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Influence of biofilm on the transport of fullerene(C60) nanoparticles in porous media

Meiping Tong a,b,*, Jiali Ding a, Yun Shen a, Pingting Zhu a,b

a The Key Laboratory of Water and Sediment Sciences, Ministry of Education, College of Environmental Sciences and Engineering,

Peking University, Beijing 100871, PR Chinab School of Environment and Urban Studies, Shenzhen Graduate School of Peking University, Shenzhen 518055, PR China

a r t i c l e i n f o

Article history:

Received 5 June 2009

Received in revised form

8 September 2009

Accepted 17 September 2009

Available online 30 September 2009

Keywords:

Fullerene C60

Biofilm

EPS

Deposition kinetics

Porous media

QCM-D

* Corresponding author: The Key Laboratoryand Engineering, Peking University, Beijing 1

E-mail address: tongmeiping@iee.pku.edu0043-1354/$ – see front matter ª 2009 Elsevidoi:10.1016/j.watres.2009.09.040

a b s t r a c t

The significance of biofilm on fullerene C60 nanoparticles transport and deposition were

examined both in porous media and quartz crystal microbalance with dissipation (QCM-D)

systems under a variety of environmentally relevant ionic strength (1–25 mM in NaCl and

0.1–5 mM in CaCl2) and flow conditions (4–8 m day�1). The magnitudes of deposition rate

coefficients (kd) were compared between porous media with and without biofilm extra-

cellular polymeric substances (EPS) coating under equivalent fluid velocities and solution

chemistries. The observed kd were greater in porous media with biofilm EPS coating relative

to those without biofilm EPS coating across the entire solution ionic strengths and fluid

velocities examined, demonstrating that the enhancement of C60 deposition by the biofilm

EPS coating is relevant to a wide range of environmental conditions. This greater deposi-

tion was also observed on silica surfaces with biofilm EPS coating in QCM-D system. The

results clearly showed that biofilm EPS have a great influence on C60 deposition. Derjaguin–

Landau–Verwey–Overbeek (DLVO) theory could not explain the enhanced C60 deposition by

biofilm EPS. Biochemical and physical characteristics of biofilm EPS were responsible for

the increased C60 deposition.

ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction Espinasse et al., 2007), solution chemistry (Chen and Eli-

Fullerene C60, with wide applications in various fields

including optics and electronic engineering (Eckert et al., 2000;

Venturini et al., 2002), biotechnology (Bosi et al., 2003), and

medical sciences (Faraji and Wipf, 2009; Yamashita et al.,

2009), would be eventually released into the natural environ-

ment. Due to their potential risk to the natural ecosystem and

human health (Colvin, 2003; Oberdorster, 2004; Sayes et al.,

2004, 2005), the fate and distribution of fullerene C60 in natural

systems especially in subsurface is therefore of great interest.

Factors such as fluid velocity (Lecoanet and Wiesner, 2004;

of Water and Sediment S00871, PR China. Tel.: þ8.cn (M. Tong).

er Ltd. All rights reserved

melech, 2006; Espinasse et al., 2007), natural organic matter

(NOM) (Espinasse et al., 2007; Chen and Elimelech, 2007, 2008),

and porous media type (Wang et al., 2008; Li et al., 2008) have

been demonstrated to have a great influence on the transport

and deposition kinetics of C60. For example, Espinasse et al.

(2007) showed that C60 deposition increased with increasing

solution ionic strength, the presence of polysaccharide-type

organic matter, and the decreasing fluid velocity. Very

recently, Chen and Elimelech (2008) examined the impact of

humic acid and alginate (NOM) on the deposition kinetics of

C60 on silica surfaces. These authors found that pre-coated

ciences, Ministry of Education, College of Environmental Sciences6 10 6275 6491; fax: þ86 10 62756526.

.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 3 1095

NOM on silica surface could either retard or enhance C60

deposition, depending on the solution ionic composition and

the physicochemical properties of the NOM macromolecules.

Biofilm, structured community of microorganisms encap-

sulated in matrix of self-developed extracellular polymeric

substances (EPS), is present every where in the natural envi-

ronment (Denyer et al., 1993). A very recent study (Liu and Li,

2008) reported that biofilm EPS had a profound impact on the

deposition and retention of Escherichia coli (E. coli) in porous

media. Biofilm EPS are also expected to play important roles in

the transport and deposition kinetics of fullerene C60. However,

to date, the influence of biofilm EPS on transport and deposition

of C60 has never been explored, and it requires investigation.

The objective of this paper is to systematically examine the

significance of biofilm EPS on the transport and deposition

kinetics of fullerene C60. E. coli, one of the most ubiquitous

bacterial strains present in natural environment, was employed

to form biofilm EPS. Packed porous media column and quartz

crystal microbalance with dissipation (QCM-D) systems were

utilized to examine the impact of biofilm EPS on C60 transport

under a variety of environmentally relevant ionic strength

(1–25 mM in NaCl and 0.1–5 mM in CaCl2) and flow conditions

(4 and 8 m day�1 in columns, 0.1 mL min�1 for QCM-D) at pH 6.8.

Our results showed that under all examined conditions in both

systems, E. coli biofilm EPS enhanced C60 deposition. Non-DLVO

interaction between biofilm EPS and C60 as well as the increased

quartz sand surface roughness with biofilm EPS coating were

responsible for the enhancement of C60 deposition.

2. Materials and methods

2.1. Preparation of fullerene C60 solutions

Fullerene powders (99.9%, purified by sublimation) were

purchased from the Materials Electronics Research Corp.

(Tuscon, AZ). The aqueous suspensions of C60 were prepared

from fullerene powders following the method provided by

Deguchi et al. (2001). A total of 6.25 mg fullerene powders were

added to a volume of 250 mL tetrahydrofuran (THF) (99.9%,

Beijing Chemical Reagents Company, China) at room

temperature (25 �C). The mixture was purged with nitrogen to

remove dissolved oxygen and stirred 24 h to allow the solution

to be fully saturated with C60, forming a clear pink solution.

Excess solid material was then filtered off from the solution

with employment of 0.22 mm nylon membrane filter.

Following that, 250 mL Milli-Q water was added to the C60-THF

solution at a rate of 500 mL min�1, while being continuously

stirred. During the process of Milli-Q water addition, the color

of C60-THF solution changed from pink to transparent yellow.

The mixed solution was then gently heated by a rotary evap-

orator. THF was continuously removed from the C60-THF

solution at a temperature of approximately 60 �C, and

approximately 275 mL solution was evaporated in 30 min. A

total of 50 mL Milli-Q water was added to the solution to

compensate the water lose during the evaporation process.

The more volatile THF and part of the water was evaporated

off at 80 �C. Finally, with the addition of 50 mL Milli-Q water,

the clear yellow solution was evaporated until a volume of

250 mL. This solution was filtered through 0.22 mm cellulose

acetate membrane filter and stored in the dark at 4 �C. The

resulting C60 nanoparticles had a mean particle diameter of

85 nm, characterized by a transmission electron microscopy

(H-9000NAR TEM, Hitachi, Japan). The concentration of C60

suspension, determined using a TOC-meter (Tekmer Fusion,

Teledyne Instruments), was 13 ppm as TOC.

Deposition experiments both in the packed porous media

column and the quartz crystal microbalance with dissipation

(QCM-D) systems were conducted at influent C60 concentra-

tion of 1 ppm (as TOC) (w2 � 109 aggregates mL�1) at ionic

strength ranging from 1 to 25 mM in NaCl and at ionic strength

ranging from 0.1 to 5 mM in CaCl2 solutions at pH of 6.8

(adjusted with 0.1 M NaOH). The corresponding electropho-

retic mobility and zeta potential of C60 particles under these

conditions were performed using Zetasizer Nano ZS90 (Mal-

vern Instruments, UK). Measurements were performed at

room temperature (25 �C) and repeated nine to 12 times.

2.2. Porous media

Quartz sand (ultrapure with 99.8% SiO2) (Hebeizhensheng

Mining Ltd., Shijiazhuang, China) with sizes ranging from 417

to 600 mm were used for fullerene C60 deposition experiments in

porous media. The procedure used for cleaning the quartz sand

is provided in the previous publication (Tong et al., 2005), as

well as in the Supplementary Information. The zeta potentials

of the crushed quartz sand were also performed in both NaCl

and CaCl2 solutions under the experimental conditions with

the employment of Zetasizer Nano ZS90. The electrophoretic

mobility measurements were repeated nine to 12 times.

2.3. Porous media experiments

Deposition experiments in porous media were conducted both

in the absence and presence of biofilm (both without and with

biofilm EPS coating). For the experiments performed without

biofilm, the cylindrical columns (20 cm long and 4.0 cm in

diameter) were wet packed with cleaned quartz sand,

whereas, for the experiments conducted with biofilm, the

columns were wet packed with quartz sand premixed with

E. coli suspensions (w108 cells mL�1) (used to grow biofilm).

Detailed information of E. coli growth were provided in Long

et al. (2009) and also given in the Supplementary Information.

Packing was performed by adding wet quartz sand (or quartz

sand premixed with cells) in small increments (w2 cm) with

mild vibration of the column to minimize any layering or air

entrapment (Tufenkji and Elimelech, 2004). Two 60 mesh

stainless steel screens were placed at each end of the column.

To spread the flow upon entry into the column, w3.5 g of

quartz sand were added to the top of the influent screen,

forming a 2 mm-thick layer that was covered by another

screen. The porosity of packed column is approximately 0.42.

After packing, for the experiments without biofilm, the

columns were pre-equilibration with 10 pore volumes of salt

solutions at desired ionic strength (NaCl or CaCl2). After pre-

equilibration, three pore volumes of C60 suspensions at

desired ionic strength were introduced into the columns. This

was followed by elution with C60-free salt solutions with the

same ionic strength (five pore volumes). All of the solutions

were introduced into the columns in an upflow mode.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 31096

For the experiments conducted with biofilm, columns were

settled for 5 h after packing to allow the cells tightly attach

to quartz sand. After that, synthetic nutrient solution

(consisted of 0.79 g of K2HPO4 $ 3H2O, 1 g of NaCl, 0.02 g of

FeSO4 $ 7H2O, 0.2 g of MgSO4 $ 7H2O, 0.2 mg of MnSO4 $ H2O,

2 mg CaCl2 $ 2H2O, 0.2 mg of ZnSO4 $ 7H2O, 0.09 mg of

CoCl2 $ 6H2O, 0.12 mg of NaMoO4 $ 2H2O, and 0.06 mg of H3BO3)

was introduced into the columns at a flow rate of 2.5 mL min�1

at room temperature (25 �C) for 80 h to supply the necessary

nutrients for the growth of E. coli biofilm in the columns. To

ensure the formation of uniform biofilm distribution inside the

column, the flow injection direction was switched between

upflow and downflow directions in every 8 h (Liu and Li, 2008).

Pre-experiments (Fig. S1) demonstrated that the uniform bio-

film was established along the column following this biofilm

growth procedure. After the formation of biofilm, at least 15

pore volumes of Milli-Q water was injected into the columns to

fully remove the residual synthetic nutrient solutions. Subse-

quently, columns were followed with the same experimental

procedure as that conducted in columns without biofilm, that

is pre-equilibration (10 pore volumes), injection of C60 suspen-

sion (three pore volumes), and then elution with C60-free salt

solution (five pore volumes).

Samples from the column effluent were collected continu-

ously (w12 mL each sample) in 15 mL sterile centrifuge tubes.

Aqueous effluent samples were analyzed using TOC-meter.

The packed porous media experiments were conducted in both

NaCl (ranging from 1 to 25 mM) and CaCl2 (ranging from 0.1 to

5 mM) solutions. The flow rate was varied between experi-

ments to produce pore water velocities of 4 and 8 m day�1.

To quantitatively compare the deposition kinetics of C60 in

porous media under different conditions, the deposition rate

coefficient (kd) was determined using the following equation

(Kretzschmar et al., 1999):

kd ¼ �vqL

lnCCo

(1)

where v is the flow velocity, q is the porosity, L is the length of

porous media, C is the concentration of C60 in the aqueous

effluent samples, Co is influent C60 concentration, and C/Co is

the normalized breakthrough concentration relevant to clean

bed conditions, and can be obtained from the plateau of

breakthrough curves.

2.4. Quartz crystal microbalance with dissipation(QCM-D) experiments

Since EPS dominated the biofilm surfaces (Liu and Li, 2008), the

influence of biofilm (EPS) on C60 deposition can also be inves-

tigated in a QCM-D system by pre-coating the silica surfaces

with EPS extracted from E. coli (detailed EPS extraction proce-

dure and zeta potential measurements can be found in Long

et al. (2009) and also provided in Supplementary Information).

The deposition kinetics of C60 on bare and EPS-coated silica

surfaces were examined in both NaCl (four ionic strengths

ranging from 1 to 25 mM) and CaCl2 (four ionic strengths

ranging from 0.1 to 5 mM) solutions by the employment of

a QCM-D E1 system (Q-Sense AB, Gothenburg, Sweden). QCM-

D experiments were performed with 5 MHz AT-cut quartz

sensor crystals with silica-coated surface (Batch 070624-10).

Before each measurement, the crystals were soaked 30 min in

a 2% SDS solution, rinsed thoroughly with Milli-Q water, dried

with ultrahigh-purity N2 gas, and then oxidized for 30 min in

a UV/O3 chamber (Bioforce nanosciences, Inc., Ames, IA).

The fullerene C60 deposition experiments in the QCM-D

system were performed in a flow-through mode, using a peri-

staltic pump (ISMATEC, Switzerland) operating in a clockwise

mode. Specifically, the pump was connected to the sensor

crystal outlet, and the studied solutions, stored in a sterilized

50 mL polypropylene conical tube (Becton Dickinson, NJ)

connected to the sensor crystal inlet, were fed through the

crystal sensor chamber at a flow rate of 0.1 mL min�1.

For deposition experiments on bare silica surfaces, the

QCM-D system was pre-equilibrated with salt solution at

a desired ionic strength (NaCl or CaCl2) for a minimum of

30 min to establish a stable baseline (the drift of average

normalized frequency was less than 0.2 Hz within 30 min).

After pre-equilibration, C60 suspension with the same ionic

strength and pH was injected into the crystal chamber. For

deposition experiments conducted on EPS-coated silica

surfaces, the bare silica surfaces were first modified with

a layer of positively charged poly-L-lysine (PLL) hydrobromide

(molecular weight of 70,000–150,000) (P-1274, Sigma–Aldrich,

St. Louis, MO). Detailed information about surface modifica-

tion with PLL was provided in Supplementary Information.

After PLL coating, HEPES buffer was introduced into the

chamber for 20 min at a flow rate of 0.1 mL min�1 to remove

the unadsorbed PLL. Following that, a 1 mM NaCl solution was

then flowed through the crystal chamber to fully remove the

HEPES buffer at the same flow rate. After these two elutions,

a layer of EPS was adsorbed on the PLL layer by flowing 2 mL of

EPS (60 mg L�1 TOC) prepared in 1 mM NaCl solution across

the PLL-coated surface. The fast adsorption of EPS under such

conditions leads to sharp frequency shifts Df3 of about 13 Hz.

The baseline stabilized after 5 min when PLL layer was

exposed to the EPS solutions, indicating complete coverage of

EPS on the PLL layer. The formed layer of EPS was then rinsed

with 1 mM NaCl solution for 20 min to remove unadsorbed

EPS. Finally, the salt solution of interest was flowed through

the EPS-coated surface until a constant baseline was obtained

prior to the introducing of the C60 suspensions with the same

solution ionic strength for the deposition experiment.

For the experiments performed on both bare and EPS-

coated silica surfaces, the deposition rate (kf) can be deter-

mined from the slope of the initial (linear) portion of the

change in normalized frequency Df3 versus time curve (Chen

and Elimelech, 2006; Nguyen and Elimelech, 2007), since the

changes in resonance and overtone frequencies are propor-

tional to the mass deposited on the crystal:

kf ¼d Df3

dt(2)

3. Results and discussion

3.1. Zeta potentials of fullerene C60, quartz sand, E. coli,and EPS

The influence of solution ionic composition and ionic

strength on the electrophoretic mobilities and zeta

Fig. 1 – Breakthrough-elution curves for fullerene C60 in the absence (solid triangle) and presence of E. coli biofilm (open

triangle) in NaCl solutions at fluid velocity of 8 m dayL1 and pH 6.8 (adjusted with 0.1 M NaOH) as a function of solution ionic

strength in quartz sand. Duplicate measurements were conducted over entire ionic strength range, with error bars

representing standard deviations.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 3 1097

potentials of fullerene C60, crushed quartz sand, E. coli, and

EPS extracted from E. coli were examined and presented in

Fig. S2. Zeta potentials of all materials were negative at pH

6.8 and became less negative with increasing ionic strength

in both NaCl (Fig. S2, top) and CaCl2 solutions (Fig. S2,

bottom) due to compression of the electrostatic double

layer. Under the same ionic strength (1 and 5 mM), zeta

potentials of all materials in CaCl2 solutions (Fig. S2, bottom)

were less negative relative to those in NaCl solutions

(Fig. S2, top). This was true over all examined ionic strengths

range. This observation was possibly due to the adsorption

of calcium ions to the material surfaces resulting in the

neutralization of surface charge. Under the same ionic

strength, zeta potentials of C60 was less negative comparing

to those of EPS, E. coli, and quartz sand. This held true over

entire ionic strength range in both NaCl and CaCl2 solutions.

Fig. S2 also showed that under all ionic strengths, zeta

potentials of EPS, E. coli, and quartz sand were comparable

in both NaCl (top) and CaCl2 (bottom) solutions. This

observation suggested that the covering of E. coli biofilm on

Fig. 2 – Breakthrough-elution curves for fullerene C60 in the absence (solid triangle) and presence of E. coli biofilm (open

triangle) in CaCl2 solutions at fluid velocity of 8 m dayL1 and pH 6.8 (adjusted with 0.1 M NaOH) as a function of solution

ionic strength in quartz sand. Duplicate measurements were conducted over entire ionic strength range, with error bars

representing standard deviations.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 31098

quartz sand would not have a great influence on electroki-

netic properties of quartz sand.

3.2. Influence of biofilm on fullerene C60 transport

To examine the influence of biofilm on the transport behavior

of fullerene C60, C60 transport experiments were performed in

packed quartz sand columns both with and without biofilm

coating in both NaCl (five ionic strengths ranging from 1 to

25 mM) and CaCl2 (four ionic strengths ranging from 0.1 to

5 mM) solutions. The effluent breakthrough-elution curves

obtained at fluid velocity of 8 m day�1 in NaCl and CaCl2 solu-

tions were presented in Figs. 1 and 2, respectively. At all

examined ionic strengths in both NaCl and CaCl2 solutions at

8 m day�1, the steady-state breakthrough plateaus without

biofilm coating were flat (Figs. 1 and 2, solid triangle), indicating

temporal constancy of the deposition rate coefficient (negli-

gible blocking or ripening) during the course of the experi-

ments. This was also true in the presence of biofilm in both

NaCl and CaCl2 solutions (Figs. 1 and 2, open triangle). In the

absence of biofilm, steady-state breakthrough plateaus

decreased with the increase of ionic strength in both NaCl and

CaCl2 solutions (Figs. 1 and 2, solid triangle), consistent with

many previous studies (Espinasse et al., 2007; Wang et al., 2008).

The same trend was also observed in the presence of biofilm in

both NaCl and CaCl2 solutions. At the same ionic strengths

(1 and 5 mM) both with and without biofilm, the breakthrough

plateaus in CaCl2 solutions (Fig. 2) were lower than those in

NaCl solutions (Fig. 1) at the same fluid velocity (8 m day�1).

Fullerene C60 column experiments both with and without

biofilm coating were also performed in both NaCl and CaCl2solutions at a lower fluid velocity, 4 m day�1. The above

observations acquired at fluid velocity of 8 m day�1 were also

true at fluid velocity of 4 m day�1 (Figs. 3 and 4). Close

inspection of breakthrough curves obtained at 8 m day�1

versus those obtained at 4 m day�1 in the absence of biofilm

showed that over all examined ionic strength range in both

NaCl and CaCl2 solutions, the breakthrough plateaus at 4 m

day�1 (Figs. 3 and 4) were lower than those at 8 m day�1 at the

same ionic strength (Figs. 1 and 2), consistent with previous

study (Espinasse et al., 2007). The same results were also

observed in the presence of biofilm over entire ionic strength

range in both NaCl and CaCl2 solutions.

More noteworthy observation is that the steady-state

breakthrough plateaus in the presence of biofilm (open

triangle) were lower than those in the absence of biofilm (solid

triangle) across the entire ionic strength range examined in

both NaCl and CaCl2 solutions at 8 m day�1 (Figs. 1 and 2). The

same results were also observed at a fluid velocity of 4 m day�1

in both NaCl and CaCl2 solutions (Figs. 3 and 4). These results

strongly demonstrated that coating the quartz sand surfaces

with biofilm affected the transport behavior of C60.

Fig. 3 – Breakthrough-elution curves for fullerene C60 in the absence (solid triangle) and presence of E. coli biofilm (open

triangle) in NaCl solutions at fluid velocity of 4 m dayL1 and pH 6.8 (adjusted with 0.1 M NaOH) as a function of solution ionic

strength in quartz sand. Duplicate measurements were conducted over entire ionic strength range, with error bars

representing standard deviations.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 3 1099

To quantitatively compare the deposition (transport)

kinetics of C60 both in the absence and presence of biofilm at

different solution ionic strengths and at different fluid veloc-

ities, deposition rate coefficient (kd) was derived from the

steady-state plateaus by employment of Eq. (1). In both NaCl

and CaCl2 solutions at both 4 and 8 m day�1, kd in the absence

of biofilm increased with the increase of ionic strength (Fig. 5,

white bar). This observation was also consistent with the

trends of zeta potentials of C60 (and quartz sand) versus ionic

strength (Fig. S2) and thus generally agreed with classic DLVO

theory. The increase of solution ionic strength compresses

electrostatic double layer between C60 and quartz sand

surfaces, and thus results in greater C60 deposition. The

increase of kd with increasing ionic strength was also true in

the presence of biofilm in both NaCl and CaCl2 solutions at two

fluid velocities (4 and 8 m day�1), which can also be explained

by the less negative zeta potentials of C60 and E. coli at higher

ionic strength (Fig. S2), resulting in the compression of the

Fig. 4 – Breakthrough-elution curves for fullerene C60 in the absence (solid triangle) and presence of E. coli biofilm (open

triangle) in CaCl2 solutions at fluid velocity of 4 m dayL1 and pH 6.8 (adjusted with 0.1 M NaOH) as a function of solution

ionic strength in quartz sand. Duplicate measurements were conducted over entire ionic strength range, with error bars

representing standard deviations.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 31100

electrostatic double layer between C60 and E. coli biofilm

surfaces. Fig. 5 also showed that at the same ionic strength

(1 and 5 mM) both in the absence and presence of biofilm at

two fluid velocities, kd in CaCl2 solutions (Fig. 5, right) were

factors of about 1.6–4.8 greater than those in NaCl solutions

(Fig. 5, left). These results agreed with the less negative zeta

potentials of C60 in CaCl2 solutions relative to those in NaCl

solutions under the same conditions (Fig. S2). The presence of

Ca2þ also reduces the charge of the quartz sand and E. coli

biofilm surfaces, as shown by the less negative zeta potentials

of crushed quartz sand and E. coli in CaCl2 solutions relative to

those in NaCl solutions at the same ionic strength (Fig. S2). A

greater reduction of electrostatic repulsion between C60 and

quartz sand (or E. coli biofilm) surfaces in CaCl2 solutions

relative to that in NaCl solutions leads to a greater C60 depo-

sition in CaCl2 solutions.

Comparison of kd obtained in the presence of biofilm with

those acquired in the absence of biofilm clearly showed that kd

in the presence of biofilm (Fig. 5, black bar, left) were greater

(factors of 1.4–1.6) relative to those in the absence of biofilm

(Fig. 5, white bar, left) in NaCl solutions at 8 m day�1 (Fig. 5, top,

left). This was also true in CaCl2 solutions (factors of 1.6–1.8

greater) (Fig. 5, top, right). The same results were also observed

both in NaCl and CaCl2 solutions at lower flow rate 4 m day�1

(factors of 1.4–1.9 greater) (Fig. 5, bottom). These results

strongly demonstrated that the presence of biofilm on sand

surfaces increased C60 deposition in columns under all

examined conditions. However, zeta potential measurements

showed that under all ionic strengths, zeta potentials of E. coli

and quartz sand (as well as EPS extracted from E. coli) were

comparable at the same ionic strength in both NaCl and CaCl2solutions (Fig. S2). Clearly, coverage of quartz sand surfaces

with E. coli biofilm would not significantly change the elec-

trokinetic properties of the sand surfaces, which was also

confirmed by the observed equivalent zeta potentials of

crushed quartz sand both in the absence and presence of

background E. coli (or EPS) in solutions under the same solution

conditions (data not shown). Thus negligible differences in

deposition behaviors of C60 on quartz sand and those on bio-

film-coated sand would be expected according to DLVO theory.

The obvious greater kd (lower breakthrough plateaus) obtained

in the presence of biofilm cannot be only explained by DLVO

interactions. Biochemical and physical characteristics of bio-

film might be responsible for the increased C60 deposition.

Since the biofilm surfaces are dominated by EPS, the enhanced

C60 deposition by the presence of biofilm can be attributed to

the interactions (non-DLVO interactions) between EPS and C60,

and the surface property of EPS. Examination of the influence

of EPS on C60 deposition is therefore necessary.

3.3. Influence of EPS on fullerene C60 deposition

To systematically examine the influence of EPS on the depo-

sition kinetics of fullerene C60, deposition experiments were

Fig. 5 – Deposition rate coefficient (kd) of fullerene C60 in the absence (white bar) and presence of E. coli biofilm (black bar) in

both NaCl (left) and CaCl2 (right) solutions at fluid velocities of 8 m dayL1 (top) and 4 m dayL1 (bottom) at pH 6.8 (adjusted

with 0.1 M NaOH) as a function of ionic strength in quartz sand.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 3 1101

performed on both bare and EPS-coated silica surfaces in both

NaCl (four ionic strengths ranging from 1 to 25 mM) and CaCl2(four ionic strengths ranging from 0.1 to 5 mM) solutions by

employment of a QCM-D system. It should be noted that EPS

were extracted from E. coli. In agreement with the obvious

increase of deposition rate coefficient with the increase of

ionic strength observed in packed quartz sand columns

(Fig. 5), the deposition rate (kf) on both bare and EPS-coated

silica surfaces also increased with increasing ionic strength in

NaCl solutions (Fig. 6, top). The same trend was also observed

in CaCl2 solutions (Fig. 6, bottom). These observations were

also consistent with the trends of zeta potentials of C60, silica

(can be represented by quartz sand), and EPS versus ionic

strength (Fig. S2) and thus generally agreed with classic DLVO

theory. At the same solution ionic strength (1 and 5 mM) on

bare silica surfaces, kf in CaCl2 solutions were greater (factor of

about 7.5) than those in NaCl solutions, consistent with

previous studies (Chen and Elimelech, 2006, 2007, 2008). On

EPS-coated surfaces, kf in CaCl2 solutions were also greater

(factors 8.6–10.0) relative to those in NaCl solutions. These

results were also agreed with observations in packed

columns.

Close comparison of kf on EPS-coated surfaces versus those

on bare silica surfaces yielded that kf on EPS-coated surfaces

were greater relative to on bare silica in both NaCl (factors of

2.7–3.8 greater) and CaCl2 (factors of 2.7–5.0 greater) solutions

across entire ionic strength range examined, which was

consistent with the observations in packed quartz sand

columns. The results strongly demonstrated that pre-coating

silica surfaces with EPS enhanced the deposition of C60 under

all examined conditions, which deviated from the DLVO

prediction based on the observed comparable zeta potentials

of EPS and crushed quartz sand under the same conditions

(Fig. S2). These results also demonstrated that other mecha-

nisms (in addition to DLVO interactions) could play important

roles in C60 deposition (agreed with column experiments).

Analysis of the biochemical components of EPS showed that

E. coli EPS were mainly comprised of proteins and poly-

saccharides as well as a small amount of other organic

constitutions (Zhu et al., 2009). These components would form

different attractive interactions (non-DLVO interactions) with

C60, resulting in greater C60 deposition. For example, hydro-

phobic groups in EPS such as non-polar groups in proteins

could attract C60 to approach EPS surfaces closely. Previous

study showed the attractive interaction was present between

protein and C60 (Deguchi et al., 2007). The attractive protein-

C60 interactions could enhance C60 deposition. In addition,

silica surface roughness was increased with EPS (biofilm)

coating (Siegrist and Gujer, 1985; Shellenberger and Logan,

2002; Liu and Li, 2008). The much rougher surface would allow

for the greater C60 deposition. The greater deposition rate of

C60 due to the increased surface roughness has been

Fig. 6 – Deposition rate (kf) of fullerene C60 on bare (white

bar) and EPS-coated silica surface (black bar) in NaCl (top)

and CaCl2 (bottom) solutions at pH 6.8 (adjusted with

0.1 M NaOH) as a function of ionic strength in QCM-D.

Duplicate measurements were conducted over entire ionic

strength range, with error bars representing standard

deviations.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 1 0 9 4 – 1 1 0 31102

previously demonstrated (Chen and Elimelech, 2008). The

results suggested that the enhanced C60 observed in packed

columns in the presence of biofilm was also possibly caused

by the increased quartz sand surface roughness with biofilm

coating as well as the non-DLVO interactions present between

biofilm surfaces and C60.

4. Conclusion

This study demonstrated that biofilm, ubiquitous biomass in

natural environment, have a great influence on the transport

behavior of fullerene C60 under solution conditions relevant to

subsurface environment. The deposition kinetics of C60 on

biofilm-coated surfaces was not only controlled by DLVO

interactions. Other mechanisms: non-DLVO interactions

between biofilm surfaces and C60 and the surface modification

with biofilm coating also have a great contribution to the

enhanced deposition. Our results indicate that the C60 trans-

port distance in the natural environment could be greatly

inhibited by the presence of biofilm.

Acknowledgement

This work was supported by the National Natural Science

Foundation of China-Young Scientists Fund under grant No.

40802054. The authors wish to acknowledge Dr. Kai Loon Chen

from the Department of Geography and Environmental

Engineering at Johns Hopkins University for his useful

suggestions.

Appendix A. Supplementary Information

Distribution of retained biofilm bacteria in biofilm-coated

column (Fig. S1); electrophoretic mobilities and zeta potentials

of fullerene C60, quartz sand, E. coli, and EPS (Fig. S2); also

presented are details about quartz sand cleaning procedure;

growth and harvest protocol for E. coli; EPS extraction

methods; and protocol for silica surfaces modification with

PLL. Supplementary information for this manuscript can be

downloaded at doi:10.1016/j.watres.2009.09.040.

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