surface modification of polymeric membranes with …
TRANSCRIPT
SURFACE MODIFICATION OF POLYMERIC MEMBRANES
WITH THIN FILMS AND SILVER NANOPARTICLES FOR
BIOFOULING MITIGATION
by
Li Tang
A dissertation submitted to Johns Hopkins University in conformity with the
requirements for the degree of Doctor of Philosophy
Baltimore, Maryland
February 2015
2015 Li Tang
ii
Abstract
Membrane filtration is a highly efficient water treatment technique with
substantial potential for helping overcome the global water crisis. However, biofouling,
or the formation of biofilms on membranes, currently hinders the sustainable application
of membrane filtration and is, in fact, widely considered to be one of the most
challenging obstacles to overcome. Therefore, an effective membrane biofouling
mitigation technique is urgently needed.
The objective of this dissertation work was to investigate the influence of the
surface modifications of membranes with polymeric thin films and silver nanoparticles
(AgNPs) for biofouling mitigation. The surface-modified membrane’s anti-biofouling
properties can be evaluated through quantitative assessments of the membrane’s bacterial
anti-adhesive properties and antimicrobial properties. The first part of the dissertation
effort focused on surface modifications with AgNPs and 2-bilayer PAH/PAA PEMs on a
commercial polysulfone (PSU) microfiltration (MF) membrane. The membrane’s
bacterial anti-adhesive properties were highly enhanced after PEM- and AgNP/PEM-
modifications. Specifically, the deposition kinetics of Escherichia coli cells on the PEM-
and AgNP/PEM-modified membranes were reduced and the removal efficiencies were
significantly enhanced compared to those of the base membrane. Interaction force
measurements demonstrated that the bacterial anti-adhesive properties exhibited by the
membrane modified with a PEM film could be attributed to the highly swollen and
hydrated PEMs that inhibit the direct contact or close approach of bacteria to the
underlying membrane. AgNPs that were immobilized on the membrane surface imparted
antimicrobial properties to the membrane and the degree of bacterial inactivation
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increased as a function of AgNP mass loading. In addition, the AgNP mass loading
required for the inhibition of bacterial growth in our study was significantly lower than
the AgNP loadings reported in other studies for membranes with AgNPs dispersed within
the membrane matrix, hence implying that the distribution of AgNPs within the
membrane plays an important role in controlling the membrane’s antimicrobial
properties.
The second part of this dissertation focused on surface modifications with PDA
and AgNPs formed in situ on a laboratory-cast PSU MF membrane. AgNPs could be
generated on the membrane surface through Ag+ ion reduction by the catechol groups in
PDA by simply soaking the membrane in a AgNO3 solution. The AgNP mass loading
was found to increase with increasing soaking time. The PDA film increased the surface
hydrophilicity of the membrane and the PDA- and PDA/AgNP-modified membranes
exhibited bacterial anti-adhesive properties. The AgNPs that were immobilized on the
membrane through metal coordination imparted strong antimicrobial properties to the
membrane. This technique for membrane surface modification paves a way to mitigate
membrane biofouling by enhancing the membrane’s bacterial anti-adhesive and
antimicrobial properties simultaneously and also provides a feasible method to replenish
AgNPs on the membrane in situ in water treatment processes.
Advisor: Kai Loon Chen
Committee Members: Edward J. Bouwer, William P. Ball, and D. Howard Fairbrother
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank my advisor, Dr. Kai Loon Chen, for
providing me with such a great opportunity to study for a Ph.D. degree with him at Johns
Hopkins University (JHU) and guided me through the journey of Ph.D. study. He always
encourages me to be persevering and optimistic and to push myself to overcome the
challenges I encountered in my research. He has been always available whenever I need
advice and support, showing his understanding and patience when I made mistakes, and
unselfishly passing on the skills and knowledge to me. I have been benefiting so much
from the Ph.D. study with Kai Loon, and I believe this valuable research experience will
definitely lay a solid foundation to my future career development.
I would like to sincerely thank my dissertation committee members: Drs. Edward
Bouwer, Bill Ball, and Howard Fairbrother for spending time to read through my
dissertation and proving me with insightful feedback during my Ph.D. study. I would
like to thank Drs. Haiou Huang and Patricia McGuiggan for serving on my Graduate
Board Oral Examination, and Drs. Alan Stone and Markus Hilpert for serving on my
Department Qualifying Examination. I have been benefiting a lot from their challenging
questions that help me improve the quality of my dissertation work. I also would like to
thank Drs. Ken Livi and Michael McCaffery from the Integrated Imaging Center at JHU
for spending time to help me capture backscattered and environmental SEM imaging and
performing EDX analysis. They always showed their kindness and patience and
provided me with valuable suggestions during our collaboration.
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I am so grateful to be a member of the Department of Geography and
Environmental Engineering (DoGEE), where I received a warmth of home and lifetime
friendship. I have been enjoying and benefiting from the insightful discussions in every
class I had in Dogee with Drs. Edward Bouwer, Alan Stone, Bill Ball, and Lynn Roberts.
My Ph.D. research could not be completed without the help from the Dogee staff
members, such as Keith, Denise, Adena, Joyce, Rob, Mike, Jessica, and Christine. I
would like to express my many thanks to Keith, who helped me a lot for setting up my
experiment platform during my initial research period.
I would like to express my appreciation to the help and friendship with the
members in Dr. Chen’s research group, such as Peng, An, Myunghee, Xin, Xitong,
Wenyu, Ji Yeon, Yeunook, Davide, Qiaoying, Yiping, Margaret, and Mathieu. I would
also like to thank my colleagues Jin, Pavlo, Jessica, Katie, Nate, Xiaomeng, Qian, Philip,
Mike, and Stephanie. I thank for the help from the members in Dr. Howard Fairbrother’s
research group, such as Julie, Miranda, and Mike, for performing the ATR-IR, contact
angle, and XPS measurements.
I would like to thank my parents Qingling Tang and Shouyan Wang who provide
me with unconditional love and support throughout my life. They raise me up and offer
me with everything they could give to me to make who I am today. I would like to show
my special thanks to my forever friends Jianling Wang and Yan Cai. My Ph.D. study
cannot be completed without them. Jianling accompanies me through the challenging
Ph.D. life. I cannot forget the sincere encouragements and warmly care from Yan from
time to time throughout the most difficult year in my Ph.D. study. Yan turns my life
around. Those words will be always in my heart.
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LIST OF CONTENTS LIST OF CONTENTS ................................................................................................................. vi
LIST OF FIGURES .................................................................................................................... viii
LIST OF TABLES ........................................................................................................................ xi
Chapter 1. Introduction ......................................................................................................... 1 1.1. Water Resources in the World ........................................................................................... 2 1.2. Membrane Filtration in Water and Wastewater Treatment ................................... 2 1.3. Membrane Biofouling ........................................................................................................... 4 1.4. Membrane Surface Modification for Biofouling Mitigation .................................... 5 1.5. Polyelectrolyte Multilayers................................................................................................. 6 1.6. Polydopamine Films .............................................................................................................. 8 1.7. Silver Nanoparticles As An Antimicrobial Agent ...................................................... 10 1.8. Objective and Scope of Dissertation ............................................................................. 13 1.9. Dissertation Organization ................................................................................................ 14 1.10. References ........................................................................................................................... 16
Chapter 2. Bacterial Anti-Adhesive Properties of Polysulfone Membranes Modified with Polyelectrolyte Multilayers *................................................................. 25
2.1. Introduction .......................................................................................................................... 26 2.2. Materials and Methods ...................................................................................................... 30
2.2.1. Base membranes ......................................................................................................................... 30 2.2.2. Polyelectrolytes ........................................................................................................................... 30 2.2.3. Membrane modification by layer-by-layer adsorption technique ......................... 30 2.2.4. ATR-IR analysis ............................................................................................................................ 32 2.2.5. XPS analysis ................................................................................................................................... 32 2.2.6. Bacteria for membrane filtration experiments .............................................................. 33 2.2.7. Direct microscopic observation membrane filtration system ................................. 34 2.2.8. Direct microscopic observation of bacterial deposition and release .................... 36 2.2.9. Interaction force measurements .......................................................................................... 37
2.3. Results and Discussions .................................................................................................... 38 2.3.1. Characterization of E. coli cells and PSU membranes modified with PAH/PAA multilayers ..................................................................................................................................................... 38 2.3.2. XPS analysis of PEM-modified membranes ...................................................................... 44 2.3.3. Influence of PEM modification on bacterial deposition kinetics ............................. 46 2.3.4. Effect of PEM modification on reversibility of bacterial deposition ...................... 51 2.3.5. Proposed mechanism for anti-adhesive properties of PEM-modified membranes .................................................................................................................................................... 55 2.3.6. Influence of calcium on anti-adhesive properties of PEM-modified membranes 61
2.4. Conclusions ............................................................................................................................ 65 2.5. Acknowledgements ............................................................................................................. 67 2.6. References .............................................................................................................................. 67
Chapter 3. Imparting Antimicrobial and Anti-Adhesive Properties to Polysulfone Membranes through Modification with Silver Nanoparticles and Polyelectrolyte Multilayers * ............................................................................................. 71
3.1. Introduction .......................................................................................................................... 72
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3.2. Materials and Methods ...................................................................................................... 74 3.2.1. Base Membranes ......................................................................................................................... 74 3.2.2. Silver Nanoparticles and Polyelectrolyte Multilayers ................................................. 75 3.2.3. Membrane Modification with AgNPs and PEMs ............................................................. 76 3.2.4. Bacteria for the evaluation of antimicrobial and anti-adhesive properties of membranes .................................................................................................................................................... 78 3.2.5. Evaluation of antimicrobial properties of membranes ............................................... 78 3.2.6. Evaluation of antimicrobial properties of membranes ............................................... 80 3.2.7. Direct observation during three cycles of filtration and rinsing ............................. 82 3.2.8. Silver leaching test...................................................................................................................... 82
3.3. Results and Discussion ...................................................................................................... 83 3.3.1. Characterization of membranes modified with PEMs and AgNPs .......................... 83 3.3.2. Effect of AgNP/PEM-modification on membranes’ antimicrobial properties ... 87 3.3.3. Influence of AgNP/PEM-modification on kinetics and reversibility of bacterial deposition ....................................................................................................................................................... 90 3.3.4. Bacterial deposition and release over three cycles of filtration and rinsing...... 94
3.4. Conclusion .............................................................................................................................. 99 3.5. Acknowledgements ........................................................................................................... 100 3.6. References ............................................................................................................................ 100
Chapter 4. Polysulfone Membranes Modified with Bioinspired Polydopamine and Silver Nanoparticles Formed in situ to Mitigate Biofouling * ...................... 104
4.1. Introduction ........................................................................................................................ 105 4.2. Materials and Methods .................................................................................................... 106
4.2.1. Polysulfone membrane fabrication .................................................................................. 106 4.2.2. Membrane modification with polydopamine ............................................................... 107 4.2.3. In situ formation of AgNPs on polydopamine-modified membranes ................. 108 4.2.4. Membrane characterization ................................................................................................ 109 4.2.5. Anti-adhesive properties of membranes ....................................................................... 111 4.2.6. Antimicrobial properties of membranes ........................................................................ 113 4.2.7. Stability of AgNPs immobilized on membranes .......................................................... 113
4.3. Results and Discussion .................................................................................................... 114 4.3.1. AgNP Mass Loading Increases with Exposure Time to AgNO3 solutions .......... 114 4.3.2. Surface modifications enhance anti-adhesive properties ....................................... 119 4.3.3. In situ generated AgNPs inhibited bacterial growth on membranes.................. 121 4.3.4. Stability of AgNPs immobilized on membranes .......................................................... 123
4.4. Conclusion ............................................................................................................................ 124 4.5. Acknowledgments ............................................................................................................. 125 4.6. References ............................................................................................................................ 125
Chapter 5. Conclusions, Key Contributions, and Implications ............................ 129 5.1. Summary of Key Findings and Conclusions ............................................................. 130 5.2. Principle Contributions ................................................................................................... 134 5.3. Implications for Practice ................................................................................................ 137 5.4. Recommendations for Future Work ........................................................................... 138 5.5. References ............................................................................................................................ 140
Curriculum Vitae ................................................................................................................. 142
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LIST OF FIGURES
Figure 2.1 Schematic of the direct microscopic observation membrane filtration system.
........................................................................................................................................................... 35 Figure 2.2 ATR-IR data for the PSU base membrane. Above are the PSU structure and
plotted ATR-IR data from 3500 to 2500 cm-1 and 1700 to 700 cm-1. Below is the
table of assigned IR peaks. ........................................................................................................ 40 Figure 2.3 SEM images of (a) a PSU base membrane and (b) a PSU membrane modified
with 2 bilayers of PAH and PAA. ........................................................................................... 42 Figure 2.4 AFM images of (a) a PSU base membrane and (b) a PSU membrane modified
with 2 bilayers of PAH and PAA. ........................................................................................... 43 Figure 2.5 Evolution of the C(1s), N(1s), and S(2p) XP spectra for a PSU membrane
coated with increasing numbers of PAH/PAA bilayers. Below the XP spectra is an
illustration of the structure and chemical composition of each bilayer. ....................... 45 Figure 2.6 Images of deposited bacteria (0.446 mm × 0.333 mm) on (a) a base membrane
and (b) a PEM-modified membrane after the membrane was exposed to a bacterial
suspension prepared at 10 mM NaCl and pH 7.0 for 1 h in the absence of permeate
flux. (c) Number of bacteria deposited on the base and PEM-modified membranes
(per mm2). Error bars represent standard deviations. ....................................................... 47 Figure 2.7 Number of bacteria on (a) a PSU base membrane and (b) a PEM-modified
membrane during the deposition and release stages. The deposition experiment was
conducted at 10 mM NaCl and a permeate flow rate of 30 µm/s. The membrane was
subsequently rinsed with a 10 mM NaCl solution, followed by a 1 mM NaCl
solution, in the absence of permeate flow. For all the deposition and release stages,
the pH was maintained at 7.0. .................................................................................................. 50 Figure 2.8 Bacterial deposition rates, kobs, for base and PEM-modified membranes at 10
mM NaCl and 1 mM CaCl2 + 7 mM NaCl. The pH during the deposition process
was 7.0. The permeate flow rate was 30 µm/s. Error bars represent standard
deviations. ...................................................................................................................................... 51 Figure 2.9 Bacterial removal efficiencies for base and PEM-modified membranes after
deposition at (a) 10 mM NaCl and (b) 1 mM CaCl2 + 7 mM NaCl. The release
experiments were conducted in two stages. In Stage 1, the membranes were rinsed
with the same solutions that were used for bacterial deposition (either 10 mM NaCl
or 1 mM CaCl2 + 7 mM NaCl). In Stage 2, the membranes were rinsed with 1 mM
NaCl solutions. The pH for both release stages was 7.0. Error bars represent
standard deviations. ..................................................................................................................... 53 Figure 2.10 Representative approach interaction force curves between a CML colloid
probe and membrane surface at 10 mM NaCl and pH 7.0. Force curves are
presented in the form of (a) linear and (b) semi-log plots. ............................................... 57 Figure 2.11 Representative retract interaction force curves between a CML colloid probe
and membrane surface at (a) 10 mM NaCl and (b) 1 mM CaCl2 + 7 mM NaCl. The
pH was 7.0. Positive (blue) and negative (red) work of adhesion are presented in
(b). .................................................................................................................................................... 60 Figure 2.12 Work of adhesion distributions for (a) base membrane at 10 mM NaCl, (b)
PEM-modified membrane at 10 mM NaCl, (c) base membrane at 1 mM CaCl2 + 7
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mM NaCl, and (d) PEM-modified membrane at 1 mM CaCl2 + 7 mM NaCl. All
measurements were conducted at pH 7.0. Red and black bars represent repulsive
(negative) and attractive (positive) interactions between CML colloid probe and
membrane surface. ....................................................................................................................... 62 Figure 2.13 The PAH/PAA multilayer is highly hydrated and swollen in the absence of
calcium (top). In the presence of calcium, the PAH/PAA multilayer becomes less
hydrated and less swollen (bottom). The schematics are not drawn to scale and are
for illustrative purposes only. ................................................................................................... 65 Figure 3.1 Photographs of (a) the vacuum filtration setup and (b) a membrane coupon
placed on the agar plate. ............................................................................................................. 80 Figure 3.2 SEM images of (a) PSU base membrane, (b) Membrane P, (c) Membrane P20,
and (d) Membrane P43. .............................................................................................................. 85 Figure 3.3 Number of bacterial colonies (or CFUs) on Membrane P, Membrane P5,
Membrane P20, and Membrane P43. Error bars represent standard deviations. * No
colonies were present on Membrane P43. ............................................................................ 88 Figure 3.4 (a) Number of bacteria on Membrane P43 during the deposition and release
stages. The deposition experiment was conducted at 10 mM NaCl and a permeate
flow rate of 30 µm/s. The membrane was subsequently rinsed with a 10 mM NaCl
solution, followed by a 1 mM NaCl solution, in the absence of permeate flow. For
the deposition and release stages, the pH was maintained at 7.0. (b) Bacterial
deposition rates, kobs, for base membrane, Membrane P, Membrane P20, and
Membrane P43. (c) Bacterial removal efficiencies for base membrane, Membrane
P, Membrane P20, and Membrane P43 after deposition when rinsed with 10 mM
NaCl and 1 mM NaCl solutions. Error bars represent standard deviations. .............. 93 Figure 3.5 Number of bacteria on (a) base membrane and (b) Membranes P and P43 over
three-cycles of bacterial deposition and release. For each cycle, bacterial deposition
took place at 10 mM NaCl in the presence of a permeate flow rate of 30 µm/s. The
membrane was subsequently rinsed at 10 mM NaCl in the absence of permeate flow.
The pH was maintained at 7.0 over the three cycles of deposition and release. ........ 96 Figure 3.6 Number of bacteria on Membrane PM over three-cycles of bacterial deposition
and release. For each cycle, bacterial deposition took place at 10 mM NaCl in the
presence of a permeate flow rate of 30 µm/s. The membrane was subsequently
rinsed at 1 mM NaCl in the absence of permeate flow. The pH was maintained at
7.0 over the three cycles of deposition and release. ........................................................... 99 Figure 4.1 Chemical structure of dopamine………………………………………………...………...108
Figure 4.2 Schematic diagram of PDA modification and in situ formation of AgNPs on
the membrane surface and environmental SEM image of PDA-720 membrane.
White scale bar represents 5 μm……………………………………………….............................108
Figure 4.3 N(1s), S(2p), and Ag(3d) XP spectra of the surface of the base and modified
membranes……………………………………………………………………………………………..….115
Figure 4.4 BSE SEM images of base and modified membranes. Recording contrast and
brightness levels where held constant for all images in order to insure proper BSE
intensity comparisons between samples. White scale bars represent 2 μm…………117 Figure 4.5 BSE SEM imaging and EDX analysis of PDA-1440 membrane. (a) BSE SEM
image of PDA-1440 membrane with white circle indicating location for EDX
analysis (bright spot). (b) EDX spectrum of bright spot. (c) BSE SEM image of
x
PDA-1440 membrane with white circle indicating location for EDX analysis (dark
spot). (b) EDX spectrum of dark spot………………………………………………………........118
Figure 4.6 Mass loadings of AgNPs on modified membrane surfaces………………...…….119
Figure 4.7 Contact angle measurements of selected membranes………………………………120 Figure 4.8 Bacterial deposition rate coefficients, kobs, for selected membranes…………..121 Figure 4.9 CFUs on base and modified membranes. The symbols indicate that
no colony was present on the membranes. Error bars in b, c, and d represent standard
deviations…………………………………………………………………………………………………..123
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LIST OF TABLES
Table 3.1 Designations and modification conditions of PEM- and AgNP/PEM-modified
membranes. .................................................................................................................................... 83
1
Chapter 1. Introduction
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1.1. Water Resources in the World
Clean water shortage is a global problem that impacts human health and living
conditions and impedes social development.1 Freshwater constitutes 2.5 % of the Earth’s
water, saltwater in the seas and oceans constitutes 96.5 %, and the brackish or saline
water trapped in the subsurface estuaries or ground aquifer constitutes the remaining 1
%.2 Moreover, only 1.2 % of freshwater is surface water while 98.8 % of freshwater is
locked up in the ground or in ice. Thus, only a small fraction of freshwater is accessible
to humans.2 At the same time, the global population is still growing rapidly, from 7
billion in 2011 to a projected 9 billion in 2050.3 This population growth will inevitably
increase the water consumption dramatically due to human domestic use, food supply,
and industrial activities, such as hydrocarbon resource extraction, power generation, and
vehicle manufacturing, and thus further worsen the water crisis. Furthermore, the limited
freshwater resource is being constantly polluted by a variety of contaminants resulting
from human and industrial activities, such as heavy metals, disinfection byproducts,
persistent organic pollutants, and endocrine disrupters.1 The contaminated water is not
safe enough to drink and poses a huge threat to human health. In some poor and less
developed regions, the lack of adequate sanitation results in the outbreak of waterborne
diseases like diarrheal, mostly due to unclean water supplies.4
1.2. Membrane Filtration in Water and Wastewater Treatment
With the increasing pressure on clean water supply, the conventional water supply
from the surface water and ground water is not able to meet the increasing water demand.
As a result, efficient water purification techniques are required to exploit the
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unconventional water resources that have been inaccessible in the past. Currently,
membrane filtration technique has been emerging as a popular approach to overcome the
clean water supply crisis. The vast amount of seawater in the oceans, constituting 96.5 %
of Earth’s water, could potentially become a huge resource to supply drinking water.
Membrane distillation and reserve osmosis (RO) membrane filtration are two commonly
employed techniques to desalinate and purify seawater. Another potential
unconventional water supply resource is wastewater from human domestic and industrial
activities. Membrane filtration technique, including microfiltration (MF), ultrafiltration
(UF), nanofiltration (NF), and RO, is considered as one of the most efficient techniques
available for wastewater reuse and reclamation. The NEWater program in Singapore and
the Orange County Wastewater Purification System in California are two successful
examples of wastewater treatment plants that utilize the membrane filtration for
reclaiming the wastewater for industrial and/or potable use.5-6
MF and UF membranes are porous membranes that can be operated at relatively
low transmembrane pressures (TMPs) and, as a result, they are often referred to as low
pressure membranes (LPMs).7-9 Particulate matter in water, including suspended solids,
colloids, virus, and bacteria, can be effectively removed by LPMs through the size
exclusion mechanism. The size of particular matter removed by LPMs is between 10-3 to
10 μm.10 Currently, LPMs have been widely employed for wastewater treatment to meet
the discharge requirements for wastewater reuse for agricultural and industrial purposes.
In addition, LPMs have been intensively utilized in the pharmaceutical, food and
beverage, and electronics industries to provide high quality water that satisfies various
industrial applications.
4
Compared to conventional drinking water treatment techniques, LPM filtration
technique offers several advantages.7 First, LPM filtration provides an excellent barrier
for particulate matter and pathogens to ensure a safe water to drink. Second, compact
membrane filtration unit has a smaller footprint, which is a great advantage in regions
where land resource is limited. Third, the incremental addition of capital investments to
expand treatment capacity in the membrane plant is typically smaller than that in a
conventional treatment plant, which is especially attractive for fast-growing utilities to
meet the increasing demand for clean water. In addition, the product water quality of
membrane filtration is less sensitive to fluctuations of the raw water quality compared to
that in conventional water treatment processes.7
1.3. Membrane Biofouling
As LPM is continuously used to filter water and wastewater effluents,
microorganisms in the water will inevitably deposit on the membrane surface and form a
biofilm and “bio-foul” the membrane. Membrane biofouling can result in severely
compromised membrane permeability, deteriorated product water quality, increased
energy consumption, frequent backwashing, costly chemical cleaning, and shortened
membrane lifespan.11 The first step to form a biofilm is the transport of microorganisms
to the membrane surface through advection (resulting from the permeate flow), Brownian
motion, and/or chemotaxis.12-13 The next step is the attachment of microorganisms on the
membrane surface through electrostatic and/or hydrophobic interactions.14-15 The
microorganisms and associated biopolymers have a high propensity to interact with
polymeric membranes, since most of the polymers used to synthesize the membranes are
hydrophobic in nature.16-17 Finally, the deposited microorganisms will grow, proliferate,
5
and form a biofilm on the membrane surface due to the nutrient-rich conditions on the
membrane surface.18 Biofouling is considered as the most serious type of membrane
fouling (among organic, inorganic, and colloidal fouling) because even a small number of
bacteria deposited on the membrane surface can result in a dramatic growth of biofilm
and a considerably large decline in membrane filtration performance.19 Moreover,
microorganisms deposited on the membrane surface will secrete extracellular polymeric
substances (EPS) and thus the microorganisms embedded in a biofilm are well protected
by the matrix of EPS and are more resistant to biocides or chemical cleaning than
planktonic microorganisms.20-21 Consequently, it is extremely difficult to clean LPMs
that have been impacted by biofouling. In addition, the permeance of the biofouled
membrane cannot be restored to its original level even after cleaning. Therefore, an
effective membrane biofouling mitigation strategy is required for the sustainable
application of membrane filtration for water treatment.
1.4. Membrane Surface Modification for Biofouling Mitigation
Currently, membrane modifications through the enhancement of surface
hydrophilicity have been intensively investigated to mitigate biofouling.12, 22-24 The
membrane surface can be tailored to become more hydrophilic. Hydrophilic membranes
have a strong ability to bind a thin film of water on the membrane surface to form a
hydration layer that can reduce bacterial adhesion.25-27 To date, various techniques for
membrane surface modifications have been developed to enhance surface hydrophilicity.
These techniques include incorporating or blending of hydrophilic polymers into the
membrane matrix,27-31 plasma treatment of the membrane surface,32-39 and grafting or
coating of highly hydrophilic materials on the membrane surface.32, 40-46 The
6
incorporation or blending of hydrophilic polymers has been shown to be very effective in
enhancing the membrane hydrophilicity. However, this method requires the dissolving of
two polymers with significantly different hydrophilicity in the same solvent and thus its
application is limited by the miscibility of the two polymers.47-48 The major drawback of
plasma treatment is that the hydrophilic character of the modified surface can gradually
change with time and under high temperature. This phenomenon is often referred to as
“hydrophobic recovery.”17, 49-50 Because of this drawback, plasma treatment is not an
ideal technique for membrane surface modification. Therefore, the grafting or coating of
highly hydrophilic materials on membranes has been the preferred approach for the
modification of membrane surface.
By grafting or coating of highly hydrophilic materials on the membrane surface, a
polymeric thin film assembled on top of the membrane can impart the membrane with
strong anti-fouling properties.32, 40-45, 51-54 The grafting technique includes plasma-
induced, photo-initiated, and chemical grafting of hydrophilic materials.32, 40-42, 55-57 The
coating technique includes coating with hydrophilic polymers51-52, 58-60 or assembling
polyelectrolyte multilayers (PEMs),44, 54, 61 through both noncovalent interactions (i.e.,
electrostatic and van der Waals interactions)44, 51-52, 54 and covalent interactions.58-60
1.5. Polyelectrolyte Multilayers
PEMs are films that are composed of multilayers of positively and negatively
charged polyelectrolytes assembled via a layer-by-layer (LbL) adsorption technique.
PEMs were first introduced by Decher in the early 1990s.62-64 Specifically, the LbL
adsorption process involves exposing a substratum, e.g., a membrane, to oppositely
charged polyelectrolyte solutions in a sequential manner, which allows for the
7
electrostatic-driven adsorption of polyelectrolyte films on the substratum through the
overcompensation of surface charge with the adsorption of each polyelectrolyte layer.65
The PEM-modified membrane surface properties, such as hydrophobicity, can be tuned
by adjusting the constituent polyelectrolytes, the number of layers, the sequence of
polyelectrolytes assembled into the films, and the buffer solutions that are used to prepare
the polyelectrolyte solutions (e.g., pH and ionic strength).44, 53, 66-68
Surface modification with PEMs has shown to enhance the surface’s resistance to
the adhesion of mammalian and microbial cells.53, 61, 66-70 For example, Mendelsohn at
al.68 assembled highly hydrated PEM films comprised of poly(allylamine hydrochloride)
(PAH) and poly(acrylic acid) (PAA) on a polystyrene substratum. The modified surface
demonstrated remarkable resistant to the adhesion of an extremely adhesive murine
fibroblast cell line. The authors attributed the anti-adhesive properties of PEMs to the
weakly ionically stitched structure, which can swell substantially under neutral pH
conditions to form a highly hydrated layer. Lichter et al.66 attributed the significant
resistance of PAH/PAA PEMs to bacterial adhesion to their mechanoselective ability,
that is, bacteria prefer to adhere onto the materials exhibiting higher mechanical stiffness.
To date, only a few studies have reported the use of PEM modification to impart
bacterial anti-adhesion properties to polymeric membranes for water treatment. For
instance, Diagne et al.53 assembled 1.5 bilayers of poly(styrene sulfonate) (PSS) and
poly(diallyldimethylammonium chloride) (PDADMA) on the polyethersulfone (PES)
membrane and observed that the PSS/PDADMA multilayer-modified membrane was
more resistant to fouling by humic acid and Escherichia coli cells compared to the
unmodified membrane. The authors attributed the anti-fouling properties of the PEM-
8
modified membrane to the enhancement of surface charge and hydrophilicity of the
modified membranes. Rahaman et al.61 modified the thin-film composite (TFC)
polyamide RO membranes with 10 bilayers of polyethylene amine (PEI) and PAA and
further functionalized the membrane by grafting of hydrophilic poly(sulfobetaine). The
modified membranes exhibited a considerably reduction in E. coli cell adhesion relative
to the unmodified membrane because of the increased hydrophilicity.
1.6. Polydopamine Films
Polydopamine (PDA) is a bioinspired material with a chemical structure similar to
3,4-dihydroxl-L-phenylalanine, a chemical secreted by mussels that helps mussels form
the strong bindings to diverse substrates.71-72 PDA has been used as a novel and universal
coating material for surface modifications since 2007, because PDA is able to coat
virtually all kinds of surfaces.73 PDA can be formed by oxidization and self-
polymerization of dopamine monomer under the alkaline condition with oxygen as the
oxidant.74 However, the molecular mechanism behind the formation as well as the exact
structure of PDA is still under scientific debate.74-75 The mechanism for the formation of
PDA was once believed to be the covalent polymerization of the aryl rings of
monomers.76-77 Specifically, it was proposed that the dopamine is first oxidized to
dopamine-quinone followed by intramolecular cyclization via 1,4 Michael-type addition.
The products can be further oxidized, arranged, branched, and polymerized to the cross-
linked PDA.78 However, there is no sound experimental evidence to prove this pathway.
Recently, several new pathways have been proposed based on the solid-spectroscopic and
crystallographic techniques, as well as the high performance liquid chromatography
coupled with mass spectrometry analysis.79-80 These new studies proposed the
9
mechanisms for PDA formation to involve either the aggregation of monomers through
noncovalent forces, such as hydrogen bindings, charge transfer, and π-stacking, or the
combination of self-assembly and covalent polymerization of dopamine monomers.
Recently, PDA has been used to enhance the hydrophilicity of membrane
surfaces.58-60, 81-82 The PDA film can impart hydrophilic properties to the membrane
surface due to the presence of functional groups that have a high affinity for water
molecules in PDA, such as catechol and quinone.58, 82-83 Recent studies have provided
evidence that the membrane surface modification by PDA film can enhance a
membrane’s anti-fouling properties by increasing the surface hydrophilicity.58-60, 81-82
McCloskey et al.58 modified two PSU UF membranes, a poly(vinylidene fluoride)
(PVDF) MF membrane, and a polyamide (PA) RO membrane with PDA and all the
modified membranes exhibited a systematic reduction in protein (i.e., bovine serum
albumin (BSA)) adhesion relative to the unmodified membrane. In a following study,
McCloskey et al.59 coated various membrane surfaces with PDA, including
polypropylene MF, poly(tetrafluoroethylene) MF, PVDF MF, poly(arylene ether sulfone)
UF, PSU UF, polyamide (PA) NF, and PA RO membranes, and found that the modified
membranes showed highly improved fouling resistance during oil/water filtration due to
the increased surface hydrophilicity. The normalized decline in the permeate flux of the
modified membranes was highly reduced compared to that of the unmodified membrane.
Miller et al.60 demonstrated in their study that the PDA-modified PSU UF membrane
showed a considerably reduced adhesion of BSA and Pseudomonas aeruginosa cells in
their static adhesion tests since the PDA increased membrane hydrophilicity.
10
In addition to the enhancement in surface hydrophilicity, PDA film on membranes
can serve as a platform that facilitates various chemical reactions since PDA possesses
strong grafting and reducing abilities due to its catechol, amine, imine, and quinone
functional groups. Therefore, after coating the surface with PDA, the surface serves as a
new platform for the secondary reactions with a variety of materials (e.g., polyethylene
glycol (PEG), graphite oxide, and transition metal ions).60, 84-86 For example, Ag+ ions
can be reduced by the catechol groups in PDA to form AgNPs.86 Therefore, PDA coating
can impart diverse hybrid properties to various surfaces and thus has been widely applied
in the fields of energy, catalysis, biomedical engineering, water treatment, and sensing.83,
87-92
1.7. Silver Nanoparticles As An Antimicrobial Agent
Bacterial deposition on the membrane surface is inevitable due to the ubiquitous
permeation drag force caused by the continuous convective permeation during the
membrane filtration.12, 28 Those bacteria deposited on the membrane surface can grow,
proliferate, and form a biofilm.20, 93 Therefore, it is highly desirable to impart the
membrane with strong antimicrobial properties to inactivate the deposited bacteria and
prevent the formation of biofilms. With the fast development of nanomaterials in recent
years, the application of the antimicrobial nanomaterials in the production of the
antimicrobial membrane has been gaining popularity.54, 94-100 Silver nanoparticles
(AgNPs) are among the most popularly used antimicrobial agents.101 The exact
mechanisms of antimicrobial properties of AgNPs are still under scientific debate. To
date, three mechanisms have been proposed: (1) Ag+ ions that are released from
dissolving AgNPs can be uptaken by microorganisms, which may damage the cell
11
membranes and disrupt ATP production and DNA replication;102-105 (2) reactive oxygen
species that are generated by AgNPs can trigger oxidative stress, which may interfere
with the metabolic pathways and the reproduction of microorganisms;106-109 and (3) direct
damage of cell membranes by AgNPs.107, 110-112 Among these mechanisms, the first
AgNP antimicrobial mechanism is the most commonly accepted one.102-105
A widely used method to incorporate AgNPs into polymeric membranes is to blend
the AgNPs into the polymer solution and then cast the nanocomposite membrane through
the wet phase-inversion method.100, 113 Experimental results from these studies confirmed
that the membrane’s resistance to biofouling was highly enhanced after the incorporation
of antimicrobial AgNPs into the membrane matrix. Importantly, the membrane’s
resistance to biofouling was observed to be higher when the AgNPs were located near the
feed solution side of the membrane.100, 113 Several studies have provided evidence that
positioning AgNPs close to the membrane’s feed side enables the direct contact or the
close proximity between AgNPs and bacterial cells and thus can greatly enhance the
antimicrobial effects of the AgNPs.98-99, 114-117 However, it is difficult to control the
spatial distribution of AgNPs within the membrane matrix through the wet phase-
inversion method.
An alternative method is to immobilize AgNPs on the membrane surface with the
use of PEMs. This approach will enhance the opportunities for direct contact or close
proximity between the AgNPs and bacterial cells that are deposited on the membrane
surface. To date, several studies have reported that the membranes’ antimicrobial
properties were considerably enhanced when the AgNPs were immobilized on the
membrane surfaces with PEMs.53, 61, 94, 118-119 For example, Diagne at el.53 incorporated
12
AgNPs into the top PSS layer of the 1.5 bilayer PSS/PDADMA assembly on the PES
membrane and their experimental results showed that the PEM-modified membrane
containing AgNPs exhibited stronger antimicrobial activities to E. coli cells compared to
the PEM-modified membrane without AgNPs. Liu et al.94 synthesized NF and FO
membranes by assembling 2.5 bilayers of PAH and PSS on the polyacrylonitrile (PAN)
membranes with AgNPs dispersed into either polyelectrolyte solutions or rinsing
solutions. The incorporated AgNPs enhanced the membranes’ antibacterial properties
against both Gram-positive Bacillus subtills and Gram-negative E. coli cells compared to
that of the base PAN membrane. Karkhaneichi et al.118 modified RO membranes with 3
bilayers of PAH and PSS with AgNPs embedded in the PEMs and covered the PEMs
with an amphiphilic 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer with
2-aminoethyl methacrylate (AEMA) as a polyzwitterion top layer. This modified
membrane exhibited an increased killing ratio of both Pseudomonas putida and E. coli
cells and thus showed substantially improved antibacterial properties relative to the base
membrane. Rahaman et al.61 coated polyamide RO membranes with PEMs that
comprised of PEI-AgNPs and PAA and the modified membranes exhibited the E. coli
inactivation efficiency of over 95%.
Another emerging technique to immobilize AgNPs on membranes is to generate
AgNPs in situ on the membrane surface. Two recent studies have shown that AgNPs
formed in situ could impart the membrane with significantly enhanced antimicrobial
properties.96-97 For example, Ben-Sasson et al.96 presented a facile method to generate
and immobilize AgNPs on the thin film composite RO membrane surfaces. AgNPs were
formed in situ by exposing RO membrane’s active surface in AgNO3 solution for 10 min
13
and then contacting with NaBH4 solution for 5 min. This modification resulted in more
than 75 % reduction in the number of live bacteria (i.e., E. coli BW 26437, P. aeruginosa
ATCC 27853, and Staphylococcus aureus 8325) attached to the membrane surface.
Moreover, confocal microscopy analysis revealed that the modification dramatically
suppressed biofilm formation, with 41 % reduction in total biovolume and significant
reduction in EPS, as well as dead and live bacteria, on the membrane. In the study of
Cao et al.,97 Ag+ ions were adsorbed on the surface sulfonic groups of the sulfonated PES
membrane through metal coordination and AgNPs were generated on the membranes
through the reduction of Ag+ ions using vitamin C. The modified membranes showed
strong antimicrobial properties compared to the unmodified ones: one order of magnitude
reduction in the bacterial colony growth (i.e., E. coli, S. aureus, and Staphylococcus
albus) was observed.
1.8. Objective and Scope of Dissertation
The overall goals of this dissertation work were to investigate the use of thin films
and antimicrobial AgNPs for the modification of membrane surface to enhance the
bacterial anti-adhesive and antimicrobial properties of membranes. Specifically, this
dissertation work will investigate the effectiveness of two emerging nanocomposite thin
films –– PEMs and PDA –– that contain AgNPs. The membranes’ anti-biofouling
properties will be examined through the quantitative assessment of their bacterial anti-
adhesive properties and antimicrobial properties. PEMs that were composed of two
bilayers of PAH and PAA were assembled on top of a commercial PSU membrane
surface. PDA film was coated on a laboratory-cast PSU membrane surface by circulating
the PDA solution under alkaline condition. AgNPs were either synthesized ex situ
14
through the reduction of Tollens’ reagent or generated in situ on the membrane surface by
the strong reducing catechol groups in PDA. The specific objectives were to:
investigate the influence of PAH/PAA PEM modification on the bacterial anti-
adhesive properties of a PSU MF membrane;
study the mechanism of the bacterial anti-adhesive properties of PAH/PAA
PEMs;
investigate the influence of AgNPs and the PAH/PAA PEM modifications on the
bacterial anti-adhesive and antimicrobial properties of a PSU MF membrane;
investigate the influence of PDA films and in situ formation of AgNPs on the
bacterial anti-adhesive and antimicrobial properties of a PSU MF membrane.
1.9. Dissertation Organization
Chapter 2 focuses on the investigation on the influence of PAH/PAA PEMs on the
bacterial anti-adhesive properties of a commercial PSU MF membrane. Two bilayers of
PAH and PAA were assembled on the surface of a commercial PSU membrane using the
LbL adsorption technique with the employment of a custom-built cross flow cell. X-ray
photoelectron spectroscopy (XPS) analysis of 1–8 of bilayers of PAH/PAA PEMs
assembled on the PSU membrane was performed to confirm the successful assembly of
the PAH/PAA PEMs. Using a direct microscopic observation membrane filtration
(DMOMF) system, the deposition kinetics and removal efficiencies of E. coli cells on the
membrane surface were determined at 10 mM NaCl and pH 7.0. In addition, the E. coli
deposition kinetics and removal efficiencies were examined at 1 mM CaCl2 (+ 7 mM
NaCl) and pH 7.0 to investigate the influence of Ca2+ ions on the membrane’s bacterial
anti-adhesive properties. Furthermore, atomic force microscopy was used to conduct the
15
interaction measurements to elucidate the mechanism for the enhanced bacterial anti-
adhesive properties of the PEM-modified membranes.
Chapter 3 presents the results from the investigation on the influence of AgNPs and
2 bilayers of PAH/PAA PEMs on the bacterial anti-adhesive and antimicrobial properties
of commercial PSU MF membranes. The AgNPs were synthesized ex situ through the
reduction of Tollens’ reagents by glucose in an ultrasonication bath and then coated with
citrate as a capping agent to prevent AgNP aggregation. The AgNPs were deposited on
the membrane surface by filtering a diluted AgNP solution through the membrane with a
flow cell in the dead-end mode. PEMs were assembled on top of the AgNPs using the
LbL adsorption technique, similar to that employed in Chapter 2. The anti-adhesive
properties of the membrane were examined using the DMOMF system with a similar
procedure described in Chapter 2. The antimicrobial properties of the modified
membranes were evaluated using a colony forming unit (CFU) enumeration method. A
solution (10 mM NaCl and pH 7.0) was circulated through the DMOMF system for 1 h
and the total Ag concentration in the solution was measured using inductively coupled
plasma mass spectrometry (ICP-MS) to determine the degree of Ag leaching.
In Chapter 4, a PSU membrane was coated with a thin layer of PDA and the AgNPs
formed in situ on the membrane surface to enhance the membrane’s bacterial anti-
adhesive properties and impart antimicrobial properties to the membrane. The PSU
membrane was cast through the phase inversion process. The PSU MF membrane
surface was modified with PDA by circulating a PDA solution across the membrane
surface for 6 h. AgNPs were formed in situ on the membrane surface by exposing the
membrane to a 50 mM AgNO3 solution (pH unadjusted). The membrane soaking time in
16
AgNO3 solutions was varied (1 min, 1 h, 2 h, 12 h, and 24 h) to examine the influence of
the soaking time on the AgNP mass loading and the membrane’s bacterial anti-adhesive
and antimicrobial properties. An optical contact angle meter was used to measure the
contact angle of DI water droplets on the membrane surface to evaluate the membrane
hydrophilicity after modification. XPS analysis was performed to investigate the surface
elemental composition to confirm the formation of PDA film and AgNPs. Scanning
electron microscopy (SEM) imaging and energy-dispersive X-ray (EDX) analysis was
performed to confirm the formation and observe the distribution of AgNPs on the
membrane surface. The anti-adhesive properties of the membranes were examined by
measuring the deposition kinetics of E. coli cells on the membranes during filtration. The
membranes’ antimicrobial properties were evaluated using the CFU enumeration method,
similar to that described in Chapter 3. In addition, the antimicrobial test was carried out
on selected modified membrane coupons after prolonged DI water filtration to evaluate
the stability of AgNPs formed on the membrane surface. The Ag concentrations in the
permeate were quantified by ICP-MS to assess the degree of Ag leaching.
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25
Chapter 2. Bacterial Anti-Adhesive Properties of
Polysulfone Membranes Modified with
Polyelectrolyte Multilayers *
* All the sections in this chapter have been published as: Tang, L., Gu, W.Y., Yi, P.,
Bitter, J.L., Hong, J.Y., Fairbrother, D.H. and Chen, K.L., Bacterial Anti-adhesive
Properties of Polysulfone Membranes Modified with Polyelectrolyte Multilayers, Journal
of Membrane Science, 2013, 446, 201-211. Co-author Wenyu Gu provided assistance
during the membrane modification process and helped with AFM data analysis. Co-
author Peng Yi helped with the training of AFM force measurement and helped with the
discussions of AFM data analysis. Co-author Julie Bitter helped perform the membrane
characterization and data analysis with XPS and ATR-IR. Co-author Ji Yeon Hong
helped with the membrane modification for XPS analysis. Co-author Howard Fairbrother
helped with the discussions for the membrane characterization with XPS and ATR-IR.
Co-author Kai Loon Chen helped with experimental data interpretation and manuscript
editing.
26
2.1. Introduction
Low pressure membranes (LPMs) are porous membranes that can be used at
relatively low transmembrane pressures (less than 200 kPa). Microfiltration (MF) and
ultrafiltration (UF) membranes are examples of LPMs, and MF and UF membranes have
pore sizes of ca. 0.1–1.0 µm and 0.01–0.10 µm, respectively. LPM processes have
gained popularity in drinking water treatment and wastewater reuse because of their small
footprint, relatively low costs, and effectiveness in removing pathogenic microorganisms
and particulate matter.1
One of the key challenges of LPM processes is biofouling, or the formation of a
biofilm on the membrane surface. As LPM filtration is continuously employed to filter
water and wastewater effluents, planktonic bacteria in the bulk suspension may be
transported to the membrane by the convective permeate flow, and some of the bacteria
may deposit on the membrane surface. The deposited bacteria then produce extracellular
polymeric substances (EPS) on the membrane surface and proliferate to form
microcolonies which will grow and coalesce into a biofilm. The formation of a biofilm
on a LPM surface will result in higher operating pressures, poorer product water quality,
frequent chemical cleaning, and shortened membrane life.2
Among the different types of membrane fouling for LPM processes (colloidal,
organic, and biological), biofouling is arguably the most serious because even a small
amount of biofilm growth results in a significant loss in clean water flux.3 Moreover,
biofilms tend to be very resistant to biocides because the microorganisms are protected
by the matrix of EPS. Thus, it is extremely difficult to clean membranes that have been
fouled by biofilms.4 Currently, efforts to retard biofouling have centered on the use of
27
disinfectants (e.g., chlorine). However, disinfectants are not always successful in
controlling biofouling since it is impossible to inactivate all microorganisms in the
influent waters and only a small number of microorganisms are required to form a
biofilm.2, 4 Furthermore, the prolonged exposure of membranes to disinfectants can
damage membrane structures which will result in the decline in the membranes’ ability to
reject contaminants.5-6 The use of disinfectants can also lead to the generation of
potentially carcinogenic disinfection byproducts.7
The initial bacterial deposition and adhesion play a critical role in the
development of biofilms on membrane surfaces.8 While the initial transport of
planktonic bacteria to a membrane surface is mainly controlled by hydrodynamic factors
(such as cross-flow velocity and permeate flow rate), the initial adhesion of bacteria on
the membrane surface is governed by both hydrodynamic factors and interfacial
interactions between the bacteria and membrane. It is generally understood that bacteria–
membrane interfacial interactions can be comprised of electric double layer, van der
Waals, steric (or electrosteric), and hydrophobic interactions. Past studies have
demonstrated that membranes that are highly negatively charged or hydrophilic tend to
exhibit strong bacterial anti-adhesive properties.2, 9-10
Several techniques have been explored, in recent years, to modify membrane
surfaces with the goal of developing anti-biofouling membranes. One of the emerging
techniques is the modification of membranes with polyelectrolyte multilayers (PEMs).
PEMs can be assembled on a substratum through the layer-by-layer (LbL) adsorption
technique.11-13 This technique involves exposing the substratum, e.g., a membrane, to
oppositely charged polyelectrolytes in a sequential manner, thus resulting in the
28
electrostatic-driven adsorption of polyelectrolyte films on the substratum through the
overcompensation of surface charge with the adsorption of each polyelectrolyte layer.14
The main advantage of PEM modification is that it allows for the convenient construction
of surface coatings with nanoscale control over the film thickness, composition, and
surface chemistry.15 In addition, PEM modification has been used to inhibit the
attachment of cells on material surfaces.15-16 For instance, Mendelsohn et al.16 assembled
highly hydrated PEM films comprised of poly(allylamine hydrochloride) (PAH) and
poly(acrylic acid) (PAA) on polystyrene substrata that were remarkably resistant to the
adhesion of an extremely adhesive murine fibroblast cell line.
While the modification of membranes with PEMs has been found to enhance the
selectivity of ion rejection,17-21 only a few studies, to date, have reported the application
of PEM modification to enhance membranes’ resistance to fouling. Shan et al.22 reported
the reduction in fouling by silica colloids when polyethersulfone (PES) membranes were
modified with PEMs comprised of PAH and poly(styrene sulfonate) (PSS). Wang et al.20
demonstrated in their study that the modification of polyacrylonitrile (PAN) membranes
with PEMs comprising fewer than 5 bilayers of sulfonated poly(ether ether ketone) and
branched polyethyleneimine can retard organic fouling by bovine serum albumin, sodium
alginate, and humic acid due to the hydrophilic nature of the PEMs. Diagne et al.23
assembled 1.5 bilayers of PSS and poly(diallyldimethylammonium chloride) (PDADMA)
on PES membranes, and they observed that the PSS/PDADMA multilayer-modified
membranes were more resistant to fouling by humic acid, as well as the adhesion of
Escherichia coli cells, compared to unmodified membranes. The authors attributed the
anti-fouling properties of the PEM-modified membranes to the enhancement of surface
29
charge and hydrophilicity of the modified membranes. Qi et al.24 showed that the
assembly of 3 PAH/PSS bilayers on the feed-solution side of a PAN forward osmosis
membrane can enhance its resistance to fouling by dextran and alginate. In addition,
PEMs have been used to incorporate biocidal agents, e.g., silver nanoparticles, on the
membrane surfaces to inactivate deposited bacteria.23, 25
The objective of this research is to examine the bacterial anti-adhesive properties
of polysulfone (PSU) MF membranes that are modified with PEMs comprised of PAH
and PAA. In this study, PSU base membranes are modified with PAH/PAA PEMs using
the LbL adsorption technique with the employment of a flow cell. A direct microscopic
observation membrane filtration system is used to observe the deposition of fluorescent
E. coli cells on PEM-modified membranes under a constant permeate flux and a constant
cross-flow velocity in the absence and presence of calcium cations (10 mM NaCl and 1
mM CaCl2 + 7 mM NaCl, respectively). After the deposition stage of each filtration
experiment, the reversibility of bacterial deposition on the PEM-modified membrane is
evaluated by first rinsing the membrane with the same solution that is used for bacterial
deposition, followed by a low-ionic strength solution (1 mM NaCl), in the absence of
permeate flux. The deposition kinetics and reversibility of bacterial deposition for the
PEM-modified membranes are then compared to the values for the PSU base membranes
in order to assess the anti-adhesive properties of the modified membranes. Our results
show that the modification of PSU membranes with PAH/PAA PEMs can reduce the
bacterial deposition kinetics and significantly enhance the reversibility of bacterial
deposition both in the absence and presence of calcium. Interaction forces between a
carboxylate modified latex (CML) colloid probe and the membrane surfaces are
30
measured through atomic force microscopy (AFM) to elucidate the mechanisms for the
bacterial anti-adhesive properties of the PEM-modified membranes.
2.2. Materials and Methods
2.2.1. Base membranes
In this study, PSU membranes (Pall Corporation, Ann Arbor, MI) were used as
the base membranes on which PEMs were assembled. The MF membranes are
asymmetric in structure and have a nominal pore size of 0.2 μm on the active side. The
membranes were received as flat sheets and cut into smaller coupons. The membrane
coupons were then rinsed and soaked in deionized (DI) water (Millipore, Billerica, MA)
for at least three days at 4 °C before use.
2.2.2. Polyelectrolytes
PAH (Mw = 15,000) and PAA (Mw = 50,000) were purchased from Sigma-Aldrich
(St. Louis, MO) and Polysciences, Inc. (Warrington, PA), respectively. Both
polyelectrolytes were used as received without any further purification. The PAH and
PAA solutions that were used to modify the membrane surfaces were prepared by
dissolving the polyelectrolytes in DI water and were used within eight days after
preparation. The concentration of both polyelectrolyte solutions was 20 mM (based on
the repeat unit molecular weight). The ionic strength of both polyelectrolyte solutions
were adjusted to 150 mM with NaCl, and the pH was adjusted to 3.0 with either 1 M HCl
or 1 M NaOH.
2.2.3. Membrane modification by layer-by-layer adsorption technique
PSU membranes were modified with PAH/PAA multilayers through the LbL
adsorption technique using an approach similar to that of Mendelsohn et al.16 A custom-
31
made flow cell was used for membrane modification. The flow cell comprised two
polycarbonate plates, and the cross-flow channel was 89.0 mm in length, 45.0 mm in
width, and 2.5 mm in height. The PSU membrane to be modified was held tightly
between the top and bottom plates with double O-rings, with the active side facing the top
plate, to provide a leak-proof seal. To assemble a single bilayer of PAH and PAA on the
PSU membrane surface, the active side of the PSU membrane was first rinsed with a
PAH solution for 12 min using a gear pump (Cole-Parmer, Vernon Hills, IL). After that,
it was flushed with a 150 mM NaCl and pH 3.0 rinse solution (with no polyelectrolytes)
for 12 min to flush away the excess or loosely bound polyelectrolytes from the membrane
surface. The adsorption of PAA on the membrane was then achieved by rinsing the
membrane with a PAA solution before being flushed with a rinse solution. This process
is then repeated n – 1 times in order to achieve a total of n bilayers on the membrane.
The cross-flow velocities used for polyelectrolyte adsorption and for flushing with a rinse
solution were 0.75 mm/s and 2.25 mm/s, respectively. 1–8 PAH/PAA bilayers were
assembled for X-ray photoelectron spectroscopy (XPS) analysis, while 2 bilayers were
assembled for the bacterial deposition and release experiments.
Surface morphologies of the base and PEM-modified membranes were acquired
using scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR). The
membranes for SEM analysis were vacuum-dried in a desiccator overnight and examined
under the low-vacuum mode. The vacuum-dried membranes were also imaged using an
atomic force microscope (AFM, Multimode NanoScope IIId, Bruker Nano Inc.) in the
tapping mode to obtain the surface topology and surface roughness. The imaging was
conducted using a silicon cantilever (Bruker, Camarillo, CA) at a scan rate of 0.5 Hz.
32
The root mean square (RMS) roughness of the membranes was determined for a scan size
of 5 μm by 5 μm of the sample.
The average hydraulic resistances of the base and PEM-modified membranes
were determined by using a laboratory-built dead-end membrane filtration set-up to
measure the permeate fluxes of DI water over a range of transmembrane pressures
(TMPs) (up to ca. 160 kPa).
2.2.4. ATR-IR analysis
The PSU membrane used in this study was characterized as-received using ATR-
IR. Infrared spectra on PSU membranes were acquired using a Mattson Infinity Series
FTIR spectrometer with a mercury cadmium telluride detector (4 cm-1 resolution) and an
ATR device (Pike Technologies MIRacle) equipped with a diamond crystal in single
reflection mode. ATR spectra represent an average of 500 scans.
2.2.5. XPS analysis
X-ray photoelectron spectroscopy (XPS) was conducted on PSU membrane and
membranes that were sequentially rinsed with PAH and PAA solutions, as described in
Section 2.3, to confirm the formation of PEMs on the membrane surfaces. The XPS
analysis was performed with a PHI 5400 XPS system (base pressure < 5 × 10–8 Torr)
using Mg Kα X-rays (1253.6 eV). Samples were prepared by pressing a cut out of each
membrane onto double sided copper tape (1 × 1 cm2) so that no copper was visible.
Photoelectrons ejected from each sample were measured with a precision high energy
electron analyzer operating at constant pass-energy. Survey scans performed to
determine elemental composition were completed using a pass-energy of 178.95 eV at a
scan rate of 0.250 eV/step. The regions containing the desired elements of interest were
33
analyzed at a scan rate of 0.125 eV/step using two different pass-energies: 89.45 eV for
quantification purposes, and 22.36 eV to determine lineshapes and peak positions. XP
spectra were processed with commercially available software (CasaXPS), and atomic
concentrations were quantified by integration of the relevant photoelectron peaks.
2.2.6. Bacteria for membrane filtration experiments
E. coli K12 MG1655 was used as the model bacteria in this study.26 The bacteria
carry the antibiotic resistance gene and are labeled with the green fluorescent protein
which allows them to be observed under an epifluorescence microscope. The E. coli cells
were incubated in a Luria Bertani broth (25 g/L, Fisher Scientific) that contained 50 mg/L
kanamycin (Aldrich Chemical) at 37 °C for ca. 3 hours to allow the cells to reach the
exponential growth phase. The cells were then harvested through centrifugation at
4,200g for 10 min at 4 °C (Avanti centrifuge J-20 XPI, Beckman Coulter, Brea, CA),
decantation of the supernatant, and re-suspension of the cell pellet in a 154 mM NaCl and
pH 7.0 (buffered with 0.15 mM NaHCO3) solution. The suspension was then centrifuged
at 4,200g for 5 min, the supernatant was decanted, and the cell pellet was re-suspended in
a 154 mM NaCl and pH 7.0 solution. This washing process was repeated once, but after
the centrifugation step, the cell pellet was re-suspended in a 10 mM NaCl and pH 7.0
solution. The cell suspension was briefly vortexed before it was used to prepare the
suspension for a bacterial deposition and release experiment. For all the bacterial
deposition and release experiments, the cell concentration in the feed suspension was ca.
1.4 × 107 cells/L.
A ZetaPALS analyzer (Brookhaven, Holtsville, NY) was used to measure the
electrophoretic mobility of E. coli cells at 25 °C and the zeta (ζ) potentials of the cells
34
were calculated using the Smoluchowski equation.27 Three cell samples were used for
each solution chemistry, and 10 measurements were performed for each sample.
2.2.7. Direct microscopic observation membrane filtration system
The direct microscopic observation membrane filtration system used in this study
was similar to the systems used in other studies.2, 10, 26 The closed-loop filtration system
was operated under the cross-flow mode. It comprised four main components: (1) cross-
flow membrane filtration (CMF) cell, (2) pressure vessel, (3) pumping and tubing system,
and (4) epifluorescence microscope and digital camera. A schematic of the membrane
filtration system is presented in Figure 2.1. The CMF cell comprised two polycarbonate
plates. A 3-mm thick glass window was inserted into the top plate to allow for the direct
microscopic observation of bacterial deposition on and release from the membrane
surface. The cross-flow channel inside the CMF cell was 76.0 mm in length, 25.0 mm in
width, and 1.0 mm in height. The membrane to be tested was held tightly between the
top and bottom plates, with the active side facing the top plate, with double O-rings to
provide a leak-proof seal. A permeate spacer (McMaster-Carr, Aurora, OH) was placed
below the membrane in a shallow insert of the bottom plate.
35
Figure 2.1 Schematic of the direct microscopic observation membrane filtration system.
The feed bacterial suspension (volume of 2 L) was contained in a stainless steel
pressure vessel (Alloy Products, Waukesha, WI) that was pressurized to ca. 170 kPa. A
gear pump (Cole-Parmer, Vernon Hills, IL) was used to circulate the feed suspension
through the CMF unit at a cross-flow velocity of 10 cm/s (Re = 96.2, shear rate = 600.0 s–
1). The flow rate of the feed suspension entering the CMF unit was kept constant at 0.15
L/min, and it was monitored using a rotameter mounted on the feed line (Blue-White,
Huntington Beach, CA). The permeate flux was maintained constant at 30 μm/s during
the deposition experiment using an 8-roller digital peristaltic pump (Cole-Parmer, Vernon
Hills, IL) mounted on the permeate line. The permeate was circulated back into the
pressure vessel, and the permeate flow rate was monitored using a digital flow meter
(Cole-Parmer, Vernon Hills, IL).
The CMF unit was placed on the stage of an epifluorescence microscope (Nikon
Eclipse E600W, Japan) that was equipped with a 10× objective lens. During the filtration
experiment, digital images of E. coli cells on the membrane surface were acquired with a
4
5
3
FEED
1
PERMEATE
RETENTATE
2
6
7
8
1 : Pressure Vessel
2 : Gear Pump
3 : Rotameter
4 : CMF Cell
5 : Digital Flowmeter
6 : Peristaltic Pump
7 : Microscope and Camera
8 : Computer
36
0NA
kk
m
obs
CCD camera (Roper Scientific, Photometrics CoolSnap ES, Germany) in real time. The
deposited E. coli cells in the images were enumerated manually after each experiment in
order to obtain the deposited cell densities as a function of time.
2.2.8. Direct microscopic observation of bacterial deposition and release
The bacterial deposition experiments were conducted under two different solution
chemistries: (1) 10 mM NaCl and (2) 1 mM CaCl2 + 7 mM NaCl (total ionic strength of
10 mM). The pH of all solutions was adjusted to 7.0 (buffered with 0.15 mM NaHCO3).
All salts used in the experiments were ACS grade (Fisher Scientific) and electrolyte stock
solutions were prepared by dissolving the salts in DI water. All experiments were
conducted at room temperature (23 °C).
Before each deposition experiment, the membrane was first equilibrated at a
permeate flux of 30–40 μm/s for at least 40 min with the same electrolyte solution (with
no bacteria) as that to be used in the deposition experiment. Just before the start of the
deposition experiment, the permeate flux was adjusted to 30 μm/s. The E. coli cell
suspension was then injected into the pressure vessel using a syringe pump (Harvard,
Holliston, MA) to initiate the deposition experiment. During the deposition experiment,
an image of the central part of the membrane surface was captured every 3 min. The
number of deposited E. coli cells within the field of view of the microscope was plotted
as a function of time, and the rate of increase in deposited bacteria within the field of
view, k, can be obtained by determining the slope of the graph. The deposition rate
coefficient of E. coli cells, kobs, was then calculated by using the equation2, 26:
(2.1)
37
where Am is the area of the field of view of the microscope (between 11,900 and 150,500
µm2) and N0 is the number concentration of E. coli cells in the feed suspension (1.4 × 107
cells/L).
After each deposition experiment, a bacterial release experiment was conducted in
two stages. In Stage 1, the membrane with deposited bacteria was rinsed with the same
solution (with no bacteria) as that used in the deposition experiment in the absence of
permeate flow. In Stage 2, the membrane was rinsed with a 1 mM NaCl solution (with
no bacteria) in the absence of permeate flow. This decrease in the ionic strength of the
rinse solution in Stage 2 is expected to increase the electric double layer repulsion
between the bacteria and membranes and thus, to enhance the chances of bacterial
release. For both release stages, the cross-flow velocity was maintained constant at 10
cm/s. At the end of each release stage, an image of the membrane was captured. The
removal efficiency was calculated by normalizing the number of deposited cells
remaining on the membrane after each release stage to the number of deposited cells
immediately after the deposition experiment. The bacterial deposition and release
experiments were carried out at least three times for each solution chemistry.
2.2.9. Interaction force measurements
In order to investigate the effects of PEM modification on the interactions
between E. coli cells and membrane surfaces, the interaction forces between a CML
colloid probe and membrane surfaces were measured using an AFM (Multimode
NanoScope IIId, Bruker Nano Inc.).28-32 The CML colloid was used as a surrogate for E.
coli cells because both CML colloids and E. coli cells carry carboxylic acid functional
groups.26 The CML colloid probe was prepared by attaching a CML colloid (Invitrogen,
38
Eugene, Oregon) with a diameter of 16 μm to a 0.06 N/m tipless silicon-nitride cantilever
(Bruker, Camarillo, CA) using an epoxy adhesive (Henkel Corporation, Rocky Hill, CT).
Immediately before the interaction force measurements, the colloid probes were oxidized
in a UV-ozone chamber (ProcleanerTM 110, BioForce Nanosciences, Inc., Ames, IA) for
15 min to remove any possible organic contaminants on the probe.
All AFM force measurements between the colloid probes and membrane surfaces
were conducted in a glass fluid cell. The fluid cell was rinsed with ethanol, followed by
DI water, and then blow-dried with ultrapure nitrogen before and after use. The solution
of interest was degassed through ultrasonication for 15 min and stored in a water bath at
27 °C before use. The solution of interest was slowly injected into the fluid cell with a
syringe and force measurements were conducted by bringing the colloid probes toward
the membrane surfaces and then retracting the probes upon contact. A scan rate of 0.49
Hz and ramp size of 1.0 μm were employed and the average cantilever approach and
retract velocity was calculated to be 0.98 μm/s. Force–separation curves were derived
from the cantilever deflection and piezo displacement obtained from the AFM
measurements. For each solution chemistry, force measurements were conducted at 13–
15 locations on the membrane surface and 5 measurements were taken at each location.
2.3. Results and Discussions
2.3.1. Characterization of E. coli cells and PSU membranes modified with PAH/PAA
multilayers
The zeta potentials of E. coli cells were determined to be –36.9 (±2.6) mV at 10
mM NaCl and –25.7 (±1.2) mV at 1 mM CaCl2 + 7 mM NaCl, both at pH 7.0. The zeta
potential of the E. coli cells in the presence of calcium was less negative than that in the
39
absence of calcium due to the neutralization of bacterial surface charge by calcium ions.33
In the absence of calcium, indifferent sodium ions can only screen the surface charge of
E. coli cells.
The ATR data for the base PSU membrane is presented in Figure 2.2. The data
showed peaks corresponding to methyl groups at 2969, 1104, and 833 cm-1. Peaks
typically seen with a conjugated π system such as aromatic ring stretches at 1585 and
1503 cm-1 and ring vibrations at 1017 cm-1 are indicative of the benzene rings along the
polysulfone backbone. Sulfone group stretches appear at 1324, 1291 and 1149 cm-1.
This ATR data is consistent with typical PSU membranes.34-35 In Figure 2.2, the
lineshape in the C(1s) region with zero bilayers shows a dominant peak at 284.5 eV due
to the C-C/C-H species as well as a shoulder centered at 286.5 eV which can be ascribed
to the C-O species present in PSU. A small broad peak at 291.5 eV can also be seen,
representing a π- π* shake up feature associated with carbon atoms in a conjugated
system. Spectral intensity is also observed in the O(1s) region (data not shown) and a
peak at 167.5 eV in the S(2p) region due to the sulfone species.
40
Wavenumber (cm-1
)
10001500
% T
ran
sm
itta
nce
60
65
70
75
80
85
90
95
100
Wavenumber (cm-1
)
250030003500
% T
ran
sm
itta
nce
60
65
70
75
80
85
90
95
100
n
Figure 2.2 ATR-IR data for the PSU base membrane. Above are the PSU structure and
plotted ATR-IR data from 3500 to 2500 cm-1 and 1700 to 700 cm-1. Below is the table of
assigned IR peaks.
Infrared Peaks (cm-1) Peak Assignments
2969 Aliphatic asymmetric C-H stretch
1585, 1503 Aromatic C C ring stretch
1489 CH3-C-CH3 (C-C) stretch
1324, 1291 Asymmetric O=S=O stretch
1241 Asymmetric C-O-C aryl ether stretch
1149 Asymmetric O=S=O stretch
1104 In-plane C-H bend
1017 In-plane C-H bend/C C ring vibrations
875 Methyl C-H bend
833 Methyl C-H rock
715 Methyl C-H rock
41
SEM and AFM images of a PSU base membrane and a membrane modified with
2 PAH/PAA bilayers are shown in Figure 2.3 and Figure 2.4, respectively. Generally, the
morphologies of the base and PEM-modified membranes looked similar, albeit some
pores on the modified membrane, as observed under the SEM, seemed to be blocked by
the PEM. The hydraulic resistances of the base membranes and membranes modified
with 2 bilayers were 5.5 × 1010 m–1 and 2.3 × 1011 m–1, respectively. The increase in
hydraulic resistance of the membrane after PEM modification implies that the pore size
of the base membrane, especially at the entrance of the pores, may be reduced when the
PEM was assembled on the membrane surface. Although the hydraulic resistance of the
PEM-modified membrane was about four times of that of the base membrane, it was still
within the typical range of hydraulic resistances for MF membranes. From AFM
imaging, the RMS value of the base membrane was 45.0 ± 3.8 nm, while that of the
PEM-modified membrane was 46.8 ± 7.6 nm. Therefore, the surface roughness of the
membrane after modification did not change too much from that of the base membrane.
42
(a)
(b)
Figure 2.3 SEM images of (a) a PSU base membrane and (b) a PSU membrane modified
with 2 bilayers of PAH and PAA.
43
(a)
Figure 2.4 AFM images of (a) a PSU base membrane and (b) a PSU membrane modified
with 2 bilayers of PAH and PAA.
(b)
44
2.3.2. XPS analysis of PEM-modified membranes
XPS analysis of the virgin PSU membrane corroborates the characterization data
from ATR, and XPS analysis of PSU membranes with increasing numbers (1–5) of
PAH/PAA bilayers added to the surface is shown in Figure 2.5. As the number of
bilayers increased, there was a systematic decrease in the signal intensity of the S(2p)
region as the sulfone groups present in the original PSU membrane were covered by a
thicker overlayer. Conversely, a signal intensity appeared in the N(1s) region at 401.2 eV
due to the presence of nitrogen atoms in PAH. As the number of bilayers increased, the
signal intensity in the N(1s) region steadily increased as well. The addition of the
PAH/PAA bilayers also changed the C(1s) lineshape, shifting from that of a pure PSU
membrane to a spectral envelope which exhibited features of the two bilayer components,
PAH and PAA. These features include a noticeable and systematic decrease in C-O and
π- π* peaks as the number of PAH/PAA bilayers increased, accompanied by a steady
growth of a carboxyl peak centered at 288.4 eV due to the addition of PAA. Thus, these
observations demonstrate that the PEMs can be successfully assembled on PSU
membranes through the LbL adsorption technique and that the composition of the PEMs
on membrane surface can be controlled by varying the number of sequential exposures to
the PAH and PAA solutions. Analysis of Figure 2.3 also shows that after approximately
4 bilayers (membranes with up to 8 bilayers were measured), the XPS spectra remains
essentially unchanged because the near surface region is now determined exclusively by
the composition of the bilayers.
45
Binding Energy (eV)
282285288291294
C(1s) C-C/C-H
C=O(-OH)
*
C-O
398400402404
x 8N(1s)
164166168170
S(2p) x 17Number
of Bilayers
0
1
2
3
4
5
Polyacrylic acid
Polyallylamine
1 bilayer
Figure 2.5 Evolution of the C(1s), N(1s), and S(2p) XP spectra for a PSU membrane
coated with increasing numbers of PAH/PAA bilayers. Below the XP spectra is an
illustration of the structure and chemical composition of each bilayer.
46
2.3.3. Influence of PEM modification on bacterial deposition kinetics
The anti-adhesive properties of the PEM-modified membranes were first tested by
performing bacterial deposition experiments using the direct microscopic observation
membrane filtration system at 10 mM NaCl in the absence of permeate flow. For these
experiments, the permeate outlet of the CMF cell was sealed to prevent water from
permeating through the membrane. The base and PEM-modified membranes were
exposed to the bacterial suspensions at a cross-flow velocity of 10 cm/s for an hour, and
the images of the E. coli cells deposited on the membranes were captured at the end of
the experiments (Figures 2.6a and 2.6b). The number of E. coli cells deposited on the
PEM-modified membranes was three orders of magnitude lower than that on the base
membranes (Figure 2.6c). This result shows that the modification of membranes with
PAH/PAA PEMs can significantly enhance their resistance to bacterial attachment when
the effects of permeate drag force are absent.
47
0 2010
0
101
102
103
104
De
po
site
d B
acte
ria
(#
/mm
2)
Base
Membrane Modified
Membrane
(c)
(a) (b)
Figure 2.6 Images of deposited bacteria (0.446 mm × 0.333 mm) on (a) a base membrane
and (b) a PEM-modified membrane after the membrane was exposed to a bacterial
suspension prepared at 10 mM NaCl and pH 7.0 for 1 h in the absence of permeate flux.
(c) Number of bacteria deposited on the base and PEM-modified membranes (per mm2).
Error bars represent standard deviations.
48
In order to test the anti-adhesive properties of the PEM-modified membranes in
the presence of permeate flow, deposition experiments were conducted by using the
membrane filtration system at a permeate flux of 30 μm/s and cross-flow velocity of 10
cm/s. Figures 2.7a and 2.7b present the number of deposited E. coli cells (per mm2) on
the base and PEM-modified membrane surfaces, respectively, when bacterial deposition
first took place at 10 mM NaCl and the membranes were subsequently rinsed with 10
mM NaCl and 1 mM NaCl solutions. For the base membrane, 5429 E. coli cells
deposited on 1 mm2 within a time period of 20 min. In comparison, only 2884 E. coli
cells deposited on 1 mm2 of the PEM-modified membrane within the same period of
time. By determining the rate of increase of deposited bacteria in the deposition stage,
the deposition rate coefficients, kobs, were calculated using Equation 2.1 and presented in
Figure 2.7. The average kobs value for the PEM-modified membranes (17.3 µm/s) was
smaller than that for the base membranes (35.8 µm/s), indicating that PEM modification
was effective in enhancing the membranes’ resistance to bacterial attachment even in the
presence of permeate drag force. The kinetics of bacterial deposition on membrane
surfaces are known to be governed by the interfacial interactions between bacteria and
membrane surfaces, as well as the drag force resulting from the permeate flow.2, 26, 36
Since the permeate flux was maintained constant at 30 μm/s for both experiments
conducted using the base and PEM-modified membranes, the permeate drag force exerted
on the bacteria was the same for both experiments. Therefore, the lower deposition
kinetics observed for the PEM-modified membranes in the deposition experiments
conducted in the absence and presence of permeate flow implies that the interfacial
interaction between the bacteria and membrane surfaces was more repulsive (or less
49
0 20 40 60 80 1000
1000
2000
3000
4000
5000
6000
7000
Flush with
1 mM NaCl
Deposited B
acte
ria (
#/m
m2)
Time (min)
Flush with
10 mM NaCl
(a)
0 10 20 30 40 500
1000
2000
3000
4000
Flush with
1 mM NaCl
Flush with
10 mM NaCl
Deposited B
acte
ria (
#/m
m2)
Time (min)
(b)
adhesive) for the PEM-modified membranes compared to the base membranes at 10 mM
NaCl.
50
Figure 2.7 Number of bacteria on (a) a PSU base membrane and (b) a PEM-modified
membrane during the deposition and release stages. The deposition experiment was
conducted at 10 mM NaCl and a permeate flow rate of 30 µm/s. The membrane was
subsequently rinsed with a 10 mM NaCl solution, followed by a 1 mM NaCl solution, in
the absence of permeate flow. For all the deposition and release stages, the pH was
maintained at 7.0.
In order to study the effects of calcium, deposition experiments were conducted
on the base and PEM-modified membranes at 1 mM CaCl2 + 7 mM NaCl and a permeate
flux of 30 μm/s. The deposition rate coefficients obtained in the presence of calcium are
presented in Figure 2.8. The results show that, analogous to what was previously
observed in the presence of 10 mM NaCl, the deposition kinetics of E. coli cells on the
PEM-modified membranes (15.3 µm/s) were lower than the kinetics on base membranes
(36.4 µm/s) in the presence of calcium. This result demonstrates that the modification of
membranes with PAH/PAA PEMs is just as effective in reducing bacterial deposition
kinetics in the presence of calcium.
51
3 40
10
20
30
40
50
kobs (m
/s)
Base Membrane
Modified Membrane
10 mM NaCl 1 mM CaCl2
+ 7 mM NaCl
Figure 2.8 Bacterial deposition rates, kobs, for base and PEM-modified membranes at 10
mM NaCl and 1 mM CaCl2 + 7 mM NaCl. The pH during the deposition process was 7.0.
The permeate flow rate was 30 µm/s. Error bars represent standard deviations.
2.3.4. Effect of PEM modification on reversibility of bacterial deposition
In addition to deposition kinetics, the reversibility of bacterial deposition can
serve as a suitable gauge of a membrane’s resistance to bacterial adhesion.26 To examine
the effects of membrane modification with PEMs on the reversibility of bacterial
deposition, the removal efficiencies of the bacteria that were deposited at 10 mM NaCl
were obtained for both the release stages. After bacterial deposition had taken place on a
base membrane at 10 mM NaCl, the membrane was first rinsed with the same bacteria-
free solution for 30 min, followed by a 1 mM NaCl solution for 30 min (Figure 2.7a). In
both release stages, hardly any release of deposited bacteria was observed. In stark
contrast, when a PEM-modified membrane with bacteria that were deposited at 10 mM
NaCl was rinsed with the same solution for only 10 min, 97% of the deposited bacteria
52
were released (Figure 2.7b). A subsequent rinse with a 1 mM NaCl solution for 10 min
resulted in almost complete (99%) removal of deposited bacteria. Note that the time for
each release stage for the PEM-modified membranes was only 10 min (compared to 30
min for base membranes) due to the fast and significant degree of bacterial release that
was observed for the modified membranes. The removal efficiencies obtained from both
the release stages for the base and PEM-modified membranes are shown in Figure 2.9a.
The significant increase in the removal efficiencies (from <10% to close to 100%)
evidently demonstrated that the modification of membrane surfaces with PEMs can
dramatically enhance the reversibility of bacterial deposition in the presence of NaCl.
53
1 20
20
40
60
80
100
Rem
oval E
ffic
iency (
%)
Base Membrane
Modified Membrane
Stage 1 Release:
10 mM NaCl
Stage 2 Release:
1 mM NaCl
(a)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
Rem
oval E
ffic
iency (
%)
Base Membrane
Modified Membrane
Stage 2 Release:
1 mM NaCl
Stage 1 Release:
1mM CaCl2
+ 7 mM NaCl
(b)
Figure 2.9 Bacterial removal efficiencies for base and PEM-modified membranes after
deposition at (a) 10 mM NaCl and (b) 1 mM CaCl2 + 7 mM NaCl. The release
experiments were conducted in two stages. In Stage 1, the membranes were rinsed with
the same solutions that were used for bacterial deposition (either 10 mM NaCl or 1 mM
CaCl2 + 7 mM NaCl). In Stage 2, the membranes were rinsed with 1 mM NaCl solutions.
The pH for both release stages was 7.0. Error bars represent standard deviations.
54
Furthermore, just before the start of the release stages (i.e., when both the cross
flow and permeate flux were stopped by switching off the gear and peristaltic pumps), the
E. coli cells deposited on PEM-modified membranes were observed under the
microscope to be wriggling in their fixed positions. When the cross flow was started
(with no permeate flow) to initiate the first stage of the release experiment, a considerable
number of the initially deposited bacteria were swept away instantly. In contrast, the E.
coli cells deposited on the base membranes were motionless and did not wriggle on the
membrane surface in the absence of cross flow and permeate flow. Also, no bacteria
were observed to be released from the base membrane surface when the cross flow was
initiated. These observations suggest that the modification of the PSU membranes with
PEM can substantially weaken the attachment of bacteria to the membrane surface.
The removal efficiencies obtained after the E. coli cells were deposited on the
base and PEM-modified membranes at 1 mM CaCl2 + 7 mM NaCl are presented in
Figure 2.9b. Similar to the results obtained when bacterial deposition took place on the
base membranes at 10 mM NaCl, no significant release of E. coli cells occurred (<3%)
when the base membranes were rinsed with a 1 mM CaCl2 + 7 mM NaCl solution,
followed by a 1 mM NaCl solution. In the case of the PEM-modified membranes, in
contrast, a substantially larger degree of release was observed when the membranes were
rinsed with the two rinse solutions (59% and 68% in Stages 1 and 2, respectively). These
removal efficiencies were lower than the efficiencies obtained when the bacterial
deposition took place at 10 mM NaCl (97% and 99% in Stages 1 and 2, respectively).
Possible reasons for the lower removal efficiencies will be discussed in the following
section. Nevertheless, the modification of membrane surfaces with PEMs is shown from
55
these results to significantly enhance the reversibility of bacterial adhesion after
deposition in the presence of calcium.
2.3.5. Proposed mechanism for anti-adhesive properties of PEM-modified membranes
Representative retraction (or pull-off) force–separation curves for the base and
PEM-modified membranes obtained at 10 mM NaCl are presented in Figure 2.10a.
Positive and negative forces represent repulsive and attractive forces, respectively. In the
case of the base membrane, the CML probe experienced a maximum adhesive force of –
0.33 nN when it was retracted from the membrane surface. When the CML colloid probe
was retracted from the PEM-modified membrane, in contrast, a large and long-ranged
repulsive force was detected up to a separation distance of ca. 100 nm.
In order to test if the repulsion observed for the PEM-modified membrane was
due to electric double layer interactions, the decay lengths for base and PEM-modified
membranes, κ–1, were calculated by fitting their respective approach force–separation
curves obtained at 10 mM NaCl with the equation37:
hBF exp (2.2)
where F is the interaction force between the CML colloid probe and membrane surface, B
is a pre-exponential constant, and h is the separation distance. The approach force–
separation curves (Figure 2.10) show that repulsive forces were observed when the CML
colloid probe was brought towards both membranes. The decay length for the base
membrane was 3.0 nm (average of 10 measurements), which is equal to the theoretical
Debye screening length at an ionic strength of 10 mM.27, 38 The identical experimental
and theoretical values indicate that the interaction forces between the CML colloid probe
and base membrane, both negatively charged at pH 7.0, were dominated by electric
56
double layer interactions. In the case of the PEM-modified membrane, the approach
force–separation curves overlapped with the retract force–separation curves, and the
repulsive forces were markedly longer-ranged compared to the forces for the base
membrane. The decay length of the PEM-modified membrane was 31.6 nm (average of
10 measurements), which was considerably longer than that of the theoretical Debye
length. This discrepancy implies that another force, other than electrostatic repulsion,
dominated the interactions between the CML colloid probe and PEM-modified
membrane. This repulsive force is likely due to the compression of the highly hydrated
and swollen PAH/PAA PEM by the CML colloid probe as the probe was brought close to
the PEM-modified membrane.
57
0 40 80 120 160
0
1
2
3
4
Inte
ractio
n F
orc
e (
nN
)
Separation (nm)
Modified Membrane
Base Membrane
(a)
0 10 20 30 40 50 60
-3
-2
-1
0
1
ln (
Inte
ractio
n F
orc
e)
Separation (nm)
Modified Membrane
Base Membrane
(b)
Figure 2.10 Representative approach interaction force curves between a CML colloid
probe and membrane surface at 10 mM NaCl and pH 7.0. Force curves are presented in
the form of (a) linear and (b) semi-log plots.
58
In their study of the cellular anti-adhesive properties of PEMs, Mendelsohn et
al.16 demonstrated that PEMs comprising PAH and PAA will swell and become hydrated
when they are first assembled under low pH conditions (pH = 2.0) and then exposed to
neutral pH conditions (pH = 7.4). In the same work,16 the authors showed that the
PAH/PAA PEMs assembled at pH 2.0 are remarkably resistant to the adhesion of a
highly adhesive fibroblast cell line at pH 7.4. Lichter et al.39 also observed that
PAH/PAA PEMs assembled at pH 2.0 are mechanically soft with a relatively low elastic
modulus (ca. 1 MPa) and are highly resistant to the attachment of both E. coli and
Staphylococcus epidermidis bacteria at neutral pH conditions. Similar to the findings of
these two groups, we showed in this study that PAH/PAA PEMs assembled on PSU
membranes at pH 3.0 can inhibit the adhesion of E. coli cells at pH 7.0.
PAH and PAA are weak polyelectrolytes which contain amine and carboxylic
acid functional groups, respectively. At a low pH of 3.0, the amines of PAH (pKa ≈ 9)
are nearly fully protonated (NH3+) and thus PAH is highly positively charged.
Conversely, at pH 3.0, most of the carboxylic acids of PAA (pKa ≈ 5) remain protonated
(COOH), resulting in PAA to be only partially negatively charged. Hence, when a PEM
comprising PAH and PAA layers is assembled on a PSU membrane at pH 3.0, there are
relatively few ionic cross-links between COO– and NH3+ and the PAA polyelectrolytes in
the PEM take a loopy conformation.16 When the PEM is subsequently exposed to a
higher pH of 7.0, the COOH groups of PAA become fully deprotonated to form COO–
groups. The unpaired COO– groups then repel each other due to electrostatic repulsion,
leading to the PAA polyelectrolytes in the PEM to take an extended conformation. This
change in PAA conformation causes the PEM to undergo considerable swelling and
59
become highly hydrated.16 The approach of a bacterium into a PEM-modified membrane
will result in the compression of the swollen PEM film and removal of some water
molecules from the PEM. The compressed PEM will exert an elastic repulsive force on
the bacterium due to osmotic stresses and push the bacterium away from the underlying
PSU membrane, hence preventing the bacterium from being held to the PSU membrane
by strong, short-ranged van der Waals attraction.27, 38 Because the hydrated PEM is
highly hydrophilic,16 the bacterium will not adhere to the PEM and can be easily removed
from the PEM upon rinsing during the release stages. In the case of a PSU base
membrane, no barrier exists to prevent the bacterium from coming into direct contact
with the membrane surface, and the bacterium can be held on the membrane surface by
strong van der Waals forces that do not allow it to be released when rinsed with the
rinsing solutions.
Representative retraction force–separation curves obtained at 1 mM CaCl2 + 7
mM NaCl for the base and PEM-modified membranes are presented in Figure 10b. In the
case of the base membrane, the CML colloid probe experienced a maximum adhesive
force of – 0.19 nN upon retraction from the membrane surface. In comparison, similar to
the observation at 10 mM NaCl (Figure 2.11a), the CML colloid probe experienced a
repulsive force when it was pulled off from the PEM-modified membrane surface.
However, it is noted that this repulsive force was smaller in magnitude and not as long-
ranged as the repulsive force at 10 mM NaCl.
60
0 20 40 60 80 100 120 140-1
0
1
2
3
4
5
Inte
ractio
n F
orc
e (
nN
)
Separation (nm)
Modified Membrane
Base Membrane (a)
0 10 20 30 40 50 60 70 80-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Positive Work
of Adhesion
Modified Membrane
Base Membrane
Inte
ractio
n F
orc
e (
nN
)
Separation (nm)
Negative Work
of Adhesion
(b)
Figure 2.11 Representative retract interaction force curves between a CML colloid probe
and membrane surface at (a) 10 mM NaCl and (b) 1 mM CaCl2 + 7 mM NaCl. The pH
was 7.0. Positive (blue) and negative (red) work of adhesion are presented in (b).
61
2.3.6. Influence of calcium on anti-adhesive properties of PEM-modified membranes
In order to quantify the propensity of the CML colloid probe to adhere to the base
and PEM-modified membranes, the work of adhesion was calculated from each of the
retraction force–separation curves. The work of adhesion is defined as the work required
to pull the CML colloid probe away from the membrane surface after contact,40-41 and is
obtained by integrating the total area under the retract force profiles, as illustrated in
Figure 2.11b. This work is positive when the force between CML colloid probe and
membrane surface is attractive and is negative when the force between the probe and
membrane surface is repulsive.
The distributions of the work of adhesion for the base and PEM-modified
membranes obtained at 10 mM NaCl are presented in Figure 2.12a and Figure 2.12b,
respectively. For the base membranes, attractive interactions between the CML colloid
probe and membrane surface were detected for 80% of the pull-off events. For the PEM-
modified membranes, in contrast, repulsive interactions were detected for 100% of the
pull-off events. These results showed that the colloid–PEM-modified membrane
interactions were strongly repulsive (average work = –105.83 × 10–18 J) while the
colloid–base membrane interactions were slightly adhesive (average work = 7.67 × 10–18
J). The PEM is highly hydrated and swollen at 10 mM NaCl and exerts a strong elastic
repulsive force on the colloidal probe which prevents it from adhering to the underlying
PSU membrane.
62
-200 -150 -100 -50 0 500
10
20
30
40
50
Base Membrane
10 mM NaCl
Average = 7.67 × 10-18
J
SD = 9.49 × 10-18
J
F
req
ue
ncy (
%)
Interaction Energy (10-18
J)
(a)
-200 -160 -120 -80 -40 0 400
10
20
30
40
50
Fre
qu
en
cy (
%)
Interaction Energy (10-18
J)
Modified Membrane
10 mM NaCl
Average = -105.83 × 10-18
J
SD = 55.80 × 10-18
J
(b)
-4 0 4 8 12 16 20 240
5
10
15
20
25
30
35Base Membrane
1 mM CaCl2 + 7 mM NaCl
Average = 1.96 × 10-18
J
SD = 4.73 × 10-18
J
Fre
qu
en
cy (
%)
Interaction Energy (10-18
J)
(c)
-4 0 4 8 12 16 20 240
5
10
15
20
25
30
35
Fre
qu
en
cy (
%)
Interaction Energy (10-18
J)
Modified Membrane
1 mM CaCl2 + 7 mM NaCl
Average = -1.98 × 10-18
J
SD = 1.90 × 10-18
J
(d)
Figure 2.12 Work of adhesion distributions for (a) base membrane at 10 mM NaCl, (b)
PEM-modified membrane at 10 mM NaCl, (c) base membrane at 1 mM CaCl2 + 7 mM
NaCl, and (d) PEM-modified membrane at 1 mM CaCl2 + 7 mM NaCl. All
measurements were conducted at pH 7.0. Red and black bars represent repulsive
(negative) and attractive (positive) interactions between CML colloid probe and
membrane surface.
63
The distributions of the work of adhesion for the base and PEM-modified
membranes obtained in the presence of calcium are shown in Figure 2.12c and Figure
2.12d, respectively. For the base membranes, adhesive interactions between the CML
colloid probe and membrane surface were detected for 85% of the pull-off events.
Conversely, for the PEM-modified membranes, repulsive interactions were observed for
93% of the pull-off events. These results indicated that, analogous to the findings at 10
mM NaCl, the colloid–base membrane interactions were generally adhesive (mean work
= 1.96 × 10–18 J) while the colloid–PEM-modified membrane interactions were largely
repulsive (mean work = –1.98 × 10–18 J) in the presence of calcium. The less favorable
interactions between the colloids and PEM-modified membranes corroborated with the
observations that the bacterial deposition on PEM-modified membranes was slower than
on base membranes and that the degree of bacterial release after deposition was
considerably higher for the PEM-modified membranes.
It is noted that, while the interaction forces between the CML colloid probe and
PEM-modified membranes were generally repulsive for both solution chemistries, the
repulsive interaction at 10 mM NaCl (average work = –105.83 × 10–18 J) was
significantly stronger than that at 1 mM CaCl2 + 7 mM NaCl (average work = –1.98 ×
10–18 J). This result is consistent with the noticeably lower removal efficiencies after
deposition in the presence of calcium (59% and 68% in Stages 1 and 2, respectively, in
Figure 2.9b) compared to the removal efficiencies after deposition in the absence of
calcium (97% and 99% in Stages 1 and 2, respectively, in Figure 2.9a). When a PEM-
modified membrane is exposed to calcium, the calcium ions can form complexes with the
carboxyl groups of the PAA42-44 that are within and on the outer surface of the PEM film
64
and thus neutralize the charges of the PAA. Furthermore, calcium can form
intermolecular and intramolecular bridges through the formation of calcium complexes
with the PAA carboxyl groups.42-44 Therefore, the PAA polyelectrolytes take a more
compact conformation, resulting in the PEM to become less swollen and less hydrated, as
illustrated in Figure 2.13. Even when the PEM-modified membrane was rinsed with a 1
mM NaCl solution in the second release stage, some residual calcium ions are likely to
remain bound to the carboxyl groups of PAA, hence preventing the PEM to return to its
fully hydrated state. Therefore, the repulsive force exerted by the less swollen/hydrated
PEMs in the presence of calcium are not as strong and long-ranged as the forces exerted
by the fully hydrated PEMs in the absence of calcium. Nevertheless, despite the effects
of calcium, the PEM modification of membranes can considerably enhance the
reversibility of bacterial attachment from ca. 2 % to 68 % (Figure 2.9b).
65
H2O PAH
10 mM NaCl, pH 7.0
H2O
Ca2+
1 mM CaCl2 + 7 mM NaCl, pH 7.0
PAA
Figure 2.13 The PAH/PAA multilayer is highly hydrated and swollen in the absence of
calcium (top). In the presence of calcium, the PAH/PAA multilayer becomes less
hydrated and less swollen (bottom). The schematics are not drawn to scale and are for
illustrative purposes only.
2.4. Conclusions
The anti-adhesive properties of PSU membranes that were modified with PEMs
comprised of PAH and PAA were investigated in this study. PEMs were assembled on
the PSU membranes using the LbL adsorption technique with the employment of a flow
cell. XPS analysis of PSU membranes modified with varying number of bilayers
66
demonstrated that the carboxylic acid and nitrogen concentrations increased with
increasing bilayers, thus confirming that PAH/PAA PEMs can be successfully assembled
on PSU membranes through the LbL technique. The anti-adhesive properties of PEM-
modified membranes were evaluated by measuring the deposition kinetics of E. coli cells
in two solution chemistries (10 mM NaCl and 1 mM CaCl2 + 7 mM NaCl) and testing the
reversibility of bacterial deposition using a direct microscopic observation membrane
filtration system. Our results show that the modification of PSU membranes with PEMs
can reduce bacterial deposition kinetics by about half both in the absence and presence of
calcium. Furthermore, the removal efficiencies were significantly increased after PEM-
modification from <10% to 99% and 68% for bacterial deposition in the absence and
presence of calcium, respectively. AFM interaction force measurements showed that the
adhesive forces between the CML colloid probe and membrane surfaces were
significantly reduced (eliminated in the absence of calcium) after PEM modification.
Instead, strong, long-ranged repulsive forces observed between the colloid probe and
PEM-modified surfaces inhibited the irreversible attachment of E. coli cells on the
membrane surfaces. The remarkable bacterial anti-adhesive properties of the PEM-
modified membranes were attributed to the highly swollen and hydrated structure of the
PEMs which prevent bacteria from being held to the underlying PSU membranes by
strong, short-ranged van der Waals attraction. Although the complex formation between
the carboxyl groups of the PAAs and calcium resulted in the PEMs to become less
hydrated, repulsive interactions between the CML colloid probe and PEM-modified
membranes were still dominant in the presence of calcium. In summary, this study
demonstrated that PSU membranes modified with PAH/PAA PEMs showed significantly
67
improved bacterial anti-adhesive properties compared to PSU base membranes. Further
investigation is required to examine the influence of PEM composition, number of
bilayers, and solution chemistries employed for PEM assembly on the anti-adhesive
properties of PEM-modified membranes.
2.5. Acknowledgements
This work was supported by the National Science Foundation (CBET-1133559)
and the Global Water Program at Johns Hopkins University (JHU). L.T. acknowledges
funding support from the Dean Robert H. Roy fellowship. We thank Dr. Menachem
Elimelech from Yale University for providing us the E. coli K12 MG 1655 strain. We
acknowledge the assistance from Hyun Sik Choi and Tiffany Wei from the Department
of Geography and Environmental Engineering (JHU) with the deposition experiments.
The SEM images of the membranes are taken by Dr. Michael McCaffery from the
Integrated Imaging Center, Department of Biology (JHU).
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Interaction forces and molecular adhesion between pre-adsorbed poly(ethylene imine)
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71
Chapter 3. Imparting Antimicrobial and Anti-
Adhesive Properties to Polysulfone Membranes
through Modification with Silver Nanoparticles
and Polyelectrolyte Multilayers *
* All the results in this chapter have been submitted as parts of a manuscript of the same
title with co-authors Khanh An Huynh and Kai Loon Chen to Journal of Colloid and
Interface Science and is currently in revision. Co-author Khanh An Huynh helped with
the ICP-MS measurements. Co-author Kai Loon Chen helped with experimental data
interpretation and manuscript editing.
72
3.1. Introduction
Membrane technology, such as microfiltration (MF), ultrafiltration, nanofiltration,
and reverse osmosis (RO), is rapidly becoming one of the most popular technologies for
drinking water and wastewater treatment because of improvements in membrane
filtration performance and decreasing membrane cost.1-4 Nevertheless, one major
obstacle that continues to impede the application of membrane technology is biofouling,
or the formation of biofilms on membrane surfaces or within the membrane matrices.5-9
It is difficult to completely remove biofilms from membranes using biocide solutions as
the protective structure of the biofilms’ extracellular polymeric substances protects the
embedded microbial cells from biochemical attack.10-12
The development of anti-biofouling membranes over the last decade has centered
on the modification of membrane surfaces through the enhancement of surface charge
and/or hydrophilicity to render them more resistant to bacterial and colloidal adhesion.13-
22 The assembly of polyelectrolyte multilayers (PEMs) on membrane surfaces through
layer-by-layer (LbL) adsorption is an emerging membrane surface modification
technique to inhibit or retard biofouling.17, 22-25 Polyethersulfone (PES) membranes
modified with PEMs comprising 1.5 bilayers of poly(styrene sulfonate) (PSS) and
poly(diallyldimethylammonium chloride) (PDADMAC) were found to be more resistant
to the adhesion of Escherichia coli bacteria compared to the unmodified membranes due
to the increase in surface charge and hydrophilicity of the modified membranes.22
Polyamide thin-film composite (TFC) RO membranes were modified with 10 bilayers of
polyethylene amine (PEI) and poly(acrylic acid) (PAA) and further functionalized
through the grafting of hydrophilic poly(sulfobetaine).25 This surface modification was
73
shown to result in a considerably reduction in E. coli cell adhesion on the membranes
because of the increased hydrophilicity.25 Similarly, our previous study showed that the
assembly of 2 bilayers of poly(allylamine hydrochloride) (PAH) and PAA on the surface
of polysulfone (PSU) membranes can significantly enhance the membranes’ bacterial
anti-adhesive properties due to the hydrated, swollen nature of the PAH/PAA PEMs.17
While the surface modification of membranes with PEMs can impart some
antifouling properties to the membranes, PEMs alone cannot completely prevent bacterial
adhesion to the membranes since the drag forces caused by the permeate flow may be
strong enough to immobilize the bacteria on the membrane surface.26 Therefore, it is also
desirable to impart antimicrobial properties to PEM-modified membranes to inactivate
bacteria that do deposit on the membrane surfaces. Recently, several studies have shown
that PEMs can be used to immobilize antimicrobial nanomaterials, such as silver
nanoparticles (AgNPs), on the membrane surfaces.22, 25, 27-29 PES membranes that were
modified with 1.5 bilayers of PSS and PDADMAC together with citrate-coated AgNPs
showed no cell growth when the nanocomposite membranes were exposed to E. coli.22
PES membranes coated with 1.5 bilayers of chitosan and poly(methacrylic acid) also
showed a considerable inhibition of E. coli cell growth when AgNPs were dispersed in
each polyelectrolyte layer.28 In another study, polyamide TFC RO membranes coated
with PEMs which were composed of PEI and PAA together with AgNPs exhibited a E.
coli inactivation efficiency of over 95%.25 While these studies have demonstrated that
AgNP/PEM assemblies on the membrane surfaces can impart antimicrobial properties to
the membranes, not many studies, to date, have been conducted to systematically
evaluate these membranes’ bacterial anti-adhesive properties. Furthermore, no studies
74
have been performed to examine the effectiveness of AgNP/PEM- and PEM-
modifications on the membranes’ bacterial anti-adhesive properties over multiple cycles
of filtration.
The objective of this research is to evaluate the antimicrobial and bacterial anti-
adhesive properties of PSU MF membranes that are modified with AgNPs and PEMs
composed of PAH and PAA. Three different AgNP mass loadings are employed to
investigate the effect of AgNPs on the PEM-modified membranes’ antimicrobial and
bacterial anti-adhesive properties. The antimicrobial properties of the modified
membranes are examined through the use of a colony forming unit (CFU) enumeration
method. The bacterial anti-adhesive properties of the modified membranes are assessed
by comparing the kinetics and reversibility of E. coli deposition on the membranes using
a direct microscopic observation membrane filtration system. Finally, the effectiveness
of PEM- and AgNP/PEM-modifications on the membranes’ bacterial anti-adhesive
properties is examined over three cycles of filtration and rinsing.
3.2. Materials and Methods
3.2.1. Base Membranes
PSU MF membranes (Pall Corporation, Ann Arbor, MI) were used as the base
membranes in this study. According to the manufacturer, the nominal pore size of the
membranes is 0.2 μm on the active side. Membrane coupons were cut from flat sheet
membranes, rinsed, and then stored in deionized (DI) water (Millipore) at 4 ºC for at least
three days before use. More information about the physicochemical properties of the
PSU membranes was provided in our previous publication.17
75
3.2.2. Silver Nanoparticles and Polyelectrolyte Multilayers
AgNPs were synthesized through the reduction of a Tollen’s reagent with the use
of glucose and then cleaned and suspended in a citrate solution.30-32 All reagents used to
prepare the AgNP stock suspensions were purchased from Sigma Aldrich (St. Louis,
MO). First, 20 mL of Tollen’s reagent (0.1 mM AgNO3, 0.8 mM NH4OH, and pH 11.5)
was prepared in a 50-mL polypropylene centrifugal tube (BD Biosciences, NJ). After
that, the Tollen’s reagent was ultrasonicated at 20 C for 45 min in an ultrasonic bath
(Branson 5510, power 180 W, frequency 40 kHz) to homogenize the reactants and
enhance their reactivity.31 80 µL of 0.5 M glucose was introduced into the Tollen’s
reagent at the beginning of the ultrasonication to initiate the formation of AgNPs (final
glucose concentration = 2 mM). The AgNP suspension obtained after ultrasonication
was centrifuged at 3,650g for 60 min at 15 °C (Avanti centrifuge J-20 XPI, Beckman
Coulter, Brea, CA). The supernatant was decanted and the settled AgNPs were re-
suspended in the same amount of 1 µM trisodium citrate solution. This cleaning process
was repeated two more times. The final suspension was ultrasonicated for 30 min and
then transferred into a Pyrex glass bottle, which was stored in the dark at 4 °C.
The average hydrodynamic diameter of AgNPs was measured to be 47.3–55.0 nm
through dynamic light scattering (DLS, BI-200SM and BI-9000AT, Brookhaven). The
total and dissolved silver concentrations of the citrate-coated AgNP stock suspensions
(three batches) were determined to be 6.66–7.14 and 0.11–0.16 mg/L, respectively,
through inductively coupled plasma mass spectrometry (ICP-MS). Specifically, to
determine the total silver concentrations, the suspensions of interest were digested with
concentrated HNO3 in a microwave digestion system (MARSXpress, CEM, NC) at 150
76
C for 5 min.33 The digested suspensions were diluted with deionized (DI) water
(Millipore, MA) to reduce the HNO3 concentration to 2.5–3.5 %. Afterward, the total
silver concentrations in diluted samples were measured by an ICP-MS instrument
(PerkinElmer Elan DRC II). To determine the dissolved silver concentrations, the
suspensions of interest were first filtered through a centrifugal filter (3 kDa molecular
weight cut off, Nanosep, Pall Corp., NY) at 12,000g (Eppendorf, NY) for 20 min. Before
being analyzed for silver concentration using the ICP-MS, the HNO3 concentration in the
filtrate was adjusted to 3.5 %. The AgNP concentration was calculated by subtracting the
dissolved silver concentration from the total silver concentration.
PAH (Mw = 15,000, Sigma-Aldrich, St. Louis, MO) and PAA (Mw = 50,000,
Polysciences, Inc., Warrington, PA) solutions were prepared in DI water. The PAH and
PAA solutions were used for membrane modification within 5 days after preparation.
The concentrations of both the PAH and PAA solutions were 5 mM (based on the repeat
unit molecular weight). The ionic strength of both solutions was adjusted to 150 mM
using NaCl and the pH of the solutions was adjusted to 3.0 using 1 M HCl.
3.2.3. Membrane Modification with AgNPs and PEMs
A custom-made polycarbonate flow cell was used to modify the PSU membrane
surfaces with AgNPs and PAH/PAA multilayers. The flow cell comprises a top plate and
a bottom plate. The dimensions of the cross-flow channel in the cell are 76.0 mm in
length, 25.0 mm in width, and 3.0 mm in height. The membrane to be modified was
clamped between the top and bottom plates and sealed with double O-rings. The flow
cell can be operated either in the dead-end filtration or cross-flow mode. The inlet valve
is in the top plate while the outlet valves are in both the top and bottom plates. In the
77
dead-end mode, the outlet valve of the top plate was closed and the outlet valve of the
bottom plate was opened. In the cross-flow mode, the outlet valve of the bottom plate
was closed and the outlet valve of the top plate was opened.
To modify the membrane surface, a diluted AgNP suspension of a desired
concentration (150 mL) was filtered through a PSU membrane under the dead-end mode
at a filtration rate of 15 mL/min. Following that, PEMs comprising two bilayers of PAH
and PAA were assembled on the surface of the AgNP-modified membrane under the
cross-flow mode in the absence of permeation. PEMs were assembled through the LbL
adsorption technique using an approach similar to that described in our previous study.17
In that study, it was verified through X-ray photoelectron spectroscopy that the PEMs can
be assembled on the PSU membranes using the LbL adsorption approach. Briefly, the
membrane was first rinsed with a PAH solution for 10 min. After that, the membrane
was rinsed with a 150 mM NaCl solution (pH 3.0) for 10 min to wash away the loosely-
bound polyelectrolytes. Following that, the membrane was rinsed with a PAA solution
for 10 min and then with a 150 mM NaCl solution (pH 3.0) for 10 min. This process was
then repeated in order to form two bilayers on the membrane. The cross-flow velocity
used for the assembly of PEMs was 2.2 mm/s. The mass loading of AgNPs on the
membranes was determined by measuring the total silver concentrations of the permeate,
polyelectrolyte solutions, and rinse solutions collected from the flow cell during
membrane modification using ICP-MS and by performing mass balance. Surface
morphologies of the base, PEM-modified, and AgNP/PEM-modified membranes were
acquired using scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR).
78
The membrane samples used for SEM analysis were vacuum-dried overnight in a
desiccator and examined under the low-vacuum mode.
3.2.4. Bacteria for the evaluation of antimicrobial and anti-adhesive properties of
membranes
The model bacteria used in this study was E. coli K12 MG 1655.26 This bacterial
strain is labeled with the green fluorescent protein which allows the bacterial cells to be
observed under an epifluorescent microscope. The E. coli cells were incubated in a
culture solution containing 25 g/L Luria Bertani (LB) broth (Fisher Scientific) and 50
mg/L kanamycin (Sigma-Aldrich) at 37 ºC for ca. 3 hours and then harvested at the
exponential growth phase. The cleaning procedure of the bacterial cells to be used for the
bacterial deposition and release experiments has been described in our previous study.17
For these experiments, the cell concentration in the feed suspension was ca. 1.4 × 107
cells/L.
3.2.5. Evaluation of antimicrobial properties of membranes
A CFU enumeration method was used to evaluate the antimicrobial properties of
the membranes modified with AgNPs and PEMs.34-35 Specifically, the membrane coupon
was first placed on top of the glass support of a vacuum filtration setup (Millipore,
Billerica, MA) with the active side facing up. A 0.1 mL E. coli suspension was serial-
diluted to 4.0 × 104 cells per mL with a 154 mM NaCl solution. 0.5 mL of the diluted
bacterial suspension was further diluted with 25 mL of a 154 mM NaCl solution to ca.
800 cells per mL and was then gently filtered through the membrane. The cell
concentration was determined through optical density (wavelength 600 nm)
measurements and using a calibration curve that was obtained for this E. coli strain. The
79
(a) (b)
filtration of the bacterial suspension took 480–660 min and the permeate flux during
filtration was 28.7–39.5 μm/s. The membrane coupon with the deposited bacterial cells
was placed on an agar plate (25 g/L LB broth, 15 g/L agar, and 50 mg/L kanamycin) with
the active side facing up (i.e., support side attached to the agar) and the agar plate was
placed in an incubator (VWR, Radnor, PA) and incubated at 37 °C for ca. 15 hours.
Photographs of the vacuum filtration setup and a membrane coupon placed on the agar
plate are provided in Figure 3.1. The CFUs on the membrane on the agar plate (area of
4.9 cm2) were enumerated. The bacterial colonies on the membranes that were modified
with AgNPs and PEMs were counted and compared with the colonies on the membranes
modified with PEMs alone. This test was carried out at least three times for each
membrane.
80
Figure 3.1 Photographs of (a) the vacuum filtration setup and (b) a membrane coupon
placed on the agar plate.
3.2.6. Evaluation of antimicrobial properties of membranes
A direct microscopic observation membrane filtration system is used in this study
to observe bacterial deposition and release during a filtration process. This system has
been described in our previous study17 and is similar to the systems used in other
studies.10, 26 Briefly, the membrane to be tested was held between the top and bottom
plates of a cross-flow membrane filtration (CMF) cell with the active side facing the
cross-flow channel. A 3-mm thick glass window was inserted into the top plate of the
CMF cell, enabling the fluorescent bacterial cells to be observed under an
epifluorescence microscope. The CMF cell was incorporated into a closed-loop filtration
system, which was operated under the cross-flow mode. A stainless steel pressure vessel
(Alloy Products, Waukesha, WI) containing 2 L of the feed bacterial suspension was
pressurized to ca. 170 kPa and the suspension was circulated through the CMF cell with
the use of a gear pump (Cole-Parmer, Vernon Hills, IL) at a cross-flow velocity of 10
cm/s. The permeate flux was maintained constant at 108 L/m2·h during the deposition
experiment using an 8-roller digital peristaltic pump (Cole-Parmer, Vernon Hills, IL) and
the permeate was circulated back into the pressure vessel. The CMF cell was placed on
the stage of an epifluorescence microscope (Nikon Eclipse E600W, Japan). The
microscope is equipped with a 10× objective lens (Nikon Plan Fluor, Japan) and an
emission filter (Nikon C-FL Endow GFP HYQ, EX 450-490, DM 495, BA 500-550).
The digital images of E. coli cells on the membrane surface were acquired with a CCD
camera (Roper Scientific, Photometrics CoolSnap ES, Germany) in real time during the
81
filtration experiment. The E. coli cells deposited on the membrane surface within the
field of view of the microscope were enumerated after each experiment in order to obtain
the deposited cell densities as a function of time.
The bacterial deposition experiments were conducted in 10 mM NaCl and at pH
7.0 (buffered with 0.15 mM NaHCO3) using a procedure similar to that in our previous
study.17 The solution chemistry represents that of a typical tertiary wastewater effluent.36-
37 Briefly, the membrane was equilibrated at a permeate flow rate of 30 μm/s for ca. 40
min. Following that, E. coli cells were introduced into the pressure vessel to initiate the
deposition experiment. The bacterial deposition experiment was carried out for 20 min
and an image of the central part of the membrane surface was acquired every 3 min. The
deposition rate coefficient of the E. coli cells, kobs, was calculated by dividing the rate of
bacterial deposition by the product of the image area and cell concentration in the
suspension.10, 17
A bacterial release (detachment) experiment was conducted in two stages after
each deposition experiment. In Stage 1, the membrane with deposited bacteria was
rinsed with a solution of 10 mM NaCl and pH 7.0 for 30 min at a cross-flow velocity of
10 cm/s and in the absence of permeate flow. In Stage 2, the membrane was rinsed with
a solution of 1 mM NaCl and pH 7.0 for another 30 min, also in the absence of permeate
flow. The solution with a lower ionic strength was used in Stage 2 to increase the electric
double repulsion between the deposited bacteria and the membrane.38 The removal
efficiencies for Stage 1 and Stage 2 were calculated by dividing the numbers of bacteria
removed during Stage 1 and during Stage 2, respectively, by the number of bacteria
deposited on the membrane immediately before Stage 1. All the salts used in the
82
experiments were ACS grade (Fisher Scientific) and electrolyte stock solutions were
prepared by dissolving the salts in DI water. All the bacterial deposition and release
experiments were conducted at room temperature (24 °C) and were triplicated for each
experimental condition.
3.2.7. Direct observation during three cycles of filtration and rinsing
The E. coli cells were allowed to undergo three cycles of deposition and release
on the modified membranes at 10 mM NaCl and pH 7.0 (buffered with 0.15 mM
NaHCO3). All the three-cycle filtration and rinsing experiments were conducted using
the direct microscopic observation membrane filtration system. Specifically, bacterial
deposition took place at a permeate flux of 30 μm/s and cross-flow velocity of 10 cm/s
for 20 min in the first cycle of filtration. After filtration, the reversibility of bacterial
deposition was evaluated by turning off the peristaltic pump for 5 min to stop the
permeate flow while the cross flow was maintained. After that, the peristaltic pump was
turned on again to initiate the second cycle of bacterial deposition. The filtration and
rinsing process was then repeated twice. The removal efficiency for each cycle was
calculated by dividing the number of the bacteria removed during each release process by
the number of the bacteria deposited on the membrane immediately before the start of the
same release process.
3.2.8. Silver leaching test
The silver leaching test was conducted for the AgNP/PEM-modified membranes
with the highest AgNP mass loading. The purpose of this test was to examine the
dissolution and release of AgNPs from the modified membranes. The leaching test was
performed using the direct microscopic observation membrane filtration system and in
83
the same solution chemistry as that for the bacterial deposition experiments (10 mM
NaCl and pH 7.0). The membrane to be tested was held between the top and bottom
plates of the CML cell with the active side facing the top plate. For this test, the 10 mM
NaCl solution was circulated through the CMF unit at a cross-flow velocity of 10 cm/s in
the absence of permeation. After 1 hour, the total silver concentration of the circulated
solution was measured using ICP-MS to determine the degree of silver leaching from the
membrane.
3.3. Results and Discussion
3.3.1. Characterization of membranes modified with PEMs and AgNPs
PSU membranes were modified with PEMs only, as well as PEMs with three
different AgNP mass loadings. The AgNP loadings of the membranes modified with
both AgNPs and PEMs were determined to be 3.6, 14.7, and 31.4 μg (0.005, 0.020, and
0.043 wt. %, respectively) over a membrane surface area of 19.4 cm2. The membrane
designations and modification conditions are summarized in Table 3.1.
Membrane ID Membrane Modification Surface Density of AgNPs
(g/cm2)
Membrane P PEMs only 0
Membrane P5 0.005 wt. % AgNPs + PEMs 0.19
Membrane P20 0.020 wt. % AgNPs + PEMs 0.76
Membrane P43 0.043 wt. % AgNPs + PEMs 1.62
Table 3.1 Designations and modification conditions of PEM- and AgNP/PEM-modified
membranes.
84
The SEM images of Membranes P, P20, and P43, as well as the PSU base
membrane, are shown in Figure 3.2. Membranes P, P20, and P43 exhibited similar
morphology as that of the base membrane. However, several pores on Membranes P,
P20, and P43 appeared to be covered by the PEM film, indicating the successful
assembling of PEMs on the membrane surface. The pKa of NH3+ groups in PAH has
been reported to be ca. 9.039 while the isoelectric point of the PSU membrane has been
reported to be ca. 3.0.40-42 Therefore, the first layer of PAH adsorbed on the PSU
membrane surface through electrostatic attraction since PAH is positively charged while
the PSU membrane surface is slightly negatively charged at pH 3.0. Additionally, in the
case of Membranes P20 and P43, we observed several white spots sparsely distributed on
the membrane surfaces which were likely AgNPs and AgNP aggregates that had
deposited on the membranes and were trapped in the membrane pores. Based on the
SEM images, the AgNP coverages on Membranes P20 and P43 were determined (using
ImageJ software, National Institutes of Health) to be 0.6 % and 1.1 %, respectively.
85
Figure 3.2 SEM images of (a) PSU base membrane, (b) Membrane P, (c) Membrane P20,
and (d) Membrane P43.
The hydraulic resistances of Membranes P, P20, and P43 were determined to be
1.4 × 1011, 1.3 × 1011, and 1.0 × 1011 m-1, respectively. These values were within the
typical range of hydraulic resistances for MF membranes (between 1 × 1011 and 1 × 1012
m-1).43 The hydraulic resistances of the membranes modified with both PEMs and
AgNPs (Membranes P20 and P43) were comparable to that of the membranes modified
with only PEMs (Membrane P), indicating that the deposited AgNPs’ contribution to the
hydraulic resistance of the AgNP/PEM-modified membranes was insignificant. This
finding confirms that the nanoparticle coverage on the membrane was extremely low
even at the highest AgNP loading, which is consistent to our observations through SEM
(a) (b)
(c) (d)
86
imaging. In comparison, we had shown in our previous study17 that the hydraulic
resistance of a membrane can be increased by over 3 times after PEM modification. The
increase in hydraulic resistance after PEM modification was likely due to the partial
coverage of the membrane pores by the film of PEMs, as observed through SEM imaging
in that study.17
The degree of silver leaching from Membrane P43 after being rinsed with a 10 mM
NaCl solution at a cross-flow velocity of 10 cm/s for 1 hour was determined to be 14.5
%. In a study by Diagne et al.22, a silver leaching test was performed by filtering DI
water through AgNP/PEM-modified membranes under a dead-end mode for 150 min and
50 % of the AgNPs were lost after filtration. Even though the surface-immobilized
AgNPs in our study experienced a much larger shear force (cross-flow velocity = 10
cm/s) compared to the AgNPs in the study of Diagne et al.22 (no cross flow), the degree
of silver leaching from Membrane P43 in our study was noticeably lower. This
observation may imply that the 2 bilayers of PAH and PAA are more robust than the 1.5
bilayers of PSS and PDADMAC employed in the study of Diagne et al.22, possibly due to
stronger electrostatic interactions between the PAH and PAA layers compared to that
between the PSS and PDADMAC layers. Additionally, it is plausible that the method
used to incorporate AgNPs into the PEMs may influence the degree of leaching of
AgNPs. In our study, the AgNPs were first deposited on the membrane surface through
filtration under a dead-end mode before the membranes were coated with the PEMs
which can form protective thin films over the deposited AgNPs. In the study of Diagne
et al.22, the AgNPs were dispersed in the PSS solution before the PSS–AgNP mixture was
used to form the top layer of the PSS/PDADMAC PEMs. This approach was likely to
87
result in the deposited AgNPs to be fully exposed to the aqueous environment and thus
more prone to detachment and dissolution.
3.3.2. Effect of AgNP/PEM-modification on membranes’ antimicrobial properties
In order to assess the antimicrobial properties of the AgNP/PEM-modified
membranes, the bacterial colonies formed on the membrane surfaces were enumerated
after the E. coli suspensions were filtered through the membranes by vacuum filtration
and after ca. 15 hours of incubation at 37 °C. The numbers of colonies on the surfaces of
Membranes P, P5, P20, and P43 were presented in Figure 3.3. While 223 colonies were
observed on Membrane P, only 144 and 14 colonies were found on Membranes P5 and
P20, respectively. Furthermore, no CFU were observed on Membrane P43. This result
clearly shows that the colonies on all the AgNP/PEM-modified membranes were lower
than those on the PEM-modified membranes. Also, the colonies on the AgNP/PEM-
modified membranes were shown to decrease as the mass loading of AgNPs was
increased. Therefore, it can be concluded that the incorporation of AgNPs into the PEMs
on the membrane surfaces can impart antimicrobial properties to the PSU membranes.
Furthermore, the membranes’ antimicrobial properties are demonstrated to have a direct
dependence on the mass loading of AgNPs.
88
6 80
50
100
150
200
250
Mem
bran
e P5
Mem
bran
e P20
Mem
bran
e P43
Mem
bran
e P
CF
U/m
em
bra
ne
Figure 3.3 Number of bacterial colonies (or CFUs) on Membrane P, Membrane P5,
Membrane P20, and Membrane P43. Error bars represent standard deviations. * No
colonies were present on Membrane P43.
In this study, the minimum AgNP mass loading that resulted in the complete
inhibition of cell growth (i.e., no colonies observed on membranes) was 0.043 wt. %
(Membrane P43). This value was much lower compared to the values reported in other
studies that applied similar methods to evaluate the antimicrobial activities of AgNP-
impregnated membranes which were casted using PSU mixtures with AgNPs dispersed
within the mixtures.34-35 Zodrow et al.34 showed that their AgNP-impregnated PSU
membranes enabled a 99 % reduction in E. coli cell growth when the AgNP
concentration in the membranes was 0.9 wt. %. In the study of Liu et al.,35 the authors
reported a similar AgNP mass loading of 0.88 wt. % in their AgNP-impregnated PSU
membranes to achieve an antibacterial efficiency of 99 % with E. coli cells. In
89
comparison, the AgNP mass loading (0.043 wt. %) that led to the complete inhibition of
bacterial colony growth on the membrane surface in our current study was about two
orders of magnitude lower than the values reported in the studies described above. This
large difference in AgNP concentrations implies that the location of AgNPs on the
membranes (either immobilized on the membrane surface or embedded in the
membranes) plays a crucial role in controlling the membranes’ antimicrobial properties.
Recently, several studies have provided evidence that the direct contact or the
close proximity between AgNPs and bacterial cells can greatly enhance the toxicity
effects of the AgNPs.44-49 The direct contact or close proximity between AgNPs and
bacteria allows the cells to be exposed to lethal concentrations of Ag+ ions that are
released from dissolving AgNPs.46, 49 The casting of membranes using a polymer
mixture with AgNPs dispersed within the mixture, as employed by Zodrow et al.34 and
Liu et al.35 was likely to result in most of the AgNPs to be embedded inside the
membrane matrix and therefore unavailable for direct contact with deposited bacterial
cells. In contrast, our approach of immobilizing AgNPs on the membrane surface
through the use of PEMs dramatically enhanced the opportunities for the direct contact or
close proximity between the AgNPs and deposited bacteria. Therefore, a much lower
AgNP mass loading is required for the inhibition of bacterial growth on the membrane
surface when AgNPs are immobilized on the membrane surface using PEMs compared to
the incorporation of AgNPs within the membrane matrix.
90
3.3.3. Influence of AgNP/PEM-modification on kinetics and reversibility of bacterial
deposition
In order to evaluate the anti-adhesive properties of the AgNP/PEM-modified
membranes, bacterial deposition experiments were conducted by using the direct
microscopic observation membrane filtration system. Figure 3.4a presents a
representative plot of the number density of deposited E. coli cells on the surface of
Membrane P43 as a function of time during a deposition experiment. Figure 3.4b
presents the deposition rate coefficients, kobs, of the base, PEM-modified, and
AgNP/PEM-modified membranes when bacterial deposition took place at 10 mM NaCl.
The experimental results showed that the AgNP/PEM-modified membranes (Membranes
P20 and P43), as well as the PEM-modified membrane (Membrane P), exhibited
considerably lower kobs values (ca. 15 µm/s) than that of the base membranes (ca. 33
µm/s). Clearly, AgNP/PEM-modifications are demonstrated to be as effective as PEM-
modification in reducing the bacterial deposition rates on the membrane surfaces and the
presence of AgNPs on the membrane surface does not affect the deposition rates.
Since the kobs values for the AgNP/PEM-modified membranes (Membranes P20
and P43) were independent of the AgNP mass loadings and comparable to that of the
PEM-modified membrane (Membrane P), it is likely that the enhancement of the
AgNP/PEM-modified membranes’ resistance to bacterial adhesion was controlled by the
PAH/PAA PEMs on the membrane surfaces. The bacterial deposition kinetics on
membrane surfaces during filtration are governed by the drag forces due to the permeate
flow and the interfacial interactions between the bacteria and membranes.10, 26 The
interfacial interactions include van der Waals, electrostatic, and hydrophobic interactions.
91
Since the drag forces exerted by the permeate flow were the same for both the base
membrane and the PEM- and AgNP/PEM-modified membranes, the reduced bacterial
deposition kinetics observed on the modified membranes indicates that the approaching
bacterial cells experienced stronger repulsive forces with the modified membranes
compared to the base membrane.
Our previous study demonstrated that the repulsive interaction between bacteria
and PEM-modified membranes resulted from the highly hydrated and swollen structure
of the PEM film.17 When the PEM film comprising PAH and PAA is assembled at low
pH (pH 3.0) and high ionic strength (150 mM) conditions, there are relatively few
crosslinking between COO- groups of PAA and NH3+ groups of PAH due to the
protonation of COO- groups and the charge screening of COO- and NH3+ groups. Hence,
the PAA polyelectrolytes in the PEM film take a loopy conformation.17, 23-24
Subsequently, when the PEM film is exposed to a higher pH (pH 7.0) and lower ionic
strength (10 mM NaCl) solution, COO- groups become fully deprotonated and the
charges of the COO- and NH3+ groups are not as highly screened as before. Thus, the
PAA polyelectrolytes within the PEMs begin to repel each other due to electrostatic
repulsion and take an extended conformation.17, 23-24 This change of PAA conformation
results in a considerably swollen and highly hydrated PEM structure.23-24 Interfacial
force measurements conducted with the use of an atomic force microscope (AFM) in our
previous study also confirmed that the interactions between carboxylate-modified latex
colloidal probes, which were used as surrogates for bacterial cells, and PAH/PAA PEM-
modified membranes were highly repulsive compared to that between the colloidal
probes and base membranes due to the hydrated, swollen structure of the PEM film.17
92
Therefore, the strong repulsion exerted by the PEM films was expected to inhibit the
strong adhesion of the bacteria to the PEM- and AgNP/PEM-modified membranes.
In addition to the determination of bacterial deposition kinetics, the reversibility
of bacterial deposition on the membrane surface was examined by comparing the removal
efficiencies between the membranes modified with PEMs and AgNPs and the base
membranes. After bacterial deposition had taken place at 10 mM NaCl, the membranes
were subsequently rinsed with a 10 mM NaCl solution, followed by a 1 mM NaCl
solution (Figure 3.4a). Figure 3.4c shows the removal efficiencies obtained from both
release stages, Stage 1 and Stage 2, for the base membrane, PEM-modified membranes
(Membrane P), and AgNP/PEM-modified membranes (Membranes P20 and P43). The
AgNP/PEM- and PEM-modified membranes exhibited very similar removal efficiencies
for Stage 1 and Stage 2. For these modified membranes, the removal efficiencies for
Stage 1 and Stage 2 were ca. 80 % and 90 %, respectively, while almost no removal of
deposited bacteria was observed for the base membranes. The significant increase in the
removal efficiencies through the AgNP/PEM-modification of the membranes clearly
demonstrates that the PEMs can substantially weaken the adhesion of bacteria to
membrane surfaces17 and that the incorporation of AgNPs in the PEMs does not impact
the anti-adhesive properties of the membranes. Depending on the AgNP concentrations
on the modified membranes, the bacteria that remained on the membrane surface will be
inactivated by the AgNPs. The combinatorial anti-adhesive and antimicrobial properties
of the modified membranes are expected to retard the development of biofilms on the
membrane surface.
93
5 6 7 80
10
20
30
40
Mem
bran
e P43
Mem
bran
e P20
Mem
bran
e P
kobs (m
/s)
Bas
e M
embr
ane
(b)
0 20 40 60 80 1000
1000
2000
3000
4000
Right before flushing
Flush with 1 mM NaCl
De
po
site
d B
acte
ria
(p
er
mm
2)
Time (min)
Flush with 10 mM NaCl
(a)
Figure 3.4 (a) Number of bacteria on Membrane P43 during the deposition and release
stages. The deposition experiment was conducted at 10 mM NaCl and a permeate flow
rate of 30 µm/s. The membrane was subsequently rinsed with a 10 mM NaCl solution,
followed by a 1 mM NaCl solution, in the absence of permeate flow. For the deposition
94
and release stages, the pH was maintained at 7.0. (b) Bacterial deposition rates, kobs, for
base membrane, Membrane P, Membrane P20, and Membrane P43. (c) Bacterial
removal efficiencies for base membrane, Membrane P, Membrane P20, and Membrane
P43 after deposition when rinsed with 10 mM NaCl and 1 mM NaCl solutions. Error
bars represent standard deviations.
3.3.4. Bacterial deposition and release over three cycles of filtration and rinsing
Three cycles of membrane filtration and rinsing were conducted to evaluate the
long-term effectiveness of PEM- and AgNP/PEM-modifications on the membrane’s anti-
adhesive properties. The number density of deposited bacteria on the base membrane
throughout the three cycles of filtration and rinsing was presented in Figure 3.5a, while
the number densities of deposited bacteria on the PEM- and AgNP/PEM-modified
membranes (Membranes P and P43, respectively) were presented in Figure 3.5b. The
result showed that there were fewer bacteria deposited on the PEM- and AgNP/PEM-
modified membranes at any time compared to the base membrane. At the end of the
three cycles of bacterial deposition and release, the number density of deposited bacteria
on the base membrane, Membrane P, and Membrane P43 was 8685, 5323, and 5062 per
mm2, respectively.
For the base membranes, no removal of deposited bacteria was observed at the
end of all three release processes. In contrast, the removal efficiencies for Membrane P
at the end of the first, second, and third release processes were 61.3 %, 40.2 %, and 21.6
%, respectively. The removal efficiencies for Membrane P43 at the end of the first,
second, and third release processes were 59.4 %, 40.0 %, and 26.0 %, respectively.
Therefore, the PEM- and AgNP/PEM-modified membranes exhibited considerably
95
higher bacterial removal efficiencies compared to the base membranes over the three
cycles of bacterial deposition and release, demonstrating that both PEM- and
AgNP/PEM-modifications have the potential to impart bacterial anti-adhesive properties
to the membranes over multiple cycles of filtration and rinsing. The bacterial removal
efficiencies on Membrane P and P43 were noted to decrease over the three cycles of
filtration and rinsing, probably due to the slight deterioration of the PEM films over the
repeated filtration and rinsing process.
96
0 10 20 30 40 50 60 70 800
2000
4000
6000
8000
10000Cycle 3
Deposited B
acte
ria (
per
mm
2)
Time (min)
Cycle 1
(a)
Cycle 2
0 10 20 30 40 50 60 70 800
2000
4000
6000
8000
10000
Cycle 3
Cycle 2
Membrane P
Membrane P43
Deposited B
acte
ria (
per
mm
2)
Time (min)
(b)
Cycle 1
Figure 3.5 Number of bacteria on (a) base membrane and (b) Membranes P and P43 over
three-cycles of bacterial deposition and release. For each cycle, bacterial deposition took
place at 10 mM NaCl in the presence of a permeate flow rate of 30 µm/s. The membrane
was subsequently rinsed at 10 mM NaCl in the absence of permeate flow. The pH was
maintained at 7.0 over the three cycles of deposition and release.
97
In order to explore the possibility of a further enhancement in bacterial removal
efficiencies for the PEM-modified membranes, the procedure for the assembly of PEMs
was slightly modified. More concentrated PAH and PAA solutions were used during the
modification, which likely resulted in the formation of a thicker PEM film on the
membrane (Membrane PM). Additionally, during the rinsing process, the deposited
bacteria were flushed with a 1 mM NaCl rinse solution instead of a 10 mM NaCl solution
in order to maximize the swelling of the PEM and to enhance the electric double
repulsive interaction between the deposited bacteria and the membrane.38
For the preparation of Membrane PM, the concentrations of PAH and PAA
solutions used for membrane modification were 20 mM based on the repeat unit
molecular weight. The ionic strength of both polyelectrolyte solutions was adjusted to
150 mM with NaCl and the pH was adjusted to 3.0 with 1 M HCl. The cross-flow
channel of the flow cell used for membrane modification was 89.0 mm in length, 45.0
mm in width, and 2.5 mm in height. The cross-flow velocity used for PAH and PAA
adsorption was 0.75 mm/s and the duration for each polyelectrolyte adsorption was 12
min. After each polyelectrolyte adsorption, the membrane was flushed with a 150 mM
NaCl and pH 3.0 rinse solution (without polyelectrolytes) at a cross-flow velocity of 2.25
mm/s for 12 min to flush away the excess polyelectrolytes from the membrane surface.
Two bilayers of PAH and PAA were assembled on the membrane surface.17
The procedure for the rinsing stages during the three-cycle bacterial deposition
and release experiment for Membrane PM is provided as follows. First, both of the gear
pump for providing the cross flow across the CMF cell and the peristaltic pump for
generating the permeate flow were turned off immediately the deposition stage.
98
0 20 40 60 80 1000
2000
4000
6000
8000
10000
Cycle 3Cycle 2
Deposited B
acte
ria (
per
mm
2)
Time (min)
Cycle 1
Following that, the bacterial suspension in the pressure vessel was replaced with a 1 mM
NaCl and pH 7.0 rinse solution (no bacteria). The system was re-pressurized and the gear
pump was turned on to initiate the release stage. The duration for each release stage was
10 min.
In this three-cycle filtration and rinsing experiment, the number density of
deposited bacteria on Membrane PM at the end of three cycles of bacterial deposition and
release was 688 per mm2 (Figure 3.6), which was significantly lower than those on the
base membrane (Figure 3.5a), as well as Membranes P and P43 (Figure 3.5b).
Furthermore, the removal efficiencies at the end of the first, second, and third release
processes were 97.6 %, 96.3 %, and 86.9 %, respectively, which were substantially
higher than the removal efficiencies for Membranes P and P43. These results imply that
the PEM modification of membranes can allow for high bacterial removal efficiencies
over multiple cycles of filtration and cleaning through the optimization of the conditions
for PEM assembly and membrane rinsing.
99
Figure 3.6 Number of bacteria on Membrane PM over three-cycles of bacterial deposition
and release. For each cycle, bacterial deposition took place at 10 mM NaCl in the
presence of a permeate flow rate of 30 µm/s. The membrane was subsequently rinsed at
1 mM NaCl in the absence of permeate flow. The pH was maintained at 7.0 over the
three cycles of deposition and release.
3.4. Conclusion
The antimicrobial and bacterial anti-adhesive properties of AgNP/PEM-modified
PSU membranes were evaluated in this study. The immobilization of low concentrations
of AgNPs (0.043 wt. %) on the membrane surface with the use of PEMs completely
inhibited the growth of bacteria colonies on the membranes. This AgNP loading was
about two orders of magnitude lower than the reported loadings for nanocomposite
membranes with AgNPs incorporated in the membrane matrix since the surface
immobilization of AgNPs with PEMs dramatically enhanced the opportunities for the
direct contact or close proximity between the AgNPs and deposited bacteria.
Furthermore, the modification of membranes with AgNPs and PEMs was shown to
reduce the bacterial deposition kinetics by about 50 % and increase the reversibility of
bacterial deposition to over 90 %, likely due to the strong repulsive forces exerted by the
hydrated and swollen PEMs on the depositing bacteria. Additionally, the PEM- and
AgNP/PEM-modified membranes exhibited considerably higher bacterial removal
efficiencies compared to the unmodified membranes over the three cycles of bacterial
deposition and release, demonstrating that both PEM- and AgNP/PEM-modifications
100
have the potential to impart bacterial anti-adhesive properties to the membranes over
multiple cycles of filtration and rinsing.
3.5. Acknowledgements
This work was funded by the National Science Foundation (CBET-1133559) and
the Johns Hopkins Water Institute. L.T. acknowledges funding support from the Dean
Robert H. Roy and Gordon Croft fellowships. We acknowledge Ji Yeon Hong from the
Department of Geography and Environmental Engineering at Johns Hopkins University
(JHU) for her assistance with the experiments. The SEM images of the membranes are
taken by Dr. Michael McCaffery from the Integrated Imaging Center, Department of
Biology (JHU).
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104
Chapter 4. Polysulfone Membranes Modified with
Bioinspired Polydopamine and Silver
Nanoparticles Formed in situ to Mitigate
Biofouling *
* Most of the sections in this chapter have been accepted as a manuscript of the same title
with co-authors Kenneth J. T. Livi and Kai Loon Chen to Environmental Science &
Technology Letters. Co-author Kenneth J. T. Livi helped with the SEM imaging and
EDX analysis. Co-author Kai Loon Chen helped with experimental data interpretation
and manuscript editing.
105
4.1. Introduction
Membrane filtration has become one of the most popular technologies for water
purification and wastewater reuse due to its efficiency and effectiveness.1-4 However,
biofouling, or the formation of biofilms on membranes, has been a major obstacle that
hinders their widespread application in water treatment.5-8 Current efforts to mitigate
biofouling have been placed on modifying membrane surfaces by enhancing their
hydrophilicity.9-13
Polydopamine (PDA) is a bioinspired polymer with a molecular structure similar
to the adhesive proteins of mussels.14 PDA is highly hydrophilic due to the presence of
catechol, quinone, and amine groups in its structure.15 In addition, PDA can adhere
firmly to a wide variety of materials in the wet environment through covalent bonding,
hydrogen binding, π-π stacking, metal coordination or chelation, and/or charge-transfer
complexing.15 These unique features of PDA have been leveraged to enhance membrane
hydrophilicity for use in membrane filtration.14-20
Due to the presence of drag forces resulting from water permeation during
membrane filtration, some microorganisms may still deposit on hydrophilic membranes.5,
21 Therefore, it is also desirable to impart strong antimicrobial properties to the
membranes in order to inactivate deposited bacteria. Recently, numerous studies have
examined the effectiveness of silver nanoparticles (AgNPs) in mitigating membrane
biofouling by taking advantage of their strong and broad-spectrum antimicrobial
properties.22-34 Interestingly, Ag+ ions can be reduced by the catechol groups of PDA,
resulting in the in situ formation of AgNPs on PDA-modified surfaces.15, 35 Furthermore,
the O- and N-sites of PDA can serve as anchors for the AgNPs through metal
106
coordination via charge-transfer.36-37. Hence, the generation and immobilization of
AgNPs on PDA-modified membranes can pave a new way to impart membranes with
both anti-adhesive and antimicrobial properties simultaneously to mitigate membrane
biofouling.
In this paper, we show for the first time that the surface modifications with PDA
and AgNPs formed in situ can reduce the polysulfone (PSU) membrane’s propensity to
bacterial adhesion and growth. Specifically, PSU membranes were modified with a PDA
film to enhance membrane hydrophilicity and reduce bacterial attachment. The PDA-
modified membranes were then exposed to AgNO3 solutions to generate AgNPs in situ
on the membrane surfaces, thus imparting the membranes with strong antimicrobial
properties. This facile and scalable membrane surface modification method using
bioinspired PDA and AgNO3 solution to enhance membranes’ bacterial anti-adhesive and
antimicrobial properties simultaneously has great potential for membrane biofouling
mitigation for water filtration processes.
4.2. Materials and Methods
4.2.1. Polysulfone membrane fabrication
PSU microfiltration membranes were fabricated using the wet phase inversion
process38 and were used as the base membranes for the preparation of PDA-modified
membranes. PSU beads (Udel P3500; Solvay Advanced Polymers) were first rinsed and
cleaned with deionized (DI) water (Millipore, Billerica) and then dried at 50 °C. To
prepare a casting solution, the PSU beads and LiCl powder (anhydrous, ≥ 99%; Sigma-
Aldrich) were dissolved in 1-methyl-2-pyrrolidinone (NMP, ≥ 99%; Sigma-Aldrich) at
55 °C by continuous stirring for at least 24 h. The final composition of the casting
107
solution was 15 % PSU, 2 % LiCl, and 83 % NMP by weight. The casting solution was
then stirred at room temperature (23 °C) until it cooled down and the solution was
degassed by allowing it to stand at room temperature for at least 72 h. To fabricate a
PSU membrane, the casting solution was spread on the edge of a glass plate and the PSU
membrane film was cast using a stainless steel casting knife (Elcometer 3530 Motorised
Film Applicator; Elcometer Asia Pte. Ltd.) at a gate height of 60 μm. The glass plate
with the thin membrane film was immediately transferred to a DI water bath at room
temperature to initiate the phase inversion process. The membrane fabricated through
this phase inversion process was then transferred and stored in fresh DI water for at least
24 h before use. Bacterial filtration tests were performed to verify that the PSU
membranes can achieve 100 % rejection of Escherichia coli cells used in this study.
4.2.2. Membrane modification with polydopamine
The modification of PSU membranes with PDA was performed using a custom-
made polycarbonate flow cell. A PSU membrane was clamped tightly between the top
and bottom plates of the flow cell with the active side of the membrane facing the cross
flow channel (90 × 38 × 2 mm). Dopamine hydrochloride powder (0.1 g; Sigma-Aldrich)
was dissolved in 100 mL of a 15 mM Trizma hydrochloride (≥ 99.0%; Sigma-Aldrich)
buffer solution with pH adjusted to 8.5. The chemical structure of dopamine is presented
in Figure 4.1. Under this condition, dopamine can be oxidized by oxygen and self-
polymerize to form PDA.15 The PDA solution was circulated across the membrane
surface using a peristaltic pump (Cole Parmer, Vernon Hills, IL) at a cross flow velocity
of 2.2 mm/s for 6 h. Following that, the membrane surface was rinsed twice (10
min/rinse) with the buffer solution at the same cross flow velocity. Finally, the
108
Base Membrane Surface
PDA/AgNP- Modified Surface
PDA-Modified Surface
Coating with PDA
Exposure to AgNO3 solution
membrane was removed from the cross flow cell and rinsed under running DI water for
30 s.
Figure 4.1 Chemical structure of dopamine.
4.2.3. In situ formation of AgNPs on polydopamine-modified membranes
In order to generate AgNPs on the membrane surface, a PDA-modified membrane
was allowed to float on a 50 mM AgNO3 solution (pH unadjusted, volume 25 mL)
contained in a petri dish, with the active side of the membrane contacting the solution.
The petri dish was covered with a piece of alumina foil to prevent exposure to light. The
exposure time to AgNO3 solution was varied (1 min, 1 h, 2 h, 12 h, and 24 h) to
investigate its effects on AgNP generation on the membranes. The membranes were then
soaked in fresh DI water three times (at least 10 min for each soaking) before use. The
membrane surface modification process is illustrated in Figure 4.1.
109
Figure 4.2 Schematic diagram of PDA modification and in situ formation of AgNPs on
the membrane surface and environmental SEM image of PDA-720 membrane. White
scale bar represents 5 μm.
4.2.4. Membrane characterization
The water contact angle measurements of PSU and modified membranes were
performed at room temperature using the sessile drop method39 on an optical CAM100
contact angle meter (KSV Instruments Ltd, Finland). The contact angle for each water
(DI) droplet was the average of the contact angles on the left and right sides of the water
droplet. Measurements for each membrane were conducted on at least four water
droplets. The volume of each water droplet was 10 μL.
X-ray photoelectron spectroscopy (XPS) analysis of the membranes was
conducted with a PHI 5600 XPS system using Mg Kα X-rays to determine the elemental
composition on the membrane surface. The pass energy used to perform the survey scans
to determine the elemental composition on the membrane surface was 58.7 eV at a scan
rate of 1.000 eV/step.
The membranes were also examined with Environmental scanning electron
microscopy (SEM) imaging to determine the distribution and morphologies of the AgNPs
on the membrane surfaces. Membrane samples were coated with 5 nm of carbon and
examined in a JEOL 8600 Superprobe at 15 kV. Backscattered electron (BSE) images
and energy-dispersive X-ray (EDX) analyses were collected on each sample. Since BSE
110
intensities are a function of mean atomic number and density, smaller particles will have
lesser intensities than larger particles. Therefore, contrast settings for BSE images were
kept constant for all images in order to compare particle density and size differences
between different membranes. The BSE detector in the JEOL 8600 is an annular detector
designed to minimize topographic effects of the BSE signal. However, this cannot be
completely removed and some of the intensity differences between samples will be due to
particle roughness that increases as particles increase in size. BSE imaging is also
capable of detecting particles that are underneath or inside the PDA coatings.
Additionally, the morphology of selected membrane surfaces were examined by
secondary electron (SE) imaging using a low-vacuum Quanta 200 Environmental SEM
(FEI, Hillsboro, OR). Briefly, membrane samples were dried in vacuum; mounted onto
aluminum stubs and imaged at room temperature at 1.8 kV, at a pressure of 100 Pa, a
working distance of 5 mm, and a spot size of 3.0. Here, the topography of particles is
emphasized and particles covered by the PDA will not be detected.
The AgNP mass loadings of the modified membranes were determined by
soaking the membrane samples in 3.5% HNO3 solution and measuring the dissolved Ag
concentrations with an atomic absorbance spectrometer (AAS).25, 34 Specifically, A
coupon (4.9 cm2) cut from a AgNP-modified membrane was soaked in a Pyrex glass
bottle containing 20 mL of 3.5 % HNO3 solution. The solution was then stirred
rigorously on a magnetic stirring plate to fully dissolve the AgNPs on the coupon.25, 34
After 8 days of stirring, samples were withdrawn from the glass bottle and filtered
through a 0.1 μm PVDF filter unit (Millex-VV, Merck Millipore Ltd.). The
concentrations of the dissolved Ag in the samples were measured with an AAS
111
(AAnalyst 100, Perkin Elmer). The AgNP mass loadings for the modified membrane
were then calculated using the dissolved Ag concentrations.
4.2.5. Anti-adhesive properties of membranes
The bacterial deposition experiments were performed with a direct microscopic
observation membrane filtration system at room temperature. The bacteria strain used
was E. coli K12 MG 1655.21 The bacteria carry the antibiotic resistance gene and are
labeled with the green fluorescent protein which enables the bacteria cells to be observed
with the aid of an epifluorescence microscope (Nikon Eclipse E600W, Japan). The E.
coli cells were incubated in a 25 g/L Luria Bertani (LB) broth (Fisher Scientific) that
contained 50 mg/L kanamycin (Sigma-Aldrich) at 37 ºC for ca. 3 h until the cells reached
the mid-exponential growth phase. To prepare the bacterial suspension for the
membrane’s antimicrobial property test, the bacterial suspension after 3 h incubation was
serial-diluted with 154 mM NaCl solution (pH unadjusted) to ca. 240 cells per mL. To
prepare the bacterial suspension for the membrane’s bacterial anti-adhesive property test,
the E. coli cells harvested at the mid-exponential growth phase were washed twice with
154 mM NaCl solutions by ultracentrifugation at 4 ºC. The detailed washing procedure
for the bacterial cells was provided in our previous study.13 The final bacterial cell
concentration in the feeding suspension for the membrane’s anti-adhesive property test
was ca. 1.4 × 107 cells/L.
A cross flow membrane filtration system coupled with an epifluorescence
microscope was employed to observe the E. coli cell deposition on the membrane surface
in real time.13 This closed-loop system was first pressurized and stabilized with
compressed nitrogen gas at ca. 25 psi. The feed solution was circulated at a cross flow
112
velocity of 10 cm/s using a gear pump (Cole-Parmer, Vernon Hills, IL) through the cross
flow cell and back to the pressure vessel (Alloy Products, Waukesha, WI). The
permeation was maintained constant and circulated back to the pressure vessel using a
peristaltic pump (Cole-Parmer, Vernon Hills, IL). The membrane to be tested was
clamped between the top and bottom plates of the cross flow cell. The active side of the
membrane faced the cross flow channel and the dimension of the flow channel was 76
mm in length, 25 mm in width, and 1 mm in height. The glass window on top of the
cross flow cell enabled the real time observation of the E. coli cell deposition on the
membrane surface. The cross flow cell was placed on the stage of the epiflorescence
microscope with a 10× objective lens. A digital camera (Roper Scientific, Photometrics
CoolSnap ES, Germany) was used to acquire the images of deposited E. coli cells on the
central part (107078 μm2) on the membrane surface in real time. The membrane was
conditioned and equilibrated with a 10 mM NaCl and pH 7.0 (adjusted with 0.15 mM
NaHCO3) solution at a cross flow velocity of 10 cm/s and permeate flux of 26 μm/s for
40–50 min. The E. coli cells were then injected into the pressurized membrane filtration
system using a syringe pump to initiate bacterial deposition in the same solution
chemistry and hydrodynamic conditions. Each deposition experiment was carried out for
20 min and the images of E. coli cells were acquired every 3 min. The deposition rate
coefficient, kobs, was calculated by normalizing the rate of bacterial deposition to the
product of membrane surface area and cell concentration in the feed solution (ca. 1.4 ×
107 cells/L).5, 13 The bacterial deposition experiments were conducted at least three times
for each type of membrane.
113
4.2.6. Antimicrobial properties of membranes
The antimicrobial properties of the modified membranes were assessed using the
colony forming unit (CFU) counting method.34 A circular membrane coupon (diameter =
4.1 cm) was placed on top of the glass support of a vacuum filtration setup (Millipore,
Billerica, MA) with the active side facing up. A diluted E. coli suspension (25 mL; ca.
240 cells/mL) prepared in 154 mM NaCl (pH unadjusted) was filtered through the
membrane by applying vacuum. The filtration took 8–10 min and the permeate flux
during filtration was 31.6–39.5 μm/s. After filtration, the membrane coupon was placed
on an agar plate (25 g/L LB, 15 g/L agar, and 50 mg/L kanamycin) with the back-side of
the membrane resting on the agar. The agar plate was incubated at 37 °C for ca. 15 h and
the CFUs on the central part of the membrane (area of 4.9 cm2) were enumerated the
following day. Triplicate tests were performed for each membrane.
4.2.7. Stability of AgNPs immobilized on membranes
A membrane was placed on top of the glass support of a vacuum filtration setup
with the active side facing up. DI water (750 mL) was filtered through the membrane for
220–340 min at an average permeate flux of 34.4 μm/s, which is in the typical range of
flux for MF processes.40 The filtrate was collected and diluted in 3.5 % HNO3. The Ag
concentration was analyzed using an inductively coupled plasma mass spectrometry
(ICP-MS) instrument (PerkinElmer ICP Mass Spectrophotometer NexION 300D). The
antimicrobial properties of selected modified membranes were assessed by the CFU
counting method after filtration. A 25 mL of diluted bacterial suspension (ca. 240
cells/mL) was filtered through the membrane and then the membrane coupon was placed
on an agar plate, with the back-side of the membrane resting on the agar. The CFU on
114
the central part of the membrane (an area of 4.9 cm2) were counted after ca. 15 h
incubation. The procedures of the diluted bacterial suspension preparation and bacterial
suspension filtration by the vacuum filtration can be found in the earlier sections 4.2.5
and 4.2.6. The leaching tests and antimicrobial tests were performed three times for each
membrane.
4.3. Results and Discussion
4.3.1. AgNP Mass Loading Increases with Exposure Time to AgNO3 solutions
The designations used in this paper are “PDA membranes” for membranes
modified with PDA only and “PDA-t membranes” for PDA-modified membranes
exposed to AgNO3 solutions for a duration of t min. The elemental composition of the
membrane surfaces was analyzed by XPS (Figure 4.2). The spectra of all the modified
membranes showed similar signal intensities in the N(1s) region, all of which were higher
than that of the base membrane. This observation confirmed the formation of a PDA film
on all the modified membranes since nitrogen is present in the PDA. All the modified
membranes also exhibited a noticeably lower signal intensities in the S(2p) region
compared to the base membrane, likely due to the sulfone groups in the base PSU
membrane being suppressed by the PDA film.
115
365 370 375 380
365 370 375 380
-100000
0
100000
200000
300000
Base
PDA
PDA-1
PDA-60
PDA-120
Ag(3d)
PDA-720
166 168 170 172
166 168 170 172
x 5S(2p)
396 399 402 405
396 399 402 405
N(1s) x 5
Figure 4.3 N(1s), S(2p), and Ag(3d) XP spectra of the surface of the base and modified
membranes.
Strong signal intensities in the Ag(3d) region were clearly observed in the XPS
spectra of all the PDA/Ag-modified membranes, except for the PDA-1 membrane in
which the Ag concentration was probably too low to be detected. Secondary electron (SE)
imaging in the Environmental SEM also revealed the presence of individual spherical
AgNPs on the surface of the modified membranes (Figure 4.2). In contrast, no detectable
signal in the Ag(3d) region was observed for the PSU and PDA membranes (Figure 4.3).
The individual particles scarcely present on the PDA membrane surface were likely to be
PDA aggregates. Additionally, the signal intensities in the Ag(3d) region for the
PDA/Ag-modified membranes increased as the membrane exposure time was increased
(Figure 4.3). These findings indicated that the AgNP mass loading can be controlled by a
simple variation of the exposure time to the AgNO3 solutions and potentially the
concentrations of the AgNO3 solutions.
Binding Energy (eV)
116
Backscattered electron (BSE) SEM images in Figure 4.4 indicated that some of
the pores of the PSU membrane appeared to be covered by the PDA film. Additionally,
bright spots were detected in the images of the PDA/Ag-modified membranes. Through
EDX analysis, the Ag signal on the bright spots was shown to be high while the Ag signal
on the dark spots was almost undetectable (Figure 4.5), thus demonstrating the bright
spots to be the locations of AgNPs. It is noteworthy that BSE imaging, which uses high
energy electrons, is capable of detecting AgNPs that are underneath or inside the PDA
film, unlike SE imaging which uses low energy electrons and can only provide images of
AgNPs exposed on the membrane surface. The SEM image of PDA-720 (Figure 4.2)
appears to show fewer AgNPs than the BSE image (Figure 4.4), which is consistent with
AgNPs located both on the surface of and inside the PDA film.
117
Figure 4.4 BSE SEM images of base and modified membranes. Recording contrast and
brightness levels where held constant for all images in order to insure proper BSE
intensity comparisons between samples. White scale bars represent 2 μm.
Base Membrane PDA Membrane PDA-1
PDA-60 PDA-120 PDA-720
PDA-1440
(a) (b)
118
Figure 4.5 BSE SEM imaging and EDX analysis of PDA-1440 membrane. (a) BSE SEM
image of PDA-1440 membrane with white circle indicating location for EDX analysis
(bright spot). (b) EDX spectrum of bright spot. (c) BSE SEM image of PDA-1440
membrane with white circle indicating location for EDX analysis (dark spot). (b) EDX
spectrum of dark spot.
From the images in Figure 4.4, the distribution of AgNPs on the modified
membrane surfaces was shown to be homogeneous. Also, the AgNPs increased in size
and number as a function of membrane exposure time to AgNO3 solutions. The AgNP
mass loading of the membranes were determined by soaking the membranes in HNO3
solutions. AAS analysis of the acid solutions showed that the AgNP concentrations in
the membranes increased as a function of exposure time to the AgNO3 solutions (graph in
(c) (d)
119
0 5 10 15 20 25
0 5 10 15 20 25
0
4
8
12
16
20
Mass D
ensity (
g/c
m2)
Exposure Time (h)
0.0
0.4
0.8
1.2
1.6
Weig
ht P
erc
enta
ge (w
t. %)
Figure 4.6), which corroborated with the results from XPS and SEM analyses (Figures
4.3 and 4.4, respectively). Specifically, the AgNP mass loading on the membrane surface
increased dramatically within 60 min and increased slowly afterwards. It is speculated
that most of the AgNPs nucleated quickly on the membrane surface because of the strong
reducing catechol groups in PDA while the increase of AgNP mass after 60 min might be
due to slower post nucleation ripening mechanisms while Ag+ is further reduced.15
Figure 4.6 Mass loadings of AgNPs on modified
membrane surfaces.
4.3.2. Surface modifications enhance anti-adhesive properties
Water contact angle measurements on the membranes showed that surface
modifications with PDA, as well as with PDA and AgNPs, reduced the membranes’
contact angles by ca. 50 % compared to that of the base membrane (Figure 4.7) and thus
effectively enhanced their hydrophilicity.41-42 Furthermore, the contact angles on the
membranes that were modified with PDA and AgNPs were independent of the AgNP
120
2 4 60
20
40
60
80
PDA-7
20
PDA-1
20
PDA-6
0
PDA-1
PDA
Conta
ct A
ngle
Bas
e
mass loadings, thus indicating that the enhanced hydrophilicity of the modified
membranes can be attributed to the PDA films.
Figure 4.7 Contact angle measurements of selected membranes.
During the bacterial deposition experiments, the hydraulic resistance of the PSU
base membranes was determined to be 3.0 × 1011 m-1, while the hydraulic resistances of
the PDA, PDA-1, PDA-60, and PDA-720 membranes were 9.6 × 1011 m-1, 1.0 × 1012 m-1,
9.8 × 1011 m-1, and 1.1 × 1012 m-1, respectively. The hydraulic resistances of all the
modified membranes were ca. 3 times higher than that of the base membrane regardless
of the Ag mass loadings. Hence, the major contributor to the increase in hydraulic
resistance was the PDA film. The deposition experiments showed that PDA and
PDA/AgNP modifications reduced the bacterial deposition kinetics on the membrane
surface by ca. 60 % (Figure 4.8). These results demonstrate that the PDA and
121
1 2 3 4 50
10
20
30
40
50
PDA-7
20
PDA-6
0
PDA-1
PDA
ko
bs (m
/s)
Bas
e
PDA/AgNP modifications considerably enhanced the membrane’s resistance to bacterial
adhesion. Additionally, the kobs values of the modified membranes were comparable and
independent of the AgNP mass loadings. Since the hydrophilicity of all the modified
membranes had increased considerably (Figure 4.7), the enhanced bacterial anti-adhesive
properties exhibited by the modified membranes is attributed to the hydrophilic PDA
films.18 A slight increase in the Ag concentration of 0.85 μg/L in the circulated solution
was detected at the end of a separate cross flow filtration experiment using a PDA-60
membrane. The leaching of Ag was investigated and will be discussed in a later section.
Figure 4.8 Bacterial deposition rate coefficients, kobs, for selected membranes.
4.3.3. In situ generated AgNPs inhibited bacterial growth on membranes
Using the CFU-counting method, 257 CFUs were observed on the PDA
membrane while only 4 CFUs were observed on the PDA-1 membrane (Figure 4.9). No
CFUs were observed on the PDA-60, PDA-120, PDA-720, and PDA-1440 membranes.
122
The result indicated that the in situ formation of AgNPs by exposing the PDA membranes
to AgNO3 solutions for 1 hour or longer can ensure the complete inactivation of bacteria
cells. Despite the incomplete inactivation of E. coli cells, the PDA-1 membrane had a
comparable antimicrobial effect (close to 99 %) with that in other two studies that used a
similar method to evaluate the membrane’s antimicrobial properties.23, 34 In both
studies,23, 34 0.9 wt. % AgNPs were embedded in the membrane matrix and their results
showed that the AgNP-impregnated membranes had a 99 % reduction of E. coli cell
growth.23, 34 In comparison, the similar antimicrobial effect achieved with a lower AgNP
weight percentage in our study (0.12 ± 0.02 wt. %) implied that the membrane’s
antimicrobial properties are greatly dependent on the location of AgNPs in the membrane
structure. Recent studies demonstrated that the direct contact or close proximity of
bacteria to AgNPs immobilized on the membrane surface maximizes the exposure of the
bacteria to AgNPs by increasing the lethal concentrations of free released Ag+ ions
dissolved from AgNPs.43-46 In contrast, not all the AgNPs embedded in the membrane
matrix can be exposed to the deposited bacteria and thus a higher amount of AgNPs is
required to achieve the same antimicrobial effect of AgNPs immobilized on the
membrane surface. Therefore, the in situ formation of AgNPs on the membrane through
the reduction of Ag+ ions by PDA has proven to be an efficient method to impart the
membranes with strong antimicrobial properties as this approach ensured that AgNPs
generated on the PDA films will have maximum opportunities for contact with deposited
bacteria.
123
2 4 60
100
200
300
400
PDA-1
440
PDA-7
20
PDA-1
20
PDA-6
0
PDA-1
CF
U
PDA
Figure 4.9 CFUs on base and modified membranes. The symbols indicate that
no colony was present on the membranes. Error bars in b, c, and d represent standard
deviations.
4.3.4. Stability of AgNPs immobilized on membranes
Ag leaching test was carried out to quantify the degree of Ag release during
filtration of DI water. The Ag concentrations in the permeates for the PDA-1 and PDA-
60 membranes were 0.29 ± 0.18 and 1.17 ± 0.60 μg/L, respectively. The average Ag
concentrations in the permeates in the Ag leaching tests from the studies of Diagne et
al.47 and Yin et al.33 on AgNP-nanocomposite membranes were 5 μg/L and 1.06 μg/L,
respectively. Therefore, the leaching Ag concentrations in our study were comparable to
those of previous studies.33, 47 The concentrations of Ag in the permeates in our study
were 2–3 orders of magnitude lower than the maximum contaminant limit of Ag in
drinking water (i.e., 100 μg/L) established by the U.S. Environmental Protection
Agency48 and also by the World Health Organization.49 Therefore, there will likely be no
124
risk to health related to Ag ingestion if this PDA/AgNP membrane modification
technique were to be applied to mitigate membrane biofouling for water filtration.
Additionally, the antimicrobial properties of the PDA-1 and PDA-60 membranes
were examined after prolonged filtration of DI water. Only 2 CFUs were observed on the
PDA-1 membrane surfaces while no CFUs were observed on the PDA-60 membrane
surfaces. These results were comparable to that on the freshly prepared PDA-1 and
PDA-60 membranes (4 and 0 CFUs, respectively). Therefore, the dissolution and loss of
AgNPs were low and the AgNPs immobilized on the membrane surface could impart the
membrane with long-lasting antimicrobial properties.
4.4. Conclusion
In summary, this study showed PSU membranes modified with PDA and AgNPs
formed in situ had enhanced resistance to biofouling over the native PSU membranes.
The PDA film was effective in reducing bacterial adhesion on the membrane surface
while the AgNPs imparted antimicrobial properties. This novel surface modification
technique paves a way to mitigating biofouling by enhancing the membrane’s anti-
adhesive and antimicrobial properties, simultaneously. Additionally, this simple and
efficient approach will enable the in situ modification of existing membranes of different
configurations (such as hollow fiber and spiral wound),50 as well as feed spacers in spiral
wound membranes,51 that are already in use in water and wastewater treatment plants.
This approach may also allow for the in situ regeneration of AgNPs after they have been
depleted through dissolution, thus enabling the sustainable application of nanocomposite
membranes for water treatment.
125
4.5. Acknowledgments
This work was funded by the National Science Foundation (CBET-1133559). L.T.
acknowledges funding support from the Gordon Croft fellowship. We acknowledge Dr.
Michael McCaffery and Anna Goodridge of the Integrated Imaging Center (JHU) for
Environmental SEM imaging and additional microscopy, and Dr. Howard Fairbrother’s
group from the Department of Chemistry (JHU) for XPS and contact angle measurements.
We thank Dr. Qiaoying Wang and Xin Liu from the Department of Geography and
Environmental Engineering (JHU) for the AAS and contact angle measurements. We
also acknowledge Dr. Khanh An Huynh from the Environmental Protection Agency, Las
Vegas, Nevada, for the ICP-MS measurements.
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Chapter 5. Conclusions, Key Contributions, and
Implications
130
5.1. Summary of Key Findings and Conclusions
The investigation of the influence of PAH/PAA multilayers on the bacterial anti-
adhesive properties of commercial PSU membranes was described in Chapter 2.
Specifically, the deposition kinetics and removal efficiencies of E. coli cells on the PEM-
modified membrane were obtained and compared with that on the base membrane. The
influence of calcium on the bacterial anti-adhesive properties of the PEM-modified
membrane was also studied in this chapter. In addition, the mechanism for the enhanced
bacterial anti-adhesive properties of the PSU membrane after PEM modification was
studied through AFM force measurements between a CML colloid (used as a bacterial
cell surrogate) and the membrane surface. The antimicrobial properties of a commercial
PSU membrane modified with PAH/PAA multilayers and AgNPs were investigated in
Chapter 3. The influence of AgNP mass loadings on the membrane’s antimicrobial and
bacterial anti-adhesive properties was also presented in this chapter. Furthermore, the
results of three-cycle filtration and rinsing experiments were provided to demonstrate the
effectiveness of the PEM- or AgNP/PEM-modifications on the bacterial adhesion over an
extended time period of use. Chapter 4 described the influence of PDA coating on a
laboratory-cast PSU membrane’s bacterial anti-adhesive properties. The effect of the in
situ formation of AgNPs on the antimicrobial and bacterial anti-adhesive properties of
PDA-modified membranes was also investigated in this chapter.
As discussed in Chapter 2, the deposition kinetics and removal efficiencies of E.
coli cells on the membrane surface were determined using a direct microscopic
observation membrane filtration system. The modification of the PSU membrane with
PAH/PAA PEMs can enhance the membrane’s bacterial anti-adhesive properties
131
compared to the base membrane. Specifically, in the presence of 10 mM NaCl and pH
7.0, PEM modification reduced bacterial deposition kinetics by about half and
dramatically increased the removal efficiencies from <10 % to 99 %. In the presence of
calcium (1 mM CaCl2 + 7 mM NaCl, pH 7.0), the effect of PEM modification on
membrane’s bacterial anti-adhesive properties was slightly reduced – the bacterial
deposition kinetics was reduced by about half while the removal efficiencies was only
increased from <10 % to 68 %. AFM force measurements showed that PEM
modification resulted in the strong, long-ranged repulsive interactions between the
colloid probe and PEM-modified surfaces while slightly adhesive interactions were
observed between the CML colloid probe and base membrane surfaces. The bacterial
anti-adhesive properties of the PEM-modified membranes were attributed to the highly
swollen and hydrated structure of the PEMs. However, the COO--Ca2+ complex
formation between the carboxyl groups of the PAAs and calcium led to a compact and
less hydrated PEM structure and thus reduced the repulsive interactions between the
bacterial cell and PEM-modified membrane.
The vacuum filtration set-up was used to evaluate the influence of AgNPs and
PAH/PAA PEM modifications on the PSU membrane’ antimicrobial properties in
Chapter 3. AgNPs were immobilized on the membrane surface with the use of PEMs and
the nanoparticles completely inhibited the growth of bacterial colonies on the
membranes, even at a low mass loading of 0.043 wt. %. This AgNP loading was about
two orders of magnitude lower than the reported loadings for nanocomposite membranes
with AgNPs incorporated in the membrane matrix. This result provided evidence that the
surface immobilization of AgNPs with PEMs can dramatically enhance the opportunities
132
for the direct contact or close proximity between the AgNPs and deposited bacteria and
thus result in a much stronger antimicrobial effect. Furthermore, the AgNP/PEM
modifications reduced the bacterial deposition kinetics by about half and increased the
removal efficiencies to over 90 %. This could be contributed to the strong repulsive
forces exerted by the hydrated and swollen PEMs on the deposited bacteria. In addition,
the PEM- and AgNP/PEM-modified membranes exhibited considerably higher bacterial
removal efficiencies compared to the unmodified membranes over the three cycles of
bacterial deposition and release, demonstrating that both PEM- and AgNP/PEM-
modifications have the potential to impart bacterial anti-adhesive properties to the
membranes over multiple cycles of filtration and rinsing.
Chapter 4 described the influence of the PDA and AgNP modifications on a
laboratory-cast PSU membrane’s ability to mitigate biofouling. By circulating a PDA
solution cross the membrane surface, the PDA film could be formed on the membrane
surface successfully, as confirmed by XPS analysis of the membrane surface. The
AgNPs can be generated in situ on the membrane surface by soaking the membrane in a
AgNO3 solution due to the strong reducing ability of catechol groups in PDA. The
formation of AgNPs was confirmed by BSE SEM imaging and EDX analysis. By
dissolving the AgNPs on the membrane in 3.5 % HNO3 for 8 days, the Ag mass loadings
on membranes were determined through ICP-MS analysis and were found to increase
with membrane soaking time. The increasing AgNP mass with increasing soaking time
was also confirmed by XPS analysis. By measuring and comparing the contact angles
using the sessile method on an optical CAM100 contact angle meter, the PDA-modified
133
membrane was demonstrated to be very hydrophilic regardless of the AgNP mass
loadings.
The bacterial deposition kinetics on the membranes modified with PDA and
PDA/AgNPs was reduced, thus demonstrating that the PDA- and PDA/AgNP-
modifications enhanced bacterial anti-adhesive properties. The reduced bacterial
deposition kinetics on the modified membranes was independent of AgNP mass loadings,
thus indicating that it is likely that the hydrophilic PDA film coated on the membrane
surface contributed to the enhanced bacterial anti-adhesive properties. AgNPs formed in
situ by PDA on the membrane surface imparted strong antimicrobial properties to the
membrane. Soaking of the PDA membrane in a AgNO3 solution even for a duration of 1
min resulted in close to 99 % bacterial inactivation efficiency. In addition, the AgNPs on
the membrane surface were shown to be relatively stable. The prolonged water filtration
(220–340 min of filtration at an average filtration rate of 34 μm/s) did not result in
significant dissolution and loss of AgNPs and the Ag concentrations in the permeates of
the PDA-1 and PDA-60 membranes were 0.29 ± 0.18 and 1.17 ± 0.60 μg/L, respectively,
which were 2–3 orders of magnitude lower than the maximum contaminant limit of Ag
(100 μg/L) in the National Secondary Drinking Water Regulations established by EPA.
Furthermore, the membranes did not lose their antimicrobial properties over a prolonged
period of filtration. In summary, this membrane surface modification technique paves a
way to mitigate membrane biofouling by enhancing membrane’s bacterial anti-adhesive
and antimicrobial properties simultaneously.
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5.2. Principle Contributions
In this dissertation work, the deposition kinetics and removal efficiencies of E. coli
cells on PSU MF membranes that were modified with polymeric thin films, specifically
PAH/PAA PEMs and PDA films, were systematically investigated to better understand
the influence of the thin films on the membranes’ bacterial anti-adhesive properties. The
bacterial deposition and release experiments were conducted in a laboratory direct
microscopic observation membrane filtration system. This system enables the direct
observation of bacterial deposition at the early stages on the membrane surface and the
release of deposited bacteria after the deposition process. Moreover, the antimicrobial
properties of the PSU membrane that was modified by AgNPs by embedding AgNPs in
the PAH/PAA PEMs or through the in situ formation of AgNPs on the PDA film were
systematically studied. Overall, the findings from this dissertation will enable a better
understanding of the application of the polymeric thin films and antimicrobial
nanomaterials for membrane biofouling mitigation in water treatment processes. Specific
contributions of this dissertation work are described below:
Demonstrated for the first time that PAH/PAA PEMs assembled on the PSU
MF membrane surface can reduce the tendency of bacteria to adhere on the
membrane surface. The results from bacterial deposition and release
experiments showed that the highly hydrated PAH/PAA PEM structure impeded
the favorable deposition of the bacterial cells on the PSU membrane.
Furthermore, the results from AFM force measurements indicated that the
interactions between the bacterial cells and the PEM-modified PSU membrane
were highly repulsive. Therefore, the PAH/PAA PEMs can be successfully
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applied on the PSU membrane surface to enhance the membrane’s bacterial anti-
adhesive properties, specifically, to reduce the bacterial deposition kinetics and
increase the removal efficiencies.
Provided evidence that the direct contact or close proximity between AgNPs
and bacterial cells can enhance the antimicrobial properties of AgNP
nanocomposite membranes. AgNPs can be embedded in PAH/PAA PEMs to
impart antimicrobial properties to the PSU membrane. AgNPs can be
immobilized by filtering a AgNP suspension through the PSU membrane and then
assembling PAH/PAA PEMs on top of the AgNPs. The PSU membrane modified
by this AgNP/PEM-assembly exhibited stronger antimicrobial properties than
those reported in other studies by blending AgNPs in the membrane matrix; the
minimum AgNP mass loading that resulted in the complete inhibition of cell
growth was much lower compared to the values reported in other studies. In
addition, this finding is useful for the design and application of antimicrobial
nanomaterials for membrane biofouling mitigation during water filtration.
Demonstrated for the first time that AgNP/PEM- and PEM-modifications
are effective over multiple cycles of filtration. Three-cycle bacterial deposition
and release experiments provided direct evidence that the PEM- and AgNP/PEM-
modifications could impart long-lasting bacterial anti-adhesive properties to the
PSU membrane. Therefore, the membrane surface modifications with PEMs and
AgNPs/PEMs can be considered a promising technique to mitigate biofouling in
the practical membrane filtration processes.
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Demonstrated for the first time that PDA and the in situ formation of AgNPs
can be employed to simultaneously reduce bacterial adhesion and inhibit
bacterial growth. The PDA modification improved the PSU membrane
hydrophilicity and thus enhanced the membrane’s bacterial anti-adhesive
properties. AgNPs generated in situ through the reduction of Ag+ ions by the
catechol groups in PDA could impart the PSU membrane with strong
antimicrobial properties. The dissolution and loss of AgNPs were not significant
and the AgNPs were stable on the membrane surface, exhibiting strong
antimicrobial properties even after prolonged filtration. Therefore, this work
provides a novel and facile technique to modify membrane surface with the
enhanced bacterial anti-adhesive and antimicrobial properties for membrane
biofouling mitigation. Moreover, this technique enables a feasible way to
replenish AgNPs on the membrane surface in situ in water treatment processes.
The list of publications and expected publications from this dissertation work is
presented below:
1) Tang, L., Gu, W. Y., Yi, P., Bitter, J. L., Hong, J. Y., Fairbrother, D. H.
and Chen, K. L., Bacterial Anti-adhesive Properties of Polysulfone
Membranes Modified with Polyelectrolyte Multilayers, Journal of
Membrane Science, 2013, 446, 201-211. (Chapter 2)
2) Tang, L., Huynh, K. A., Chen, K. L., Imparting Antimicrobial and Anti-
Adhesive Properties of Polysulfone Membranes through Modifications
with Silver Nanoparticles and Polyelectrolyte Multilayers (in revision).
(Chapter 3)
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3) Tang, L., Livi, K. J. T., Chen, K. L., Polysulfone Membranes Modified
with Bioinspired Polydopamine and Silver Nanoparticles Formed in situ to
Mitigate Biofouling (accepted by Environmental Science and Technology
Letters). (Chapter 4)
5.3. Implications for Practice
In this dissertation work, the modifications of PSU membranes with PAH/PAA
PEMs and AgNPs or PDA and AgNPs were found to be effective to reduce the bacterial
adhesion on the membrane surface and impart the membrane with strong antimicrobial
properties. These findings will have some implications for practice on the use of the
polymeric thin films (e.g., PEMs or PDA) and antimicrobial nanomaterials (e.g., AgNPs)
for membrane surface modifications to mitigate membrane biofouling in water treatment
processes. Specific implications are listed below:
The surface modifications with bacterial anti-adhesive thin films and
antimicrobial AgNPs may show effectiveness to mitigate membrane biofouling
in the long-term water filtration. The hydrated PAH/PAA PEM structure or the
highly hydrophilic PDA film was effective in retarding the favorable deposition of
the bacterial cells on the PSU membrane. In addition, the incorporation of AgNPs
greatly inhibited the growth of bacterial cells on the membrane. Therefore, the
PEMs/AgNPs and PDA/AgNPs may have great potential and effectiveness to
mitigate membrane biofouling in the long-term water filtration in the real practice.
The surface modifications with PEMs/AgNPs and PDA/AgNPs could be easy
to implement on site in the membrane filtration system. The procedure to
modify the membrane surface by coating with polymeric thin films (e.g., PEMs
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and PDA) and AgNPs is quite simple and efficient to implement. In addition, this
surface modification strategy could be easily applied on existing membranes of
different configurations, such as flat sheet, hollow fiber and spiral wound.1
Therefore, the practical application of these surface modification techniques on the
membrane surface could be feasible in the membrane filtration system that are
already in use in water and wastewater treatment plants.
Membrane surface modifications with PDA and AgNPs may allow for the in
situ regeneration of AgNPs on the membrane surface. AgNPs on the
membrane surface will be eventually depleted through dissolution. It is impossible
to regenerate the AgNPs when the pre-formed AgNPs are embedded in the
membrane matrix or grafted on the membrane surface by chemical binding.
However, the PDA film may potentially enable the regeneration of AgNPs due to
the reductive nature of PDA and thus enable the sustainable application of
nanocomposite membranes for water filtration in the real practice.
5.4. Recommendations for Future Work
Further minimize the increase in the membrane’s hydraulic resistance after
modifications with PEMs or PDA. Because the assembly of PAH/PAA PEMs
and PDA film on top of the membrane surface could increase the hydraulic
resistance of the membrane, further studies should be conducted to optimize the
membrane modification procedure and conditions to minimize the increase in
membrane’s hydraulic resistance. For PEM modifications, the possible ways
include the optimization of polyelectrolyte concentration, number of layer, and
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rinsing velocity. For PDA modifications, the PDA circulating time and PDA
concentration can be optimized.
Further enhance the robustness of PEMs on the membrane surface. The
PAH/PAA PEMs assembled on the top of PSU membrane could be flushed away
gradually during water filtration and thus their effect on bacterial anti-adhesion
could be lost. Therefore, more studies on the cross-linking of the PAH/PAA PEMs
on the membrane surface to enhance their robustness are needed. In addition, more
studies on the regeneration of the new PAH/PAA PEMs on the membrane surface
are needed so that the membrane can quickly regain the bacterial anti-adhesive
properties when the PEMs assembled previously have been fouled or washed away.
Investigate the influence of solution chemistry on the membrane’s anti-
adhesive and antimicrobial properties after modifications with PEMs or PDA.
In this dissertation work, the solution chemistries for the evaluation of the bacterial
anti-adhesive and antimicrobial properties of the membranes were fixed at 10 mM
NaCl (pH 7.0) and at 154 mM NaCl (pH unadjusted), respectively, because the
main focus of this study is to investigate the membrane’s performance to retard the
adhesion and growth bacterial cell after modifications with PEMs or PDA and
AgNPs. However, the solution chemistry is likely to impact the degree of AgNP
dissolution.2-3 Therefore, a more systematical study may be required to investigate
the influence of solution chemistry on the dissolution of AgNPs on the membrane’s
bacterial anti-adhesive and antimicrobial properties.
Investigate the effectiveness of PEM/AgNP- and PDA/AgNP-modifications on
membrane’s properties to mitigate biofoulong in the long-term water
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filtration. In this dissertation work, the influence of AgNP/PEM- and PDA/AgNP-
modifications on membrane’s bacterial anti-adhesive properties was examined by
evaluating the initial bacterial deposition kinetics and removal efficiencies on the
membrane. The membrane’s antimicrobial properties were examined separately by
quantifying the growth of bacterial colonies using a CFU enumeration method.
However, the short-term bacterial deposition and release experiments and the CFU
enumeration method cannot directly assess the membrane’s ability to mitigate long-
term biofouling. Therefore, the long-term filtration experiment using the bacterial
suspension with similar solution chemistry as the natural aquatic environment
should be conducted in the future to thoroughly evaluate the effect of the
AgNP/PEM- and PDA/AgNP-modifications on the membrane’s ability to resist
biofouling. The effectiveness of the surface modifications on the biofouling
resistance of the membranes can be evaluated by measuring the permeate flux
decline under a constant transmembrane pressure and comparing to that of the base
membrane.4 In addition, the development of biofilms on the fouled membranes can
be investigated by using a laser scanning confocal microscopy (LSCM). The
biofilm will be then analyzed to obtain images of live cells, dead cells, and EPS
within the biofilm.
5.5. References
1. Mulder, M., Basic Principles of Membrane Technology. Springer: 1996.
2. Li, X.; Lenhart, J. J.; Walker, H. W., Dissolution-accompanied aggregation
kinetics of silver nanoparticles. Langmuir : the ACS journal of surfaces and colloids
2010, 26 (22), 16690-8.
3. Huynh, K. A.; Chen, K. L., Aggregation kinetics of citrate and
polyvinylpyrrolidone coated silver nanoparticles in monovalent and divalent electrolyte
solutions. Environmental science & technology 2011, 45 (13), 5564-71.
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4. Herzberg, M.; Elimelech, M., Biofouling of reverse osmosis membranes: Role of
biofilm-enhanced osmotic pressure. J Membrane Sci 2007, 295 (1-2), 11-20.
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Curriculum Vitae
LI TANG [email protected]
Education
Johns Hopkins University (JHU), Baltimore, MD 2009 - present
Department of Geography and Environmental Engineering, Whiting School of
Engineering
Ph.D. Candidate, Environmental Engineering Expected by March 2015
M.S.E, Environmental Engineering May 2012
Harbin Institute of Technology (HIT), Harbin, China 2003 - 2009
School of Municipal and Environmental Engineering
M.S.E., Municipal Engineering July 2009
B.S.E., Water and Wastewater Engineering July 2007
Research Expericene During Ph.D. Study
Graduate Research Assistant, JHU September 2015 - present
Membrane Surface Modification with AgNPs and Polyelectrolyte Multilayers (PEMs) to
Mitigate Biofouling
Designed, assembled and implemented a direct microscopic observation membrane
filtration system to quantify bacterial deposition kinetics and reversibility on the
membrane surface during water filtration, thereby enabling the fast and accurate
evaluation of membrane’s anti-adhesive properties
Successfully modified the surface of a commercial polysulpone membrane with
AgNPs and PEMs by using the layer-by-layer technique in a laboratory-made
membrane surface modification flow cell
Significantly enhanced membrane’s bacterial anti-adhesive and antimicrobial
properties: bacterial deposition kinetics was reduced by half and deposition
reversibility was increased to ~ 100% (< 10% for unmodified membranes); bacterial
colony growth on membrane surfaces could be completely inhibited
Systematically quantified the interfacial interactions between a bacteria surrogate
colloid and the membrane surface by performing AFM force measurements, and
identified the highly swollen and hydrated PEM structure could contribute to the
enhanced bacterial anti-adhesive properties
Membrane Surface Modification with Bioinspired Polydopamine and AgNPs Formed in
situ to Mitigate Biofouling
Fabricated polysulfone membranes using the phase inversion method and optimized
the fabrication parameters and procedure
Successfully developed an effective and efficient membrane surface modification
method with polydopamine and AgNPs formed in situ
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Greatly enhanced membrane’s bacterial anti-adhesive properties: bacterial
deposition kinetics was reduced by 60%
Identified that the improved surface hydrophilicity could contribute to the enhanced
bacterial anti-adhesive properties
Imparted the membrane with strong antimicrobial properties: bacterial colony
growth on the membrane surface was completely inhibited due to AgNPs
Publications
Tang, L., Livi, K.J.T., and Chen, K.L., Polysulfone Membranes Modified with
Bioinspired Polydopamine and Silver Nanoparticles Formed in situ to Mitigate
Biofouling, Environmental Science & Technology Letters, 2015 (accepted).
Tang, L., Huynh, K. A., and Chen, K.L., Imparting Antimicrobial and Antifouling
Properties to Membranes through Modification with Polyelectrolyte Multilayers and
Silver Nanoparticles, Journal of Colloid and Interface Science, 2015 (in revision).
Tang, L., Gu, W.Y., Yi, P., Bitter, J.L., Hong, J.Y., Fairbrother, D.H., and Chen, K.L.,
Bacterial Anti-Adhesive Properties of Polysulfone Membranes Modified with
Polyelectrolyte Multilayers, Journal of Membrane Science, 2013, 446, 201-211.
Zhang, J., Ma, J., Yang, Y.X., Tang, L., Liu, B.C., and Wang, S.J., Catalytic
Ozonation of Nitrobenzene by Nanosized Rutile TiO2, China Water & Wastewater,
2010, 26 (7), 103-108.
Teaching Experience
Instructor, Ph.D. Students’ Independent Research, JHU January - February 2015
Trained four Ph.D. students on the techniques for conducting bacterial deposition
and reversibility experiments using direct microscopic observation membrane
filtration system, performing AFM force measurements, and membrane casting
Instructor, Undergraduate Summer Research Program, JHU Summer 2012
Managed two undergraduates for their summer independent research on conducting
bacterial adhesion tests on PEMs-modified membranes in a parallel flow cell
The results were summarized and included in a paper published in Journal of
Membrane Science 2013
Instructor, Master’s Student Independent Research, JHU Fall 2011
Supervised two master’s students for their independent research on membrane
modification and membrane characterization
Research focused on developing modification method using the layer-by-layer
technique
Membrane characterization results were summarized and included in the paper
published in Journal of Membrane Science 2013
Grader, JHU September 2012 - Spring 2013
Held weekly office hours to consult students on their course - related questions
Graded and provided feedback on homework assignments and exams for courses:
Environmental Colloidal Phenomena, Environmental Engineering Fundamentals I,
and Physical & Chemical Processes in Environmental Engineering