FINAL TECHNICAL PAPER
Interaction of Nanoparticles with Microbial Biofilm in Water Treatment
Facility Processes
Submitted To:
The 2014 Summer NSF REU Program
Sponsored By:
The National Science Foundation
Grant ID No.: DUE-0756921 and EEC-1004623
College of Engineering and Applied Science
University of Cincinnati
Cincinnati, Ohio
Prepared By:
Victoria Sumner – Junior, Chemical Engineering, University of Cincinnati
Stephanie Palmer – Pre-Junior, Chemical Engineering, University of Cincinnati
Dorien Clark – Sophomore, Chemical Engineering, University of Cincinnati
Report Reviewed By:
Margaret J. Kupferle, PhD, PE
REU Faculty Mentor
Program Chair, Environmental Engineering
and Associate Professor of Environmental Engineering
George A. Sorial, PhD
REU Faculty Co-Mentor
Head, Department of Biomedical, Chemical and Environmental Engineering
and Professor of Environmental Engineering
Hengye Jing
REU Graduate Research Assistant, Environmental Engineering
August 1, 2014
Abstract
Providing access to clean water is a growing international concern and is one of the Grand
Challenges of Engineering recently put forth by the National Academy of Engineering
(Committee of National Academy of Engineering, 2012). Water treatment processes are
impacted by biofilms. Biofilms are any group of microorganisms in which cells stick to each
other on a surface. When sufficient nutrients for growth are present, biofilm will grow and
form a protective matrix that can harbor harmful bacteria and is hard to disinfect. This
presents problems in water distribution pipes at water treatment facilities. Chlorination of
water in the water treatment system is used to kill pathogens within the water and provide
protection from bacterial contamination as the water is pumped through the distribution
system. However, the use of chlorine can be harmful to people in the long run due to the
formation of carcinogenic disinfection by-products. Alternative ways to decrease the use of
chlorination and control the growth of biofilm in water treatment distribution systems by
looking at its interaction with different piping materials and silver nanoparticles were
studied. Pipe material impacted biofilm growth with copper encouraging the most growth
and stainless steel the least. Silver nanoparticles sorbed to the biofilm quickly and were able
to effectively kill biofilm on the coupon materials. This suggests they could be used in future
applications to control biofilm, such as a coating inside pipelines in distribution systems.
Key Words: biofilm, water distribution, silver nanoparticles, disinfection byproducts,
adsorption
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1. Introduction.
Providing access to clean water is a growing international concern and is one of the
Grand Challenges of Engineering recently put forth by the National Academy of Engineering
(Committee of National Academy of Engineering, 2012). Water treatment processes are
impacted by biofilms. Biofilms, also known as microbial aggregates, are microorganisms that
accumulate on a solid–liquid interface and are encased in a matrix of highly hydrated
extracellular polymeric substance (EPS). EPS mainly consists of polysaccharides, proteins,
uronic acids, humic acids, DNA, and cell fragments (Späth, et al. 1998). Other forms of biofilm
are plaque that forms on teeth, algae which are present on rocks in lakes and rivers, and can
contain bacteria, archaea, protozoa, and fungi. It can form inside of medical equipment, such
as prosthetics or knee joints; it can also infect and form mucus in the lungs causing cystic
fibrosis. Biofilm grows within water treatment facilities as well as distribution pipes.
Because of the negative effects of biofilm growth on certain surfaces, its removal has been a
topic of study and research for quite some time. It is difficult to remove due to the formation
of a protective layer that is composed of EPS and water channels that serve as a permeable
barrier. (Vollmer et al., 2008; Sheng and Liu, 2011; Stewart and Franklin, 2008; SahleDemessie and Tadesse, 2011; Vu et al., 2009). Biofilm also has beneficial applications; it has
been used successfully in water and wastewater treatment for well over a century
(Cunningham, 2001-2010). If biofilm growth is consistently controlled and regulated in
specific environments, then it can be used for positive applications such as these. As previous
studies have shown, controlling where and how fast it grows is challenging.
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The water treatment process starts with the inlet feed from a water source, like a
river, flowing into holding reservoirs, where enough water for several days can be stored
and some settling of large particles occurs. Next, chemical flocculation encourages more
clumping of smaller particles for more solids to be removed in secondary sedimentation.
Then sand filtration removes any remaining particulates that got through the secondary
sedimentation process. Granular activated carbon adsorption and UV disinfection may be
used to remove organics that remain in the water. Finally, a disinfectant such as chlorine is
added through the clear wells to keep the water safe and kill any pathogens as it is
distributed to the consumers via the distribution pipelines. Biofilm can grow in these pipes
decreasing the quality of the drinking water that was just treated. Biofilm can protect and
shelter pathogens such as parasites, bacterial pathogens and intestinal viruses, which can be
released into water systems causing the deterioration of the quality of drinking water.
Currently, the majority of drinking water treatment facilities, including Greater
Cincinnati Water Works, add chlorine to drinking water to protect water from pathogens
during distribution to consumers. This reduces the amount of acute or immediate health risk
due to microbial contamination, but actually creates chronic or long term health risk to
consumers due to formation of disinfection byproducts (DPB’s) when chlorine reacts with
organic materials in the water. A major challenge that water utilities face is how to control
and limit the risks from pathogens and disinfection byproducts to minimize health risks to
the population (U.S. Environmental Protection Agency, 2013). Trihalomethanes, haloacetic
acids, bromate and chlorite are the main DBP’s that are created in distribution pipelines due
to the interaction of organic materials and chlorine. Some people who drink water containing
DBP’s in excess of the maximum containment level (MCL) over many years could experience
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liver, kidney, or central nervous system problems and increased risk of cancer (U.S.
Environmental Protection Agency, 2013). Ways to decrease the need for chlorination to
control microbial growth are of great interest to researchers. Choice of pipe materials and
the impact of nanoparticles are of interest in this application.
Different pipe materials have different roughness and surface charge properties that
may impact biofilm growth. They also may release toxic or stimulating substances into the
water that either kill or stimulate growth of biofilms. The coupon materials tested for
inhibition or stimulation of the growth of Pseudomonas fluorescens biofilm in this study
included copper, stainless steel, polyvinyl chloride (PVC) and polyethylene (PE).
Nanoparticles occur in everyday life in products such as makeup, coatings of metal
products and medical equipment. They are tiny particles that range from 1 to 100
nanometers in size. An increasingly common application is the use of silver nanoparticles for
antimicrobial coatings, and many textiles, keyboards, wound dressings, and biomedical
devices now contain silver nanoparticles that continuously release a low level of silver ions
to provide protection against bacteria (Olderburg, 2014). Due to the increasing prevalence
of Ag-NPs in consumer products, there is a large international effort underway to verify
silver nanoparticle safety and to understand the mechanism of action for antibacterial
effects.
In this project, adsorption and antibacterial effects of silver nanoparticles (Ag-NPs)
were studied to understand more about how Ag-NPs interact with Pseudomonas fluorescens
biofilms. Adsorption is the physical adherence or bonding of ions and molecules onto the
surface of another phase. It is important to know if nanoparticles adsorb to the biofilm or
pass through the system. If they are adsorbed to the biofilm, they may kill the bacteria in the
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biofilm given their reported antimicrobial effects. Some antibacterial effect studies indicate
that Ag-NPs will be highly toxic to natural bacterial communities if/when they reach the
environment. Studies have shown that Ag-NPs toxicity is size dependent at low
concentrations. One study observed that silver nanoparticles of 1-10 nm were preferentially
bound to cell membranes and were incorporated into bacteria, whereas larger nanoparticles
were not, while others indicated that toxicity was shape dependent. Additionally, it has been
shown that Ag-NPs caused pitting or corrosion on bacterial cell membranes, leading to
increased permeability and cell death (Fabrega, et. al 2009). A potential use of nanoparticles
in water systems is as a coating inside of the distribution pipes to reduce biofilm growth if
they prove to effectively decrease biofilm growth in data results.
Pseudomonas fluorescens, a gram-negative rod shaped type of bacteria, commonly
found in decaying organic material such as leaves, soil, plants and water surfaces, was used
in this study because it commonly occurs in water and grows quickly at room temperature.
It was obtained from MicroBiologics, Inc. (St. Cloud, MN). It is gram-negative because its EPS
mainly contains phosphoryl groups along with the polysaccharides. The negative charge
may impact biofilm growth on different pipe materials with different surface charges and
adsorption of nanoparticles to biofilm, depending on the charge of the particles.
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The goals of the research were to:

determine which materials will be best for preventing biofilm growth in pipelines in
water distribution systems,

understand the anti-bacterial effect of nanoparticles on biofilm and how they can be
used to prevent biofilm growth in the environment, and

learn and understand specific research methods and procedures with hands on
experience.
2. Materials and Methods
Biofilm Growth and Sampling: Biofilm reactors (see Figure 1) were used to grow
Pseudomonas fluorescens while feeding it carbon and nitrate nutrient solutions. Carbon and
nitrate nutrient solutions were pumped into the biofilm reactors at 1 mL/min. Nutrient
solutions had a pH around 7.5 to 8. For the biofilm reactor, a 1000-mL glass container with
a polyethylene lid that holds eight polypropylene coupon holders was used. A stirring vane
at the bottom of the reactor provided continuous mixing. To simulate biofilm growth in
pipelines, the biofilm was grown on four different types of coupons in one of the reactors.
The two additional reactors containing polycarbonate coupons were used for kinetic
sorption and antibacterial effect studies.
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Figure 1. Basic Biofilm Reactor Set-up
Pipe Materials Study: Biofilm growth on different coupon materials was observed by
removing coupon samples weekly for testing. The coupon materials chosen for this study
were: copper, stainless steel, polyvinyl chloride (PVC), and polyethylene (PE). Four separate
holding vials (one for each coupon material) were prepared with 10 mL of carbon solution
and 10 mL of nitrate solution so the biofilm on the coupon materials remained in the same
chemical environment prior to staining. A Laser Scanning Microscope (LSM) (Zeiss LSM710,
Carl Zeiss) at the U.S. EPA was used to measure numbers of dead and live cells from coupon
samples in both 2D and 3D. Each coupon was placed in a Petri dish and staining solution,
also known as the BacLight Live/Dead bacterial viability kit (Life Technologies Corporation),
was used to stain the samples. The stained coupons were observed using the Laser Scanning
Microscope and the thickness of biofilm on each material was recorded. Three sets of
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pictures were taken of the current biofilm growth and decay from each week and the images
were analyzed for live/dead pixels per standard surface area of 2120 µm x 2124 µm.
Surface Zeta-Potential Analysis: The Zeta-Potential Analyzer was used to determine the
overall surface electrical charge of the biofilm and the piping materials at the U.S. EPA. A
0.001 M potassium chloride solution was made for the electrolyte solution used in the
analysis. The analyzer then was rinsed with deionized water and the pH meter was
calibrated with solutions at 4, 7, and 10. The conductivity was calibrated at 1413 µs/cm and
the conductivity constant was measured to be 1.109 cm. After putting the electrolyte solution
in the container (450 mL), the first material, copper (see Figure 2), was analyzed. Before
running the machine, the gage gap was checked to make sure that the pressure was around
300 mbar and the flow rate was around 100 mL/min. The flow check needed to have a slope
that was linear with no spikes before running titration. The titration was run from a pH of 2
to 11 using potassium hydroxide and the titration was run from a pH of 12 to 2 using
hydrochloric acid. These steps were repeated with stainless steel, polyvinyl chloride, and
polyethylene.
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Figure 2. Picture of a copper coupon materials being placed to be run on the Zeta-Potential
Analyzer.
Scanning Electron Microscope: A JEM7600F (JEOL USA Peabody, MA) Scanning Electron
Microscope (SEM) was used to scan the surface of the biofilm on the materials. Christina
Bennett-Stamper at the U.S. EPA took the images using the SEM.
1.
2.
Figure 3. (1) Scanning Electron Microscope surface image of Pseudomonas fluorescens with citrate
reduced nanoparticles magnified to 3,500, with a depth of 1 µm. (2) Scanning Electron Microscope
surface image of Pseudomonas fluorescens with citrate reduced nanoparticles magnified to 8,500,
with a depth of 1 µm.
Silver Nanoparticle (Ag-NP) Synthesis and Purification: Sodium citrate (Na3C6H5O7) was
used as a reducing agent to separate the silver in a silver nitrate (AgNO3) to synthesize the
silver nanoparticles (AG-NPs). Specifically, a solution of 1x10^-3 M AgNO3 (99.99%) was
mixed with a solution of 1x10-2 M Na3C6H5O7 ·H2O (99+%) in a volume ratio of 2:1,
respectively. The mixture was heated at 70 .C for 4 hours in a water bath. The color of the
solution changed from clear to a tan color. After the first hour, the solution had to be checked
every hour for aggregation. A Tangential Flow Filtration system (see Figure 4) used to purify
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the Ag-NP from ions in the matrix. The ions were separated as permeate into the waste
container and the pH of the retentate Ag-NP solution was lowered to ~5.
Figure 4. Tangential Flow Filtration Set-up: was used to purify the previously synthesized silver
nanoparticles.
Kinetic Sorption Study: For the kinetic sorption study, the silver nanoparticle solutions were
prepared at 3 different pH levels (5, 7.5 and 9) for 7 different time intervals or 21 in total.
Polycarbonate coupons from the second biofilm reactor were dropped into the pH-adjusted
Ag-NP solution at time zero. The coupons were allowed to sit undisturbed in Ag-NP solution
and at designated sampling times, a coupon was carefully removed with tweezers from a
given sample and the remaining solution was analyzed using a UV-visible spectrometer.
Absorbance was measured at 30, 60, 120, 180, 240, 300, and 360 minutes. Absorbance was
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related to Ag-NP concentration using a standard curve and reported as a function of time for
each material.
Antibacterial Effect Study: The silver nanoparticle solution was diluted in half with deionized
water in a 1 L volumetric flask and the diluted solution was placed in a clean empty biofilm
reactor container. The biofilm coupon lid with polycarbonate coupons from one of the
growth reactors was placed in the new biofilm reactor filled with nanoparticle solution. After
designated time increments, coupon samples were removed and biofilm growth was
observed at times of 30, 60, 120, 180, 240, 300, and 360 minutes using the Laser Scanning
Microscope as described previously.
3. Results and Discussion
Pipe Material Study: A Zeta-Potential Analyzer, Scanning Electron Microscope, and Laser
Scanning Microscope were used to study the impact of pipe material on biofilm growth.

Zeta-Potential Analysis: The overall surface charge can help determine how readily
biofilm organisms will attach to the different materials. Data plotted in Figure 5 show
that the zeta potential of polyvinyl chloride is more positive around the pH of 7.5 and all
the other materials are less than zero. The pH of 7.5 was chosen as a reference point
because it is the pH used to grow the biofilm. Speculations can be made that the
negatively charged biofilm could have attracted to the positively charged PVC, which
could explain the medium to high amount of biofilm growth on the coupons.
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Copper
Stainless Steel
Polyvinyl Chloride
Polyethylene
Polycarbonate
P. fluorescens Biofilm
Figure 5. A graph of the surface charge or zeta-potential of copper, stainless steel, polyvinyl chloride,
polyethylene, polycarbonate, and the P. fluorescens biofilm at different pH levels. Results for pH level
7.5 are of interest since that was the pH of the nutrient solution.

Scanning Electron Microscope: Representative Scanning Electron Microscope photos of
the bare surfaces of the piping materials used in this study are shown in Figure 6. These
images were used to explore surface roughness and to help explain biofilm formation.
Note the large crevasses on the surface of copper, which could be one reason why it had
high biofilm growth. It also has an electrochemical structure that could have affected the
biofilm decay. In general, the polymer materials were smoother than the metals. The
rough coefficients found in the literature were all the same due to the way they were
manufactured.
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1.
2.
3.
4.
Figure 6. Scanning Electron Microscope surface images of the copper coupon (1), stainless steel
coupon (2), polyvinyl chloride (3), and polyethylene (4).

Laser Scanning Microscope: The Laser Scanning Microscope was used to record the
amount of live cells versus dead cells per coupon area each week. A sample image for
Week 4 on copper is shown in Figure 7A. A 3D plot of the thickness of the biofilm grown
is shown in Figure 7B. Figure 8 summarizes the data for the number of live cells for each
week. In general, the amount of green or alive biofilm organisms declined each week. For
copper, the growth was high for the first week, but declined each week. Speculations
could be made that this was due to the toxicity of its ions killing the biofilm. For the
stainless steel, there was not much growth at all. For PVC, there was medium to high
growth, but was patchy as seen by the error bars. For PE, it had a decreasing trend like
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copper, but the 5th week, there was a huge increase in biofilm growth. This could be due
to its structural properties.
A.
B.
Figure 7. A. 2D representation of biofilm growth on a copper coupon from week 4. B. 3D graph of the
biofilm growth on a coupon showing its thickness.
Figure 8. Graph shows a plot of the average biofilm growth on all of the material coupons over a five
week period.

Summary of Findings: Results of this study are summarized in Table 1. For copper, high
biofilm growth was expected due to its rough surface, large crevasses, and corrosive
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behavior. Stainless steel and PVC were expected to have low biofilm growth due to their
smooth surface. PE was expected to have high growth of biofilm due to its ability to
release nutrients like carbon for the biofilm to feed on. Copper and stainless steel proved
to have the amount of biofilm growth that was expected due to properties of their
surfaces, although the biofilm on the copper decreased each week. The biofilm growth
could have decreased due to the toxicity of its ions being released. The PVC coupon
however, had high biofilm growth, but was patchy. PVC had a positive surface charge at
the pH used for the study, so this may have increased growth of the gram-negative
bacteria used. There was a decreasing trend for the growth of biofilm on PE as well, until
the 5th week. Speculations could be made that the growth was due to its ability to give off
carbon as a nutrient for the biofilm to eat. These results could have been affected by the
set-up of the experiment however all four materials were in the same biofilm reactor so
the release of ions from copper or the carbon from the PE may have impacted the results.
A future study separating coupon material by reactor would help discover if this was an
effect.
Table 1.
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Nanoparticle Study: In addition to nanoparticle synthesis, two studies were conducted to
explore nanoparticle interactions with biofilm, a kinetic sorption study to see how quickly
they accumulate in biofilm and an antibacterial effect study to see if they can be used to
control biofilm.

Kinetic Sorption Study- Sorption happened when the biofilm was suspended in the silver
nanoparticles solution. As seen in Figure 9, most of the sorption occurred in the first 30
minutes of contact. As time progressed, it reached equilibrium implying that the biofilm
sorbed as much silver nanoparticles as it could.
Figure 9. Graph shown is a plot of the concentration of the nanoparticles after they’ve interacted
with the biofilm over time.
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
Anti-Bacterial Effect Study- The Ag-NP solution proved to kill the biofilm over time. A lot
of biofilm was able to grow on the materials, but they all had a trend of decreased growth
and having an increase of dead cells.
A.
B.
Figure 10. A. This is a 3D graph shown in 2D of the live cells of the biofilm over 6 hour period of time.
B. This is a 3D graph shown in 2D of the dead biofilm over a 6 hour period of time.
4. Conclusion
Conclusions can be made about what effect different materials have on the growth of
biofilm (piping material study), and about how biofilm interacts with silver nanoparticles
(nanoparticle study) from the data we collected over the past 7 weeks. For the piping
material study, it was found that stainless steel performed the best at preventing biofilm
growth. However due to its high cost, stainless steel is uneconomical to use in a water
distribution system but it may be useful in limited applications such as hospitals or in the
food industry. Copper performed very poorly and recorded the highest biofilm growth over
the 5 weeks of sampling. This could be partly because the large crevasses that are found on
its surface from the Scanning Electron Microscope results; a rough surface structure allows
the biofilm to attach to the surface of the material. The last two materials that were tested
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gave unexpected results. From literature review, Polyvinyl Chloride (PVC) was expected to
have a low growth of biofilm, due to a smooth surface structure, and Polyethylene (PE) to
have a high growth of biofilm, due to nutrients on the surface of the material. In the beginning
weeks, PVC recorded medium growth of biofilm on its surface, which may have been related
to its positive surface charge at pH 7.5. The biofilm had a negative charge, which could
explain why they were attracted to each other. For PE, the amount of biofilm on the surface
grew dramatically in the later weeks of sampling. This could have happened because it takes
time for the carbon chains on the surface of the coupon to break down, and provide nutrient
for the biofilm. All of the data collected from the piping material study show that stainless
steel would be very good for a water distribution system if the cost was not so high. PVC
(which is currently used in pipelines), would be the next best option because it is so cost
effective and can prevent biofilm well enough.
The second major study conducted during the program was the nanoparticle study.
This study was to show how biofilm interacts with silver nanoparticles. This study was
comprised of two main parts: a kinetic sorption study and an antibacterial effect study. The
size distribution of the nanoparticles was also examined and it was observed that pH affects
the size of silver nanoparticles and that slight aggregation occurs within the first 30 minutes.
The kinetic sorption study was performed to see how the biofilm sorbed and released the
silver nanoparticles over time. From the data collected, it was observed that biofilm does, in
fact, absorb silver nanoparticles with most of the uptake occurring in the first 30 minutes.
There was some variance in the data possibly be due to coupon variance. The antibacterial
effect study was conducted to visualize if the silver nanoparticles killed the biofilm once it
was absorbed. The Laser Scanning Microscope was used to visualize the thickness of live and
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dead biofilm cells over time. As time progressed, the thickness of biofilm cells decreased,
which show that the nanoparticles had predicted the anti-bacterial effect. There was a
decrease in live biofilm cells and a steady increase in dead biofilm cells over a time period of
6 hours. The data collected about the nanoparticles show that silver nanoparticles have an
antibacterial effect on biofilm. This work can be used to study the application of
nanoparticles in water distribution systems, such as coatings on pipes.
6. Acknowledgements
We would like to thank to the National Science Foundation for funding our project via two
grants (DUE-0756921 and EEC-1004623), the U.S. Environmental Protection Agency for
allowing us to use their facilities and equipment, and Christina Bennett-Stamper at U.S. EPA
for Scanning Electron Microscope photographs.
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