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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: [email protected]/$ – 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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