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Predation influences the structure of biofilm developedon ultrafiltration membranes

Nicolas Derlon a,*, Maryna Peter-Varbanets a, Andreas Scheidegger a, Wouter Pronk a,Eberhard Morgenroth a,b

aEawag, Swiss Federal Institute of Aquatic Science and Technology, Uberlandstrasse 133, P.O Box 611, CH-8600 Dubendorf, Switzerlandb Institute of Environmental Engineering, ETH Zurich, 8093 Zurich, Switzerland

a r t i c l e i n f o

Article history:

Received 15 July 2011

Received in revised form

14 March 2012

Accepted 15 March 2012

Available online 2 April 2012

Keywords:

Ultrafiltration

Predation

Biofilm structure

Permeate flux

Optical Coherence

Tomography (OCT)

Confocal Laser Scanning

Microscopy (CLSM)

Gravity-driven membrane

(GDM) filtration

* Corresponding author. Tel.: þ41 44 823 537E-mail address: nicolas.derlon@eawag.ch

0043-1354/$ e see front matter ª 2012 Elsevdoi:10.1016/j.watres.2012.03.031

a b s t r a c t

This study investigates the impact of predation by eukaryotes on the development of

specific biofilm structures in gravity-driven dead-end ultrafiltration systems. Filtration

systems were operated under ultra-low pressure conditions (65 mbar) without the control

of biofilm formation. Three different levels of predation were evaluated: (1) inhibition of

eukaryotic organisms, (2) addition of cultured protozoa (Tetrahymena pyriformis), and (3) no

modification of microbial community as a control. The system performance was evaluated

based on permeate flux and structures of the biofilm. It was found that predation had

a significant influence on both the total amount and also the structure of the biofilm. An

open and heterogeneous structure developed in systems with predation whereas a flat,

compact, and thick structure that homogeneously covered the membrane surface devel-

oped in absence of predation. Permeate flux was correlated with the structure of the bio-

film with increased fluxes for smaller membrane coverage. Permeate fluxes in the presence

or absence of the predators was 10 and 5 L m�2 h�1, respectively. It was concluded that

eukaryotic predation is a key factor influencing the performance of gravity-driven ultra-

filtration systems.

ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction osmosis, intensive pre-treatment is used. Practical experi-

Biofouling reduces the water flux in membrane filtration

systems and significant efforts are directed towards prevent-

ing or reducing biofilm formation on the surface of

membrane. For example, in membrane bioreactors for

wastewater treatment, biofilm formation is controlled by

coarse bubble aeration resulting in increased shear stress at

the membrane surface and by chemical cleaning (Le-Clech

et al., 2006). In drinking water systems, membrane modules

are regularly back flushed and cleaned with hypochlorite

multiple times per week. In nanofiltration and reverse

8.(N. Derlon).ier Ltd. All rights reserved

ence, however, demonstrates that biofilm formation cannot

be prevented. Removal of biofilms requires significant

amounts of energy and chemicals, which significantly

increases costs of operation of membrane systems. Thus,

a different approach for the operation of membrane systems

has to be investigated, avoiding extensive cleaning and

flushing operations.

A different strategy was proposed by Peter-Varbanets et al.

(2010) where biofilm formation on the membrane is tolerated

and the focus was on maximizing the permeability of the

biofilm. In their study ultrafiltration (UF) systems were

.

Fig. 1 e Scheme of one experimental line including: the

water bath with temperature controlled to 20 �C, thestorage tank, the filtration modules containing the

polyethersulfone membrane (100 kDa) and the bottles for

permeate collection.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 33324

operated without any cross-flow and without membrane

cleaning. Under these conditions a highly permeable biofilm is

formed that allows for long-term (months) operation of the

system without cleaning or backwashing. The formation of

this heterogeneous biofilm resulted from biological processes

and was not observed when biological activity was reduced

(by cold temperature) or inhibited (by addition of sodium

azide) (Peter-Varbanets et al., 2010). However, the nature of

these processes (e.g., bacterial growth, predation) as well as

their impacts on the system performances were not clearly

identified. The current study evaluates the influence of

predation on structure and permeability of biofilms formed

during dead-end ultrafiltration.

A range of physical and biological factors influences bio-

film structure. Local biofilm growth is limited by substrate

availability and biofilm growth is balanced by external forces

resulting in biofilm detachment (van Loosdrecht et al., 1995).

In systems where the availability of substrate is mass trans-

port limited bacteria that are closer to the bulk phase have

a competitive advantage resulting in low-density heteroge-

neous structures (Picioreanu et al., 1998). High detachment

forces, however, can balance this phenomenon. As a result,

heterogeneous structures develop under low shear conditions

and/or high surface loading rate (Picioreanu et al., 2001). These

conceptual and numerical modelling studies assume that the

development of biofilm structure can solely be explained by

local availability of substrate and physical forces. This

approach explains in many cases the biofilm structures that

are experimentally observed in industrial systems such as

wastewater treatment plants. In the case of biofilms grown at

a very low substrate concentrations (e.g., stream biofilms)

factors such as quorum sensing or eukaryotic predation have

been shown to be dominant factors determining biofilm

structure (Bohme et al., 2009).

Recent studies performed in the field of stream biofilms

indeed reported that predation by eukaryotes significantly

impacts the morphology of biofilms (Bohme et al., 2009;

Weitere et al., 2005). Predators can indeed reduce the biomass

concentration and alter its morphology due to grazing

(Weitere et al., 2005), motility (Jackson, 1990), or sloughing

induced by raptorial feeders (Fenchel and Finlay, 1986). The

enhanced formation of micro-colonies and the resulting

channel network yields a rougher biofilm surface compared

with ungrazed biofilm (Bohme et al., 2009). If predation

significantly influence biofilm structure and overall amount of

biofilm then predation may be relevant factor influencing the

performance of membrane filtration systems e especially in

systems operated without cross-flow that provide very suit-

able conditions for the larger eukaryotic organisms to develop.

Theobjectivesof this studywere (1) to identify the influence

of predation on the overall amount and the structure of biofilm

during ultra-low pressure UF and (2) to correlate the biofilm

formation with membrane performance (quantified as water

flux or specific resistance). We hypothesized that eukaryotic

predation increases biofilm heterogeneity and that increased

biofilmheterogeneity in turn results in an increasedwaterflux.

To evaluate this hypothesis, three gravity-driven UF systems

were operated in parallel with (1) inhibition of eukaryotic

organisms, (2) addition of cultured protozoa (Tetrahymena pyr-

iformis), and (3) no modification as a control. Long-term (up to

three months) dead-end filtration experiments were per-

formedmonitoring total amount and spatial distribution of the

biofilm in combination with the permeate flux.

2. Materials and methods

2.1. Experimental setup and operating conditions

2.1.1. Experimental setupThree experimental lines each composed of five parallel

membrane modules were operated as shown on Fig. 1. Feed

water was pumped to a completely mixed tank that is

controlled to 20 �C and connected to a storage tank. The

storage tank was placed at a height corresponding to a pres-

sure of 65 mbar applied at the surface of the membrane. The

level of water in the water tank was kept constant using an

overflow. The tank was connected to the membrane modules

using silicon tubes (Saint-Gobain, Bompass, France). Permeate

of every module was collected in a separate plastic bottle and

quantified gravimetrically in daily intervals.

Water was withdrawn continuously from a creek (Chries-

bach, Dubendorf, Switzerland) and used as feed water. Total

organic carbon (TOC) and dissolved organic carbon (DOC) of

the feed water were measured twice per week as described in

Section 2.5.

2.1.2. MembranePolyethersulfone membrane (PBHK, Biomax Millipore, Bill-

erica, MA, USA) with a nominal cutoff of 100 kDa was used in

this study (mean pore size of approximately 10 nm). To

remove conservation agents the newmembranes were stored

for 24 h in deionized water. The deionized water was renewed

several times during this period. Membraneswere then placed

in filtrationmodules that consisted of standard polycarbonate

filter holders of 48 mm inner diameter (Whatman, Maidstone,

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 3 3325

Kent, UK). The filtration modules were operated continuously

without flushing or cleaning.

2.1.3. Predation levels and operating conditionsThree different predation levels were applied in three exper-

imental lines operated in parallel. One line was operated with

inhibition of eukaryotes (Low Predation, Low-P). Eukaryote

inhibition was achieved by continuous addition of cyclohexi-

mide, an antibiotic specific to eukaryotic organisms. A second

line was operated without control of predators (Natural

Predation, Nat.-P). A third line was inoculated with an exog-

enous source of protozoa (T. pyriformis) (High Predation, High-

P). The details of Cycloheximide and T. pyriformis addition are

presented in parts 2.2 and 2.3, respectively.

Two experimental runs were performed to identify the

impact of predation on the biofilm structure and in turn on the

system performances. Experiment 1 was operated for a period

of four weeks where inhibition of eukaryotes and addition of

T. pyriformis was done at the beginning of the experimental

run and the water temperature was controlled at 20 �Cthroughout the entire experiment. Experiment 2 was per-

formed in two phases where during the first two months all

systems were first conditioned without inhibition of eukary-

otes and addition of T. pyriformis and the systems were oper-

ated with creek water without temperature control

(approximately 7 �C). After this conditioning phase the

systems were exposed to the different treatments and the

water temperature was controlled to 20 �C. For experiment 2

the end of the conditioning phase is defined as the start of the

experiment.

2.2. Inhibition of predation

Cycloheximide solution at a concentration of 3.5 g L�1 was

injected in the five Low-P modules using a syringe-pump. The

flow rate (approximately 30mL d�1) was adjusted based on the

observed filtration flux to reach a stable concentration of

100 mg L�1 in the permeate.

2.3. Enhancement of the predation

The ciliate T. pyriformis (1630/1W, Culture Collection of Algae

and Protozoa, Dunbeg, UK) was used in the High-P case. T.

pyriformis was grown anexically in PPY (Proteose Peptone

Yeast extract) medium. PPY medium is composed of proteose

peptone (20.0 g L�1) and yeast extract (2.5 g L�1) added to 1 L of

Evian water (Evian-les-Bains, France). The medium was

autoclaved at 15 psi for 15min. 50mL of twoweeks old culture

werewas centrifuged at 4500g for a period of 10min to remove

the supernatant and then resuspended in Evian water. This

step was once repeated before injecting the culture directly

into the silicon tubes connecting the water tank with the

filtration modules. Viability of the protozoa after centrifuga-

tion and before inoculation was controlled by direct micro-

scopic observations.

2.4. Eukaryote abundance

Fluorescent In Situ Hybridization (FISH) was used to qualita-

tively evaluate the abundance of eukaryote organisms. A

eukaryote specific FISH probewas used: EUK1209 (50- GGG CAT

CAC AGA CCT G -30) (Amann et al., 1990). First, entire

membranes were sampled and fixed with formaldehyde

solution (2.5%), and then cut into subsections of around

0.25 cm2. Subsections were then placed in microtubes filled

with a hybridization buffer (750 mM NaCl, 100 mM TriseHCl

[pH 7.8], 5 mM EDTA, 0.1% sodium dodecyl sulphate). FISH

probes were then added to reach a final concentration of

250 ng mL�1. Hybridization was performed for a period of 4 h

at 45 �C. The membrane subsections were then placed in

microtubes containing Evian water filtered at 0.45 mm to stop

the hybridization process. Organisms were observed with

a Confocal Laser Scanning Microscope (CLSM) (Leica SP5,

Wetzlar, Germany) using 20� or 63� glycerol immersion Leica

objectives. Numerical Apertures (NA) were 0.7 and 1.3 for the

20� and 63� lenses, respectively.

Direct microscopic observations of the eukaryote abun-

dance were in addition performed in parallel of the FISH

analysis. For this purpose, the biofilms developed on the

membrane subsections were scratched with the help of

a scalpel and resuspended in filtered Evian water. A stereo-

microscope (Leica M205, Wetzlar, Germany) was used.

2.5. Chemical analysis

The accumulated mass of the biofilm (g C m�2) was measured

through the measurement of the Total Organic Carbon (TOC).

Membranes were sampled and the entire biofilms detached by

flushing 100 mL of nanopure water with the help of a sterile

syringe. Since biofilms were flushed with significant amounts

of nanopure water, the Dissolved Organic Carbon was insig-

nificant and thus the TOCwas equal to the particulate organic

matter. The TOC concentration of the samples was measured

using an automatic total organic carbon analyser (TOC-V,

Shimadzu, Japan). Before determination, the unfiltered

samples were homogenized with a mixer (Polytron PT 3100,

Kinematica, Bohemia (NY), USA) (2 min with 15,000 rpm),

a magnetic stirrer was added, and the sample was then closed

using parafilm. Homogenization during injection ensured

measurement is performed on a representative sample

(avoiding sedimentation during injection into the TOC analy-

ser). The analyser was calibrated with a stock solution

composed of sodium nitrate (6.068 g L�1), potassium hydro-

phtalate (2.126 g L�1), and orthophosphoric acid (85%,

2 mL L�1) dissolved in carbon-free water.

2.6. Biofilm characterization

2.6.1. Micro-scale characterization (CLSM)The micro-scale structure of the biofilm (morphology, thick-

ness) was characterized by CLSM. First, membranes were

sampled and fixed with formaldehyde solution (2.5%), washed

twice with filtered Evian water and cut in sections of around

0.25 cm2. Then, biofilm sampleswere stained, incubated in the

dark (4 h, 20 �C) andwashed again. SYBR�Gold nucleic acid gel

stain (1000 fold diluted stock solution, Invitrogen, Basel,

Switzerland) was used to detect all microorganisms. Conca-

navalin A (50 fold diluted stock solution, Invitrogen, Basel,

Switzerland) was used to stain the a-D -mannose and a-D-

glucose groups of biopolymers. These stains were applied on

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 33326

the samemembrane subsections. Dehydration of the samples

was then performed and consisted of six immersion steps of

20 min each in glycerol/water solutions with an increasing

gradient (40, 60, 80, 90, 95 and 100%). The fluorescence of

SYBR� Gold was detected by excitation at 488 nm and emis-

sion at 495e540 nm. Concanavalin A was detected with an

excitation at 514 nm and emission at 550e620 nm. The

reflection from surfaces impermeable for light was detected at

the wavelength of 633 nm using CLSM reflective mode. Ten

side-view images and five z-stacks were recorded for each

environment at weekly intervals. The position of the objec-

tives was randomly changed in the x and y directions when

recording side-view images and z-stacks to avoid subjective

impact of the user and thus to record representative data.

Biofilmmorphologies were evaluated throughout the analysis

of these images. No quantification of CLSM images (e.g. bio-

volume, biofilm thickness) was performed.

2.6.2. Optical Coherence Tomography (OCT)Optical Coherence Tomography (OCT) (model 930 nm Spectral

Domain, Thorlabs GmbH, Dachau, Germany) with a central

light source wavelength of 930 nmwas used to investigate the

meso-scale structure of the biofilm. The use of long wave-

length light allows to penetrate up to a depth of 2.7 mm with

axial and lateral resolutions of 4.4 mmand 15 mm, respectively.

For the image acquisition filtration modules were opened and

carefully placed on the OCT stage. OCT images were recorded

keeping the samples immersed in a thin layer of permeate.

Around 20 A-Scans (i.e., XZ plane pictures) of either 5 � 1 mm,

5� 0.5mm, or 3� 0.5mm (depending of the biofilm thickness)

were acquired at different time intervals and for each filtra-

tion modules. Image analysis software developed under

Matlab� (MathWorks, Natick, US) was used to analyse OCT

image. Image analysis consisted of the following steps:

(1) detecting the membraneebiofilm interface (grey-scale

gradient analysis);

(2) binarizing the image (automatic thresholding);

(3) calculating physical properties of the biofilm:mean biofilm

thickness (z in mm), absolute (Ra in mm) and relative

roughness ðR0aÞ coefficients.

These parameters were calculated according the following

equations:

z ¼ 1

n

XNi¼1

zi (1)

Ra ¼ 1n

XNi¼1

ðjzi � zjÞ (2)

R0a ¼

1n

XNi¼1

�jzi � zjz

�(3)

where N is the number of thickness measurements, zi is the

local biofilm thickness (mm), and z is the mean biofilm thick-

ness (mm).

Linear regression analysis was performed to statistically

evaluate the effect of the level of predation on the biofilm

physical structure in terms of mean biofilm thickness and

relative roughness coefficient. The linear least square func-

tion of R (R Development Core Team, 2011) version 2.13.0 was

used to fit the model. This approach consisted in comparing

the slope of the change in the mean biofilm thickness or

relative roughness to the slope of natural predation (Nat.-P).

First-order equations with qualitative variables were used for

this analysis Eq. (4):

Yt ¼ b0 þ b1$tþ b2$IðLow� PÞ$tþ b3$IðHigh� PÞ$tþ εt (4)

Where Yt is the quantitative variable that is considered (mean

biofilm thickness or relative roughness coefficient), b1 is the

slope of the Nat.-P case, b2 is the difference in slope of the

Nat.-P and Low-P and b3 the difference in slope betweenNat.-P

and High-P. I(Low-P) and I(High-P) are the indicator variables

(equal to 0 or 1 depending of the data set that is considered). T-

test and P-value calculations were then performed to statis-

tically distinguish the different slopes that were calculated.

2.6.3. Top view biofilm picturesTop views of the biofilms developed on the membranes were

recorded using an Olympus C-7070 digital camera (Olympus,

Le Mont-sur-Lausanne, Switzerland). The meso-scale char-

acterization of biofilms consisted inmeasuring themembrane

coverage as the fraction of the membrane surface that is

covered by biofilms. Top view pictures were treated using

ImageJ (http://rsb.info.nih.gov/ij/). First, the images were

converted into 8-bits pictures. A threshold was thenmanually

adjusted and applied to obtain binary images. The value of

threshold was determined in order to distinguish the relevant

structure, i.e., the biofilm and the uncovered membrane. The

effective surface of membrane was finally selected to calcu-

late the membrane coverage.

2.7. Flux and hydraulic resistance

2.7.1. Filtration fluxThe permeate flux was calculated by measuring the mass of

water collected in each bottle and dividing the results by the

filtration period and by the membrane area. The mass of

permeate was weighed daily using a scale (Ohaus Adventure

Pro�, Pine Brook (NJ), USA).

2.7.2. Hydraulic resistancesThe total resistance of the fouled membrane was calculated

according to the Darcy’s law:

J ¼ DPm$Rtotal

(5)

Where DP (Pa) is the transmembrane pressure (65 mbars in

this study), m is the viscosity (Pa s) of the filterate, Rtotal is the

total resistance (m�1), and J is the filtration flux (m s�1).

The total resistance Rtotal can be represented as the sum of

individual resistances:

Rtotal ¼ Rmembrane þ Rbiofilm þ Rirreversible (6)

Where Rmembrane is resistance of the pristine membrane,

Rbiofilm is resistance of the biofilm, and Rirreversible is resistance

caused by the irreversible fouling. Two membranes per line

were sampled to estimate Rbiofilm. First, the biofilm was

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 3 3327

detached from themembrane surface by flushing with 100mL

of deionized water using a syringe. Membranes were then

returned to filtration modules and the flux of the flushed

membrane was determined by filtering nanopure water. It is

assumed that the after flushing the resistance is the sum of

Rirreversible and Rmembrane. Based on the resistances measured

of the fouledmembrane, the resistance after flushing, and the

resistance of the virgin membrane the different resistances in

Eq. (6) can be calculated.

The biofilm resistance (Rbiofilm) can be correlated to the

thickness of a homogeneous biofilm layer (H ) following the

CarmaneKozeny equation:

Rbiofilm ¼ 75$ð1� εÞ22$ε3$a2

$H (7)

Where ε (dimensionless) is the biofilm porosity, a (m) is the

characteristic radius of the particles forming the cake layer,

and H (m) corresponds to the thickness of the biofilm

(Katsoufidou et al., 2005). During dead-end filtration all

retained material is deposited on the membrane, causing an

increase of biofilm thickness. According to Eq. (3), the resis-

tance of the biofilm is defined by its thickness of the layer and

structural parameters.

3. Results

The abundance of eukaryotic organisms was evaluated after

staining with FISH probes and imaging with CLSM (Fig. 2) after

one week of operation under the different predation levels, in

experiment 1.

Clear differences in terms of eukaryote abundance were

observed between the different treatments (Low-P, Nat.-P, and

High-P) after one week of growth. In the Low-P case eukary-

oteswere either absent or very rarely observed (Fig. 2a). On the

contrary, many eukaryotes were observed in the Nat.-P

(Fig. 2b) and High-P cases (Fig. 2c). Based on staining with

FISH probes and qualitative comparison of images the

eukaryote organism abundancewas greater in the High-P case

that had been inoculated with T. pyriformis, compared to the

Nat.-P case. These qualitative observations suggest that both

predation inhibition in the Low-P case and the protozoa

inoculation in the High-P resulted in significantly different

Fig. 2 e Eukaryote abundance based on CLSM pictures of the biofi

Nat.-P and (c) High-P conditions (experiment 1). A red signal (Re

organisms. Grey colour is related to the reflected signal of the so

this figure legend, the reader is referred to the web version of t

conditions compared to the control (Nat.-P). These results

were in addition confirmed by directmicroscopic observations

performed in experiment 1 and 2 after several weeks of

operation that revealed the presence of numerous eukaryotes

in both the Nat.- and High-P cases.

3.1. Impact of predation on the development of thebiofilm

In Fig. 3 top view pictures (Fig. 3 aec, gei) and CLSM pictures

(Fig. 3 def, jel) of the biofilms after two weeks and one month

under different predation conditions (experiment 1) are pre-

sented. Comparing the structure of the biofilms after two

weeks of operation (Fig. 3, columns 1 and 2) only a small

influence of the level of predation can be observed. For the

cases with predation (Nat.-P and High-P) the biofilms had

a thickness of around 25 mm while the biofilm for the case

without predation (Low-P) was slightly thicker (40e50 mm). For

all cases the biofilms covered the entire membrane surface

but the biofilm structure was more heterogeneous with an

increasing predation level. In absence of predation the biofilm

surface was rather smooth whereas heterogeneities, i.e.,

peaks and valleys were observed in presence of predation.

After two weeks of operation, biofilms developed in the Nat.-

and High-P cases are not homogeneous but characterized by

a varying thickness and showing mounds of biofilm, espe-

cially in the case of High-P (Fig. 3 bec). No clear distinction

was, however, visible between the Nat.-P and High-P levels at

a large scale (e.g. membrane coverage). After 4 weeks of

operation these differences in biofilm structure become even

clearer. In the Low-P case a compact biofilm thicker than

50 mm covered homogeneously the entire surface of the

membrane based on the CLSM observations (Fig. 3 gej). The

membrane coverage did not change between day 15 and day

30 and the membrane remained almost fully covered. Low

surface heterogeneity was observed for this biofilm, due to

loosely attached particulate matter, which probably accu-

mulated on top of the biofilm during filtration. Inorganic

particulatematterwas identified by the reflection signal of the

CLSM. Thus, areas with high particle density can be identified

by the shadowing effect occurring underneath of these areas.

In the Nat.-P and High-P cases, heterogeneous and open

structures were observed. Between days 14 and 28 the

lm developed after one week of growth under (a) Low-P, (b)

d-EUK1209 FISH probe) indicates the presence of eukaryote

lid surface. (For interpretation of the references to colour in

his article.)

Fig. 3 e Top view and CLSM images of an optical cross-section of biofilms developed after two weeks and one month of

growth (experiment 1) under different levels of predation: Low-P (first row), Nat.-P (second row), and High-P (third row).

Images are presented for the biofilm developed after two weeks (columns 1 and 2) and after four weeks (columns 3 and 4).

The following colours are used in the CLSM images: Green[ SYBR� Gold (all bacterial cells), red[ Concanavalin A (presence

of a-D-mannose and a-D-glucose groups of biopolymers), purple [ reflected signal (the membrane). The white arrows in def

and jel indicate the position of the membrane surface. CLSM pictures def were record using the 633 objective (NA: 1.3). The

red square shown in figures gei corresponds to a view field of 750 mm 3 750 mm indicating the location imaged using CLSM

with the 203 objective (NA: 0.7). (For interpretation of the references to colour in this figure legend, the reader is referred to

the web version of this article.)

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 33328

membrane coverage decreased significantly in both cases

(Fig. 3 b, c, h, i). After 30 days the biofilm was composed of

a thin basal layer with large and locally thick biomass

mounds, as can be observed both visually and by CLSM

images. The thickness of the basal layer ranged from several

microns to around 25 mm (Fig. 3 k and l). CLSM images also

revealed the existence of cavities in some structures devel-

oped in presence of predators (images not shown). This open

and heterogeneous structure was slightly more pronounced

in the case of a High-P than in the Nat.-P case. Due to the

thick biofilms observed after one month of growth, different

objective lenses were used to capture the entire structures in

the z-direction. 63� and 20� objectives were used after two

weeks and after one month, respectively. The lower NA of the

20� lens is associated with a lower resolution and in turns

a lower image quality. This explains the differences between

CLSM pictures captured after two weeks and after one

month.

The change in the meso-scale biofilm structure was

monitored using OCT (Fig. 4). OCT images confirmed obser-

vations with CLSM on micro-scale. As shown in Fig. 4, OCT

results confirmed that the surface heterogeneity of biofilms

increased with an increasing level of predation. Image anal-

ysis was applied to quantify OCT images and monitor the

change in the biofilm structure (Fig. 5). During the first month

of biofilm development, similar mean biofilm thicknesses

ranging from 50 to 80 mm were monitored regardless of the

level of predation. Then biofilms cultivated in presence of

predation became thicker than biofilms developed in absence

of predation: mean biofilm thicknesses measured for each

filtrationmoduleswas ranging from150 to 200 mmand from80

to 120 mm in presence and absence of predation, respectively.

Predation had even a greater impact on the relative biofilm

roughness (Fig. 5). In absence of predation (Low-P) the relative

roughness coefficient remained constant over three months

at 0.25. In presence of predation (Nat.-P and High-P cases), this

coefficient varied between 0.5 and 0.75 indicating that some

parts of the biofilms were locally very thin or very thick. In

addition, it is important to notice that the variability in flux

and structure between these parallel modules was

pronounced in the case of predation (Nat.- and High-P). This is

likely due to random natural inoculation of protists. In

absence of predation the variability between modules was

insignificant.

Fig. 4 e Typical OCT images of biofilms developed after one month under (A) Low-P, (B) Nat.-P and (C) High-P conditions.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 3 3329

Additional physical properties of the different biofilm

structures (morphology, accumulated mass, average

membrane coverage) are summarized in Table 1. The main

differences were observed between the structures developed

in absence of predation (Low-P) compared to structures

developed in presence of predation (Nat.-P and High-P). In

addition to themorphological differences described in section

3.2, the mass of the biofilm accumulated at the surface of the

membranes (g C m�2) was also influenced by predation. TOC

concentration measured in the High-P case was 30% lower

than the onemeasured in the Nat.-P case. No clear distinction

was done between the concentration measured in the Low-

and Nat.-P cases. In addition, the averagemembrane coverage

decreases with an increasing predation level. In presence of

predators the average membrane coverage was decreased by

10%. An accurate determination of the membrane coverage

was possible thanks to the good contrast between the biofilm

and the uncovered membrane.

Statistical analysis was performed to evaluate the influ-

ence of the predation level on the change in the mean biofilm

thickness and in the relative roughness coefficient (Table 2).

0

100

200

300

400

0 50 100 150

Time (d)

Mea

n bi

ofilm

thic

knes

s ( µ

m)

Low-P

Nat.-P

High-P

Fig. 5 e Change in the mean biofilm thickness and in the relativ

three grazing conditions (experiment 2). Physical properties we

Slopes of the change in the mean biofilm thickness and in the

relative roughness coefficient were different for the three

different levels of predation. P-values calculated for mean

biofilm thicknesses and relative roughness coefficients,

however, indicated that the slope for High-P is statistically not

different from the slope of Nat.-P. The biofilms with predation

(Nat.-P and High-P) were statistically different from the bio-

films developed without predation (Low-P case) indicating

that the biofilm structure formation is mainly determined by

the presence or absence of predators in the system.

3.2. Impact of predation on permeate flux and onhydraulic resistances of the biofilm

The average flux measured during experiment 1 and 2 are

shown in Fig. 6. According to the data shown in Fig. 6, a rela-

tion exists between the predation level and the average flux.

Fluxmonitored in the systemswith predators was around two

times higher than flux measured in absence of predation.

Indeed, a stable flux of 5 L m�2 h�1 was measured in both

experiment 1 and 2 for the Low-P condition. In the Nat.-P case,

0

0,25

0,5

0,75

1

0 50 100 150

Rel

ativ

ero

ughn

ess

coef

fici

ent(

%)

Time (d)

Low-P

Nat.-P

High-P

e roughness coefficient of the biofilms developed under the

re calculated by image analysis from OCT images.

Table 1 e Physical properties of the different types of biofilm structure developed in experiment 2.

Case Low-P Nat.-P High-P

Treatment Cycloheximide No treatment One-time inoculation

with T. Pyriformis

Biofilm morphology Flat structure without

significant heterogeneities

Open and heterogeneous

with biomass mounds

Open and heterogeneous

with large biomass mounds

Accumulated mass

of biofilm after

one month (g C m�2)

3.9 � 0.6 4.5 � 0.1 2.9 � 0.8

Average membrane

coverage (%)

97 � 1 89 � 7 87 � 1

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 33330

a constant but higher flux of 9 L m�2 h�1 was measured. The

average flux monitored in the High-P case evolved differently:

after twoweeks, this flux was lower than the fluxmeasured in

the Nat.-P case (15% and 27% lower than Nat.-P case in

experiment 1 and 2, respectively). Then, this flux gradually

increased and at the end of the experiment the flux for the

High-P casewas 17% and 20%higher than the flux of theNat.-P

in experiments 1 and 2, respectively.

These different fluxes measured correspond to different

hydraulic resistances of the biofilm. For example in experi-

ment 2 and after onemonth of development, the resistance of

the biofilm (Rbiofilm) in the case of a Low-P was 4.1 � 1012 m�1,

which corresponds to 95% of Rtotal. The resistances of the

biofilm in the Nat.-P and High-P cases were 2.1 � 1012 m�1 and

2.0� 1012m�1, respectively (which corresponded tomore than

95% of Rtotal). Resistances measured in experiment 1 after one

month of operation were in the same order of magnitude.

3.3. Relationship between the flux and the coverage ofthe membrane by biofilm

Significant differences in the structure of the biofilms and in

coverage of the membrane surface by the biofilm were

observed for different experimental durations and different

predation levels (Fig. 3 aec and gei). A correlation between the

membrane coverage and flux at end of experiment 2 is

provided in Fig. 7 for the different predation levels. The extent

Table 2 e Results of the statistical analysis of theinfluence of the predation level on the change in themean biofilm thickness and on the relative roughnesscoefficient. b1 is the slope of the Nat-P case, b2 is thedifference in slope of the Nat-P and Low-P and b3 thedifference in slope between Nat-P and High-P.

Estimate Standard error T-test P-value

Statistical analysis of the mean biofilm thickness data

b0 (Intercept) 55.2 14.7 3.7 0.0005

b1 1.38 0.26 5.4 2.5e�06

b2 (Low-P) �0.64 0.22 �2.9 0.005

b3 (High-P) �0.31 0.22 �1.4 0.17

Statistical analysis of the relative roughness coefficient

b0 (Intercept) 0.2107 0.0424 4.96 1.20e�05

b1 0.0048 0.0007 6.599 5.47e�08

b2 (Low-P) �0.0048 0.0006 �7.878 8.30e�10

b3 (High-P) �0.0006 0.0007 �0.900 0.373

of membrane coverage was determined from photographs

using quantitative image analysis.

Two clusters of data are highlighted in Fig. 7 indicating the

presence (Nat.- and High-P cases) or absence (Low-P case) of

predators in the system. In the case of Low-P, the biofilm

homogeneously covered the entire surface of the membrane

resulting in an observed membrane coverage larger than 95%.

This almost complete coverage of the membrane was asso-

ciated with fluxes smaller than 5 L m�2 h�1. No spreading of

the data points was observed in this case. In the case of Nat.-P,

a heterogeneous biofilm was formed resulting in a partial

coverage ranging from 87% to 94%. This reduced observed

membrane coverage resulted in fluxes of around 10 L m�2 h�1

according data recorded during experiment 1 and 2. A slightly

lower coverage of the membrane was observed for the High-P

case. In this case, membrane coverage was observed with the

highest average flux, i.e., a flux higher than 10 Lm�2 h�1. Thus,

it can be concluded that lower membrane coverage is related

to higher fluxes with an increasing level of predation.

4. Discussion

4.1. How does predation impact the biofilm structure?

Compact and flat biofilms that covered the entire membrane

surface (see schematic representation in Fig. 8a) developed in

absence of predation (Low-P case) whereas open and hetero-

geneous structures composed of a thin basal layer plus

biomass mounds were observed in presence of predation

(Nat.-P and High-P) (Fig. 8b). Formation of different biofilm

structures was thus governed by the presence/absence of

predation. The presence/absence of predation can explain the

observed differences in biofilm structures in two ways: an

indirect and a direct way. The indirect influence of predation

results from predators triggering a reaction of the bacterial

population. The formation of micro-colonies is promoted

when bacterial biofilms are developed in presence of eukary-

otes (Bohme et al., 2009), presumably induced by quorum

sensingmechanisms in the bacterial biofilm (Matz et al., 2004).

Predation stimulates the production of exo-polymeric

substances (e.g. alginate) and bacteria tend to aggregate in

dense and disparate structures. In such structures like the

biomass mounds, the bacteria are better protected from

predation. The development of micro-colonies triggered by

the presence of predators during the early stages of biofilm

Fig. 6 e Average fluxmeasured in (a) Experiment 1 and in (b) Experiment 2 under different predation levels. Bars indicate the

standard errors (n [ 5).

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 3 3331

growth potentially explains the heterogeneous structure

observed at the meso-scale.

The direct influence results from predators actively

shaping an existing structure. The presence of cavities in the

biofilm structure could be explained by the activity of raptorial

feeders such as amoeba (Jackson and Jones, 1991). Also, the

motility of predators like worms or nematodes can create

channel networks, which result in a higher porosity of the

structure. Bohme et al. (2009) reported porosity values from

1.5 to 1.8 times higher in presence of protozoa. Predation also

reduces significantly the amount of biomass and thus its

volume. The amount of biomass on the membrane surface

was 30% less in the High-P case than in the Low-P case but

similar mean biofilm thicknesses were measured, which

suggests that biofilm densities were different. A reduction of

70 and 39% of the biomass concentration was reported in the

case of Pseudomonas aeruginosa biofilms of a non-toxic alginate

overproducing strain and of a wild-type strain toxic to

protozoa, respectively (Weitere et al., 2005), confirming the

influence of predators on the biomass volume. In the present

study, T. pyriformis that were added in the High-P, were

presumably outcompeted by other protists and Metazoa that

were naturally present in the creek river water. Competition

between T. pyriformis. and other protists could explain that

Fig. 7 e Flux (L mL2 hL1) versus themembrane coverage (%)

measured at the end of experiment 2. Two clusters of data

are identified depending of the presence or absence of

predators in the system.

biofilm structures developed in the Nat.-P and High-P cases

were statistically not different. It is expected that continuous

addition of Protozoa or Metazoa can reduce the membrane

coverage to an even larger extent. Also, the use of a raptorial

feeder (i.e., amoeba) or of worms that grazes only on attached

biomass should be more effective than the use of ciliates such

as T. pyriformis in the current study. More likely, operating the

GDM systems in a way that “natural” predation is enhanced

would have a greater effect than modifying the protistan

ecology by adding an exogenous species.

Finally, all thesemechanisms yield rougher andmore open

structures comparedwith the ungrazed biofilms. In absence of

predation, the biofilms developed at the surface of

membranes are formed by accumulation of particulate

matter, physico-chemical interactions and bacterial activity.

It could be shown that these processes still cause stabilization

of flux. However without predation, they lead to the devel-

opment of flat and compact structures. In systems operated

with cross-flow like in drinking water distribution pipes, the

development of flat structures is usually explained by high

shear stress conditions and low loading (van Loosdrecht et al.,

1995). In addition to growth and detachment processes,

predation contributes significantly to the development of

biofilm structures. Therefore, we suggest that predation

should be more considered when questions related to biofilm

structure are addressed. From a process-engineering point of

view, a crucial issue is however to understand to what extent

an open and heterogeneous structure impacts the hydraulic

resistances and thus the filtration process.

4.2. How does an open and heterogeneous structureenhance the filtration flux?

The use of optical methods such as CLSM or Optical Coher-

ence Tomography has revealed the porous and heterogeneous

structure of biofilms. Mass transfer is directly influenced by

biofilm morphology. For homogeneous biofilm developed on

impermeable substratum and under cross-flow conditions,

mass transfer is governed by diffusion and results in

a gradient of concentrations over biofilm depth. For hetero-

geneous biofilms developed on impermeable substratum,

substrate diffuses only in the top of the peaks and not in the

Fig. 8 e Schematic representation of the two distinct structures developed: (a) Compact and flat structure developed in

absence of predation, (b) Open and heterogeneous structure developed in presence of predation. In both cases there is

a basal layer but in the case of predation this basal layer is much thinner compared to the homogeneous layer in a system

without predation.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 33332

valleys (Eberl et al., 2000; Picioreanu et al., 2000). In this case

convection can contribute to mass transfer in pores but does

not necessarily enhance significantly to the overall mass

transfer from the bulk liquid to the biofilms matrix. Mass

transfer in the case of heterogeneous biofilm structures

developed on permeable substratum, like in our study, is not

reported in the literature.

According to the CarmaneKozeny equation (Eq. (6)), the

hydraulic resistance of a biofilm is proportional to its thick-

ness. The hydraulic resistance of biofilms developed in pres-

ence of predation was heterogeneously distributed over the

surface of the membrane, due to the spatial variation of the

biofilms thickness. In addition, the hydraulic resistance of the

thick mounds can be expected to be significantly higher than

the hydraulic resistance of the basal layer on which they

developed. It can be postulated that the water does not

penetrate these thick biomass mounds but only follows their

surface. Then, it is likely that water passes through the

membranewhere only a very thin biofilm is present, i.e., in the

valleys between the mounds. According to this hypothesis

mass transfer would be governed by advection in the valleys

and by diffusion in the peaks. This suggests that biofilm

growth should be higher in the valleys than in the peaks and

should lead to a decrease of the biofilm roughness. However

relative roughness coefficient was increasing over time indi-

cating that predation helps to maintain large biofilm hetero-

geneities. This result reinforces the idea that predation is

a crucial process in the case of biofilm developed without

cross-flow on permeable substratum.

Also, CarmaneKozeny equation applies for systems with

homogeneous and packed bed-like biofilm structures. In our

study the presence of predation resulted in an increasing

mean biofilm thickness but not in an increase of the hydraulic

resistance. Correlating the hydraulic resistance to the mean

biofilm thickness is thus not correct in the case of biofilms that

are heterogeneous at the meso-scale. In our study the filtra-

tion performances are governed by the biofilm structure

(coverage and surface heterogeneities) and not only by the

mean biofilm thickness. A more accurate prediction of the

permeate flux requires considering biofilm heterogeneities in

the X and Y directions (e.g. the biofilm roughness, i.e., the

change in the local biofilm thickness). Further investigations

are however required to characterize the flow distribution in

the case of heterogeneous biofilms structures. Evaluation of

the flow distribution can be performed experimentally and

numerically using fluorescent micro-beads or computational

fluid dynamics, respectively.

4.3. Relevance for decentralized water treatment

Dead-end ultrafiltration operated without control of the bio-

film formation and at very low hydrostatic pressure (kwon as

Gravity-Driven Membrane (GDM) filtration) represents a rele-

vant alternative for the decentralized production of drinking

water in developing and transition countries (see http://www.

eawag.ch/membranefilter). In these countries, centralized

systems are relevant for the production of drinking water in

densely populated zones but not in rural areas. For the

production of high-quality drinking water in rural zones,

decentralized systems such as GDM systems need to be

developed. In these areas, surface waters (river water, pond

water, etc) are usually used for the production of drinking

water. It can be assumed that the microbial ecology of the

surface waters influences the biofilm structure and in turn the

performance of the GDM systems. An improved control of

grazing in GDM could eventually lead to an optimization of the

permeability in such systems and contribute to a broader

application of membrane technology for decentralized use. In

conventional membrane systems operated with control of the

biofilm and at high pressure, the effect of predation on biofilm

structure and filtration performance is negligible compared to

biofilm control strategies (e.g., chlorination and high shear).

5. Conclusions

(i) Predation by eukaryotic microorganisms influences the

structure of the biofilm developing on the ultrafiltration

membrane.

(ii) Heterogeneous biofilm structures characterized by

a reduced membrane coverage and a thin basal layer

developed in presence of predation. Flat and compact

structures that homogeneously cover the membrane

surface are developed in absence of predation. Addition of

exogenous protists did not result in a change in the bio-

film structure.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 3 2 3e3 3 3 3 3333

(iii) The presence of predation resulted in an increased

permeate flux. Permeate flux of around 10 L m�2 h�1 was

monitored in presence of eukaryotes compared to the flux

of around 5 L m�2 h�1 monitored in absence of predation.

(iv) The meso-scale heterogeneities of biofilm influence its

hydraulic resistance. A more open structure (lower

coverage by thick biomass mounds and thinner basal

layer) resulted in an increased permeate flux.

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