agreement with previous data

which report that heterotrophic fast growing bacteria develop

lower resistance towards either mechanical or chemical

disintegrationmethods (Denkhaus et al., 2007). Consequently,

extraction of EPS might be easier in such a fragile structure

leading to a higher content of proteins and polysaccharides

in extracts. Secondly, the extracted molecules were contam-

inated by soluble intracellular compounds but the level of

contamination, estimated around 10%, was not high enough

to justify the 2.5 fold increase of proteins and sugars observed

in B3 compared to B2 biofilm. As stated earlier in the discus-

sion, denitrification activity evidenced anoxic areas in the

B3 biofilm and Adav et al. (2009) recently located proteolitic

activities in anaerobic cores of bacterial granules that might

have been responsible for the occurrence of granule break-

down. Therefore, possible proteolytic activity in anaerobic

zones in B3 biofilm could partly explain the associated

unstable structure and the high content in released proteins.

Proteins were quantified in majority in all extracts with

a global protein/polysaccharide ratio of 2.9 � 0.2 for B1 and

B2 biofilms and of 3.7 � 0.2 for B3 biofilm. These data are in

agreement with those of Gao et al. (2008) showing protein/

polysaccharide ratios varying between 1.3 and 3.3 along

vertical profiles inside heterogeneous aerobic bio-filters.

According to Durmaz and Sanin (2001), the amount of

substrate converted to polymers by the cell depends on the

composition of the growth medium. Indeed, substrates with

low nitrogen content, as found in B1 biofilm may favor poly-

saccharide production, and on the other hand, substrateswith

excess nitrogen, as found in B2 and B3 biofilms, should

promote protein production. Therefore, while high protein

contents of B2 and B3 biofilms (164 mg g�1 VSS and

424 mg g�1 VSS) are in agreement with expectations, the

polysaccharide content of B1 biofilm somehow low compared

to B2 (15 mg g�1 VSS versus 54 mg g�1 VSS). This could be

explained by the low organic load applied to B1 which seems

to favor primarily cell growth and hence proteins (enzymatic

material) rather than carbon storage (polysaccharides).

4.3. EPS size fingerprinting of biofilms

In order to obtain a global molecular fingerprint of each bio-

film matrixes, EPS extracts obtained from the multi-method

extraction protocol were pooled for global analysis of the size

distribution of the extracted EPS.

Fractionation of these pooled extracts by SEC revealed

a total of five different EPS size clusters.

Three clusters were found in common between each

biofilm, of which cluster 1 (>5000 kDa) is excluded from the

column due to too high molecular weight EPS. Garnier et al.

(2005) have already shown the existence of associated

proteins/polysaccharides/mineral compounds in fractions

eluted near the size exclusion volume when characterizing

EPS extracted from activated sludge by SEC. Therefore, the

EPS size cluster 1 might probably be represented by poly-

mers eluted as a colloidal structure. Cluster 5 (<0.5 kDa) is,

on the other hand, eluted in the total inclusion volume of

the column, where the separation efficiency is reduced. This

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 8 1537

cluster 5 may either be effectively low molecular weight

organic molecules such as amino-acids and peptides, or

otherwise molecules which can interact with the column

and hence be partially retained during elution. Hydrophobic

retention of EPS on the sepharose column beads has already

been proven by Comte et al. (2007) and Garnier et al. (2005),

when eluting EPS extracted from activated sludges with 5%

methanol. However, performing similar experimental condi-

tions did not allow to evidence particular hydrophobic reten-

tion in this study (results not shown). Finally, the intermediate

3e7 kDa cluster 3 was predominant overall other clusters

and in all biofilms. The recurrence of this cluster 3 in the three

highly diversified biofilms suggests that the associated size

molecules are either associated to the heterotrophic activity

confirmed in all biofilms, or to mandatory EPS involved in

bacterial aggregate consolidation and/or adhesion.

The three recurrent EPS size clusters 1, 3 and 5, identified in

this present study, were the only components of the global

EPS fingerprint from the heterotrophic biofilm B1. By intro-

ducing nitrification and denitrification activities in B2 and B3

biofilms, another EPS size fraction appeared between 20 and

25 kDa (cluster 2). This latter fraction (cluster 2) could be

associated to the presence of bacteria involved in nitrogen

removal processes. This cluster was represented in similar

proportions within B2 and B3 biofilms (3% of the eluted EPS).

Since these latter biofilms exposed different nitrification and

denitrification levels, cluster 2 cannot be specifically related to

nitrification or denitrification microbial activities.

Concerning B3 biofilm, G6P-DH measurements indicated

natural cell lysis as well as cell breakage after EPS extraction.

About 9.7% of the proteins and sugars measured in the B3

extract may originate from the intracellular compartment.

However, due to their low proportion and to the fact that these

intracellular compounds may be natural constituents of the

biofilmmatrix, size fingerprint of B3 biofilm can be considered

as relevant. The global EPS size profile of B3 biofilm revealed

an additional size cluster between 0.5 and 2 kDa, named

cluster 4, that was not identified in the global EPS size profile

of B1 and B2 biofilms. Performing a more specific EPS size

fractionation focused on each soluble extract indicated that

cluster 4 was finally found in all three biofilms. Such a result

indicates that pooling extracts from one same biofilm sample

can hide under-represented size fractions and hence bias

final fingerprint profiles. Interestingly, the size cluster 2

(20e25 kDa) which was identified as specific to nitrogen

removal activities measured in B2 and B3 biofilms, was also

highlighted in sonication and Tween extracts of B2 and B3

biofilms whilst absent in either B1 biofilm extracts. These

results suggest that a 20e25 kDa EPS size fraction can effec-

tively be related to the presence and activity of the nitrogen

removing micro-organisms, evidenced within B2 and B3 bio-

films in spite of their diverging growth conditions and struc-

tural properties.

Still in a specific view of EPS diversity through extraction

methods, chromatographic profiles pointed out the strong

diversity of EPS size fractions in sonication and Tween

extracts in opposition to the EDTA extraction step. EDTA

extracts showed poor size diversity, although the EPS content

in these extracts were the highest compared to sonication and

Tween extracts. Therefore, EDTA extracts alone would not be

appropriate for a size diversity fingerprint study. On the other

hand, the mechanical sonication and hydrophobic Tween

methods are able to extract all size clusters (1e5) identified

previously. Interestingly, Tween extracts revealed an addi-

tional size fraction of 180 kDa in B1 and B2 biofilms only,

which was not visualized during the global study. Tween step

thus reveals the most diversified EPS size profiles although

EPS content in the extracts were the lowest compared to

sonication and EDTA extracts. These results suggest that

extraction method-specificity could be a relevant parameter

for fingerprint diagnosis.

The Tween-specific 180 kDa size fraction revealed in B1 and

B2 biofilms may be associated to the low organic load applied

to these two biofilms. In other words, the occurrence of

this size fraction may rather be related to a biochemical res-

ponse towards substrate-limiting conditions than to a specific

microbial function. B1 and B2 biofilmswere also characterized

by stable and homogeneous structures in opposition to B3

biofilm, therefore, the occurrence of this Tween-specific size

fraction might also mark the mechanical stability of both

biofilms in opposition to B3 where this 180 kDa size fraction

was absent. The specificity of this fraction towards Tween

treatments indicate that the associated EPS have hydrophobic

properties.

Hydrophobic properties of EPS might hence be implicated

in themechanical stability of biofilms. Such results could be of

interest for the understanding of attachment and detachment

processes. Authors expect that in the future, configuration of

appropriate coatings could be suitable to improve biofilm

adherence or on the other hand to prevent biofilm develop-

ment. Indeed, integrating these hydrophobic EPS fractions

inside or on top of coatings could promote molecular inter-

actions and hence biofilm strength. On the other hand, inte-

grating specific enzymes which are liable to digest these

hydrophobic EPS can also provide an alternative to toxic

biocides in order to prevent biofilm growth (e.g. for heat

exchangers, drinking water distribution systems). However,

further studies are required before hand, such as character-

izing hydrophobic EPS in different cohesive parts of biofilms.

These latter investigations are already under progress.

5. Conclusions

Characterization of EPS extracted from multi-species biofilms

was investigated using a multi-method extraction procedure

coupled with a SEC analysis. Results showed that EPS size

diversity was higher in the two mixed heterotrophic/auto-

trophic biofilms compared to the heterotrophic biofilm. The

multi-method extraction strategy provided consistent quan-

titative and qualitative EPS fractions. However, by focusing

on each extraction steps, results showed that each method

offered different quantities and different size diversity

profiles. Nevertheless, the occurrence of a 25e50 kDa size

fraction was systematically associated to biofilms exposing

nitrogen removing activities. Moreover, a 180 kDa size fraction

occurred in Tween extracts only and was associated to

mechanically stable biofilms.

This study has put forward the importance ofmethodology

in qualitative investigations of EPS in biofilms. Hydrophobic

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 2 9e1 5 3 81538

EPS seem to provide highly diversified size profiles with

a particular size category (180 kDa) which might be a print of

mechanical stability. Analysis of the hydrophobic EPS of

biofilms developed under different shear stress conditions is

currently under investigation.

r e f e r e n c e s

Adav, S.S., Lee, D.J., Lai, J.Y., 2009. Proteolytic activity in storedaerobic granular sludge and structural integrity. Bioresour.Technol. 100, 68e73.

Azeredo, J., Lazarova, V., Oliveira, R., 1999. Methods to extract theexopolymeric matrix from biofilms: a comparative study.Water Sci. Technol. 39, 243e250.

Boualam, M., Quiles, F., Mathieu, L., Block, J.-C., 2002. Monitoringthe effect of organic matter on biofilm growth in low nutritivewaters by ATR-FT-IR spectroscopy. Biofouling 18, 73e81.

Branda, S., Ashild, V., Friedman, L., Kolter, R., 2005. Biofilms: thematrix revisited. Trends Microbiol. 13, 20e26.

Chen, X., Stewart, P.S., 2002. Role of electrostatic interactions incohesion of bacterial biofilms. Appl. Microbiol. Biotechnol. 59,718e720.

Comte, S., Guibaud, G., Baudu, M., 2007. Effect of extractionmethod on EPS from activated sludge: an HPSEC investigation.J. Hazard. Mater. 140, 129e137.

Coufort, C., Derlon, N., Ochoa-Chaves, J., Line, A., Paul, E., 2007.Cohesion and detachment in biofilm systems for differentelectron acceptors anddonors.Water Sci. Technol. 55, 421e428.

Davies, D.G., Chakrabarty, A.M., Geesy, G.G., 1993.Exopolysaccharide production in biofilms: substratumactivation of alginate gene expression by Pseudomonasaeruginosa. Appl. Environ. Microbiol. 59, 1181e1186.

Denkhaus, E., Meisen, S., Telgheder, U., Wingender, J., 2007.Chemical and physical methods for characterisation ofbiofilms. Mikrochim. Acta 158, 1e27.

Derlon, N., Masse, A., Escudie, R., Bernet, N., Paul, E., 2008.Stratification in the cohesion of biofilms grown under variousenvironmental conditions. Water Res. 42, 2102e2110.

Dreywood, R., 1946. Qualitative test for carbohydrate material.Ind. Eng. Chem. 18, 499.

Durmaz, B., Sanin, F.D., 2001. Effect of carbon to nitrogen ratio onthe composition of microbial extracellular polymers inactivated sludges. Water Sci. Technol 44, 221e229.

Elenter, D., Milferstedt, K., Zhang,W., Hausner, M., Morgenroth, E.,2007. Influence of detachment on substrate removal andmicrobial ecology in a heterotrophic/autotrophic biofilm.Water Res. 41, 4657e4671.

Gao, B., Zhu, X., Xu, C., Yue, Q., Li, W., Wey, J., 2008. Influence ofextracellular polymeric substances on microbial activity andcell hydrophobicity in biofilms. J. Chem. Technol. Biotechnol.83, 227e232.

Garnier, C., Gorner, T., Lartigues, B.S., Abdelouhab, S., Donato, P.,2005. Characterization of activated sludge exopolymers fromvarious origins: a combined size-exclusion chromatographyand infrared microscopy study. Water Res. 39, 3044e3054.

Jahn, A., Nielsen, P.H., 1995. Extraction of extracellular polymericsubstances (EPS) from biofilms using cation exchange resin.Water Sci. Technol. 32, 157e164.

Jahn, A., Nielsen, P.H., 1998. Cell biomass and exopolymercomposition in sewer biofilms. Water Sci. Technol. 37,17e24.

Li, T., Bai, R., Liu, J., 2008. Distribution and composition ofextracellular substances in membrane-aerated biofilm.J. Biotechnol. 135, 52e57.

Liu, Y., Yang, S.F., Tay, J.H., 2004. Improved stability of aerobicgranules by selecting slow-growing nitrifying bacteria.J. Biotechnol. 108, 161e169.

Matsumoto, S., Terada, A., Tsuneda, S., 2007. Modeling ofmembrane-aerated biofilm: effects of COD/TKN ratio, biofilmthickness and surface loading of oxygen on feasibility ofsimultaneous nitrification and denitrification. Biochem. Eng. J.37, 98e107.

Mayer, C., Moritz, R., Kirschner, C., Borchard, W., Maibaum, R.,Wingender, J., Flemming, H.C., 1999. The role ofintermolecular interactions: studies on model systems forbacterial biofilms. Int. J. Biol. Macromol. 26, 3e16.

Miqueleto, A., Dolosic, C., Pozzi, E., Foresti, E., Zaiat, M., 2010.Influence of carbon sources and C/N ratio on EPS productionin anaerobic sequencing batch reactors for wastewatertreatment. Bioresour. Technol. 101, 1324e1330.

Morgenroth, E., Wilderer, P.A., 2000. Influence of detachmentmechanisms on competition in biofilms. Water Res. 34,416e426.

Ohashi, A., Mobarry, B., Manem, J.A., Stahl, D.A., Rittmann, B.E.,1995. Influence of substrate COD/TKN ratio on the structure ofmulti-species biofilms consisting of nitrifiers andheterotrophs. Water Sci. Technol. 32, 75e84.

Okabe, S., Hiratia, K., Ozawa, Y., Watanabe, Y., 1995. Spatialmicrobial distributions of nitrifiers and heterotrophs inmixed-population biofilms. Biotechnol. Bioeng. 50, 24e35.

Ramsay, B.A., Lomaliza, K., Chavarie, C., Dube, B., Bataille, P.,Ramsay, J.A., 1990. Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids. Appl. Environ. Microbiol. 56,2093e2098.

Ras, M., Girbal-Neuhauser, E., Paul, E., Lefebvre, D., 2008a. A highyield multi-method extraction protocol for proteinquantification in activated sludge. Bioresour. Technol. 99,7465e7471.

Ras, M., Girbal-Neuhauser, E., Paul, E., Sperandio, M., Lefebvre, D.,2008b. Protein extraction from activated sludge: an analyticalapproach. Water Res. 42, 1867e1878.

Simon, S., Paıro, B., Villain, M., D’Abzac, P., Van Hullebusch, E.,Lens, P., Guibaud, G., 2009. Evaluation of size exclusionchromatography (SEC) for the characterization of extracellularpolymeric substances (EPS) in anaerobic granular sludges.Bioresour. Technol. 100, 6258e6268.

Smith, P.K., Krohn,R.I.,Hermanson,G.T.,Mallia,A.K.,Gartner, F.H.,Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J.,Klenk, D.C., 1985. Measurement of protein using bicinchoninicacid. Anal. Biochem. 150, 76e85.

Standard Methods for the Examination of Water andWastewater,ninetienth ed., 1995 APHA, AWWA, WPCF, Washington DC,USA.

Wijeyekoon, S., Mino, T., Satoh, H., Matsuo, T., 2004. Effects ofsubstrate loading rate on biofilm structure. Water Res. 38,2479e2488.

Zhang, X.Q., Bishop, P.L., KinKle, B.K., 1999. Comparison ofextraction methods for quantifying extracellular polymers inbiofilms. Water Sci. Technol. 39, 211e216.